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Modification of alpha-adrenergic responses of small arteries by altered PCO2 and pH.

Closed-circuit television microscopy was used to measure in vivo small artery (75--140 microns) and vein (105--230 microns) diameters to determine if changes in tissue PCO2 and/or pH would alter the microvascular responses to norepinephrine. Sprague-Dawley rats were anesthetized with a combination of urethane (800 mg/kg) and alpha-chloralose (60 mg/kg). The cremaster muscle with intact circulation and innervation was suspended by sutures in a 60-ml bath which contained a modified Krebs solution (31 degrees C) that was buffered by Tris of bicarbonate. There were four groups of animals with different combinations of bath PCO2 and pH: (1) PCO2 less than 10 mm Hg and pH = 7.2, (2) PCO2 less than 10 mm Hg and pH = 6.9, (3) PCO2 = 60--70 mm Hg and pH = 7.2, and (4) PCO2 = 60--70 MM Hg and pH = 6.9. The maximal responses of the small artery and vein to norepinephrine were similar for the four groups. The artery sensitivity to norepinephrine was significantly lower for group 4 when compared to groups 1, 2 and 3, but there was no effect on small vein sensitivity. Thus, the combination of decreased pH and increased PCO2 reduces small artery sensitivity to norepinephrine in the cremaster muscle of the rat.

[Effect of preparations that alter catecholaminergic processes on the serotoninergic syndrome of head twitching in mice].

Specific neuroleptics suppressed head twitching (HT) in mice, provoked by d,1-5-hydroxytryptophane (200 mg/kg i. p.) in doses starting from 0.00025 mg/kg (spiroperidol). L-DOPA and piribedil inhibited HT in doses from 25 and 50 mg/kg respectively, whereas apomorphine and d,1-amphetamine in doses of 1 up to 10 mg/kg exerted ambivalent activity. HT was significantly attenuated by clonidine in doses from 0.25 mg/kg, whereas by noradrenaline, isoprenaline and naphthizine, injected into the brain ventricles, in doses of 1 microgram, and 0.025 microgram per mouse respectively. Destruction of brain catecholaminergic neurons by intraventricular 6-hydroxydopamine (50 micrograms per mouse) caused a strong enhancement of HT. However, partial protection of the adrenergic (but not the dopaminergic) neurons by pretreatment with desipramine or similar drug AW 15,1129 eliminated the protective effect of 6-hydroxydopamine. It is concluded that there is the dopaminergic link in the mechanism of HT and that the stimulation of the central adrenoreceptors inhibits HT.

Barrier methods of contraception: a reappraisal.

In the last two years, there has been a gradual reawakening of interest in barrier methods and an increase in their usage by both men and women. This is in large part due to concern about the sometimes serious side effects reported for other contraceptive methods. The return to these techniques is particularly important, given the current epidemics of teenage pregnancy and veneral disease. One of the major problems in relation to barrier methods today is the accurate determination of their efficacy. There are very limited data with statistical validity available to judge the exact rate of effectiveness one might obtain using one of these techniques. The National Survey of Family Growth, conducted by the National Center for Health Statistics, showed a failure rate per 100 women of 16.7 for foam, cream or jelly and 15.9 for diaphragms (22). There is a great need for new and improved barrier methods of contraception. Numerous clinical studies are being set up to test spermicidal agents and vaginal sponges for the female, as well as such things as disposable condoms for males.

Vasectomy: benefits and risks.

Bilateral occlusion of the vas deferens, vasectomy, is progressively becoming the method of choice for couples seeking permanent contraception at a younger age, with smaller families. They are apparently well-informed and view the procedure as a natural step. Vasectomy is an inexpensively performed office procedure that causes minimal disruption of routine and has a high degree of community acceptance. The risks of significant hematoma, infection, discomfort and other sequelae are within acceptable limits. Improved techniques will continue to reduce the small failure rate. Antibodies observed in half of the patients have not been linked to systemic disease, although they are a hazard for the one patient in 500 returning for a vasovasostomy. Refinements in microsurgery and availability of artificial insemination enhance vasectomy as the method of choice. Evidently, extending the minimum time of sterility confirmation permits detection of occasional recanalization from technical failures. Adequate screening of the couple's motivation and expectations can prevent the rare psychologic disturbances, the greatest risk with this procedure and a problem associated with all options. For the male, there is no competitive technique at this time. In a world striving for equal rights, where the female still carries the burden of temporary contraception, the simplicity and popularity of vasectomy for permanent contraception add the desired undertones of social equilibrium.

Interpretation of data by the clinician.

The cardinal challenges to every practicing physician are to interpret clinical data correctly and to place them in proper perspective. Clinical investigations frequently lack the rigidly controlled conditions and the careful experimental designs usually found in preclinical animal studies, and this deficiency is partially attributable to the inherent complexities of clinical medicine. Consequently, a great deal of controversy results from conflicting interpretations, extrapolations and overextension of limited data that are often equivocal. More careful appraisal of data and increased awareness of the well-known pitfalls found in retrospective and prospective studies, in which biostatistical design and clinical relevance are often incompatible, are emphasized, and personal biases and the flagrant sensationalism expounded by the media are condemned. The clinician is cautioned to sift through the data, consider the benefit/risk ratio for each patient and then to subordinate the role of critical scientist and assume the role of physician, exercising good judgment in light of the existing evidence and the immediate problems at hand.

Uterine and peripheral hematologic profiles in molar pregnancy.

The potential differences in hematologic profiles of blood samples drawn simultaneously from the right utero-ovarian vein and from the upper extremity were investigated in four patients with uncomplicated molar pregnancy in stable obstetric conditions. The patients had undergone no previous chemotherapy and were scheduled for total abdominal hysterectomies. The dominant abnormalities in uterine venous blood were prolongation of thrombin time; shortening of activated partial thromboplastin time; positive protamine sulfate test; and increase in coagulation factors II and VII, with a tendency to low values in factor V. Peripheral samples gave almost parallel results in all altered and normal tests, except in one case with very striking differences in factors II, V, VII and X. Several local and systemic influences are discussed. It is concluded that molar pregnancy seems to have important systemic mechanisms affecting the stability of the blood coagulation homeostasis, which act in addition to those at a local level.

Fate of oral neutralizing antacid and its effect on postprandial gastric secretion and emptying.

The fate and neutralizing efficiency of oral antacids (aluminum and magnesium hydroxides) as well as their effect on postprandial gastric function were quantified in 6 patients with duodenal ulcer disease. We employed a double-marker technique for measurement of gastric secretion and emptying and combined this with back-titration of the gastric samples and analysis of aluminum to trace the fate of antacid in the stomach and duodenum. These studies show that: (a) antacid therapy with aluminum and magnesium hydroxides significantly increases gastric secretion; (b) intragastric neutralization of gastric acid produces a significant and substantial decrease in net acid output (acid secreted minus acid neutralized), but the beneficial effects of neutralization are partially offset by incomplete intragastric formation of aluminum trichloride; (c) most but not all of the ingested antacid is utilized in acid neutralization in the stomach (average 78.6% in our 6 patients); and (d) antacid therapy does not modify the absolute rate of postprandial gastric emptying, but increases dilution of gastric contents, expanding the intragastric volume. Thus, the fractional gastric emptying rate declines, and this, in turn, should enhance antacid utilization by delaying its emptying.

Killing of Neisseria gonorrhoeae by human polymorphonuclear neutrophil granule extracts.

Neisseria gonorrhoeae was grown in vitro (on agar and in broth) and in vivo (in 10-day chicken embryos) and tested for its sensitivity to the bactericidal action of human neutrophil granule extracts. Under all conditions studied, type 1 and type 4 N. gonorrhoeae were killed equally well by dialyzed extracts of neutrophil granules (containing both azurophil and specific granule contents) and by the myeloperoxidase-Cl- - H2O2 bactericidal system. However, sensitivity to the bactericidal activity of granule extracts depended upon growth conditions and growth phase. Log-phase, egg-grown gonococci were the most sensitive; they were killed 100% by 250 to 300 micrograms of granule extract (60 min, 37 degrees C) per ml. N. gonorrhoeae grown on agar for 20 h (to stationary phase) were the least sensitive, being killed only 80 to 90% with 500 micrograms of granule extract per ml. Thus, susceptibility to granule extract of gonococci grown under the four conditions studied in this report decreased in the order: log phase, egg grown; log phase, broth grown; stationary phase, egg grown; and stationary phase, agar grown. Killing was time and temperature dependent; little killing occurred when incubations were done at 10 degrees C. Boiled granule extract had only minimal effects on N. gonorrhoeae viability. Addition of catalase (500 U/ml) to the granule extract bactericidal system did not protect; however, the same concentration of catalase completely inhibited the bactericidal activity of the myeloperoxidase-Cl- - H2O2 system.

Murine lymphoma alkaline phosphatase: a cell membrane carcinofetal enzyme.

Alkaline phosphatase (APase) has been shown to have a membrane-bound localization in the murine fetal thymus, in murine thymic lymphoma and in adult spleen. Since it was suggested from these previous experiments that the lymphoma APase might represent an embryonic function, a detailed biochemical comparison of the lymphoma APase with the fetal thymus, placenta, fetus and spleen APases was performed. The parameters investigated were pH optimum, activation, inhibition, heat inactivation, substrate ratios, Michaelis constant, and electrophoretic analysis in the presence and absence of neuraminidase with the substrates alpha-naphthyl phosphate, beta-glycerophosphate, and p-nitrophenyl phosphate. The results indicate that the lymphoma APase is very similar to the fetal thymus, placenta and spleen APases. Furthermore, these results lend support to the hypothesis that the APase activity which appears in thymic lymphoma might represent a derepressed embryonic function. Thus, the murine lymphoma APase may be termed a cell membrane carcinofetal enzyme.

Partial characterization of the acidic and basic polypeptides of glycinin.

The subunits of the 11 S storage protein from soybean cultivar CX635-1-1-1 were purified and characterized. Six polypeptides with acidic isoelectric points and four with basic isoelectric points were isolated from the purified storage protein. The acidic polypeptides had phenylalanine, leucine, isoleucine, and arginine at the NH2 termini, while the basic polypeptides all had glycine at the NH2 termini. Amino acid analysis indicated that certain acidic and basic polypeptides contained 3 to 6 times more methionine than the other polypeptides. Since the low nutritional quality of legume storage proteins is due to a deficiency in methionine, this observation will have significance in efforts to improve soybean quality. The purified polypeptides were further characterized by NH2-terminal sequence analysis. Considerable homology was found between the members of individual families of acidic and basic polypeptides, indicating that the members of each family arose from a common ancestral gene. This study showed that the glycinin polypeptide composition is more complex than previous reports indicated, and for the first time characterized the various polypeptides of the 11 S storage protein by structural analysis.

Effects of low electrolyte media on salt loss and hemolysis of mammalian red blood cells.

Cation loss and hemolysis of various mammalian red cells suspended in isotonic non-electrolyte media were investigated. Sucrose buffered with 10 mM Tris-Hepes, pH 7.4 was used as the non-permeable non-electrolyte. Mammals from which the red cells were derived include the human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, all of which contain K as the predominant cation species (HK type) and the dog, cat, sheep and cow, all of which possess Na as the predominant cation species (LK type). Of HK cells, a rapid efflux of K takes place from humans, rats and guinea pigs. Of LK type cells, the dog and cat exhibit an augmented membrane permeability to Na. The governing factors which influence cation permeability are the change in pH, temperature, and ionic strength. In response to increase in pH, the red cells of humans, dogs and cats become more permeable to cations, whereas the red cells of rat and rabbit are unaffected. In response to increase in temperature, HK type cells exhibit augmented K efflux, while the Na loss from the dog and cat cells manifest a well-defined maximum at near 37 degrees C. In all cases, a small substitution of sucrose by an equal number of osmoles of salts results in a dramatic decrease in cation loss. By contrast, the red cells of the rabbit, newborn calf, adult cow, newborn piglet, adult pig and sheep display no discernible increase in ion-permeability under the conditions alluded to above. In some species including the newborn calf, dog, and cat, an extensive hemolysis occurs usually within an hour in isotonic buffered sucrose solution. The osmolarity of sucrose solution affects these cells differently in that as the osmolarity increases from 200--500 mM, hemolytic rates of the calf and dog reach a saturation near 300 mM sucrose, whereas the hemolytic rate of the cat decreases progressively. Common features pertaining to this hemolysis are (1) the intracellular alkalinization process; and (2) the diminution of the cell volume which take place prior to and onset of hemolysis. SITS, a potent anion transport inhibitor, completely protects the cells from hemolysis by inhibiting chloride flux and the concomitant rise in intracellular pH.

Local xenogeneic graft-vs-host reaction: a practical assessment of T cell function among cancer patients.

The local xenogeneic graft-vs-host reaction (XGVHR) was used as a practical bioassay to assess T lymphocyte function and immunocompetence among cancer patients. Positive XGVHR was found in 99.5% of normal donors, 70% of cancer patients with early stage disease, and 30% of cancer patients with metastatic disease (p less than 0.001). A minimum of 4.5 x 10(6) immunocompetent T lymphocytes are necessary in order to elicit a positive XGVHR. Negative reactions among cancer patients are characterized by the lack of edema fluid accumulation and the appearance of the most basophils at the test site. This suggests that insufficient amounts of lymphokines are being released by the incompetent T lymphocytes, whereas the host is capable of mounting a rejection reaction as evidenced by the appearance of the basophils. Preliminary evidence suggests that the immunologic defect detected by the XGVHR cannot be corrected by monocyte depletion. The identification of putative suppressor T cell subsets may bear immunotherapeutic implications in the future.

The relation between dicarbocyanine dye fluorescence and the membrane potential of human red blood cells set at varying Donnan equilibria.

The fluorescence, F, of two dicarbocyanine dyes, diS-C3(5) and diI-C3(5), depends both on the membrane potential, E, and on the intracellular pH, pHc, or human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc varies at constant E. Dye fluorescence measurements in these suspensions yield calibrations of +1.7 % delta F/mV for diS-C3(5) and +0.6 % delta F/mV for diI-C3 (5). While pHo does not affect F of either dye, changes in pHc of 0.1 unit at constant E cause changes of F equivalent to those induced by 2--3mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pHc co-vary. The relation of F to E also depends in a complex way on the type and concentration of cells and dye, and the wavelengths employed. The equilibrium calibration of dye fluorescence, when applied to diffusion potentials induced by 1 microM valinomycin, yields a value for the permeability ratio, PK.VAL/PCl, of 20 +/- 5, in agreement with previous estimates by other methods. The calibration of F is identical both for diffusion potentials and for equilibrium potentials, implying that diC-C3(5) responds to changes in voltage independently of ionic fluxes across the red cell membrane. Changes in the absorption spectra of dye in the presence of red cells in response to changes in E show that formation of nonfluorescent dimers contributes to fluorescence quenching of diS-C3(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-C3(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.

CNS Modulation of adrenal tyrosine hydroxylase in Parkinson's disease and metabolic encephalopathies.

Tyrosine hydroxylase (TH) activity was assayed radioenzymatically in various regions of post-mortem brains of human individuals without neurologic disorders (controls), with Parkinson's disease, senile dementia, hypertensive encephalopathy, hepatic and diabetic coma, liver cirrhosis without coma, and hepatic coma treated with parenteral administration of L-valine. In addition TH activity of the post-mortem adrenal medulla was assayed in controls, in Parkinson's disease, senile dementia and hypertensive encephalopathy. In Parkinson's disease TH activity was significantly decreased in the nigrostriatal system, and less severe in other brainstem areas, while the raphé-reticular formation and limbic system showed normal values. In addition, there was significant decrease in the TH activity of the adrenal medulla, suggesting that Parkinson's disease is a generalized disorder not limited to distinct CNS areas, and that impairment of the dopaminergic niggro-striatal system may involve the TH activity in the adrenal medulla, thus inducing disorders of the peripheral sympathetic system. Senile brain atrophy showed no definite changes in brain, except the striatum, and adrenomedullary TH, while in one case of hypertensive encephalopathy due to long-term corticosteroid treatment normal TH activity in the adrenal medulla was opposed by decreased striatal TH activity, probably due to cerebral ischemia. TH activity in the caudate nucleus of individuals with both hepatic and diabetic coma were within normal ranges, suggesting a sufficient energy supply of the brain during such metabolic catastrophes, while reduced brain TH activity in patients with hepatic coma who died of acute gastrointestinal bleeding is probably due to severe final cerebral ischemia. No correlative data on brain and adrenomedullary TH activities in metabolic encephalopathies are available so far.

On two-dimensional passive random walk in lipid bilayers and fluid pathways in biomembranes.

The lateral mobility of pyrene, pyrene decanoic acid, and 1-palmitoyl-2-pyrene decanoyl-phosphatidyl choline (pyrene lecithin) in lipid bilayers is determined by the excimer formation technique. This method is applied to vesicles of lecithins differing in chain length and in the degree of saturation of the hydrocarbon chains. These values are compared with results in cephalins of different chain length and in dipalmitoyl phosphatidic acid at variable pH. The influence of cholesterol is investigated. The results are analyzed in terms of the Montroll model of two-dimensional random walk. The jump frequency of the probe molecule within the lipid lattice is obtained. The advantage of this measure of transport in lipid layers is that it does not involve lipid lattice parameters. The main results of the present work are: (i) The lateral mobility of a given solute molecule in lamellae of saturated lecithins is independent of hydrocarbon chain length and rather a universal function of temperature. (ii) In unsaturated dioleyl lecithin the amphiphatic molecules have lateral mobilities of the same size as in saturated lipids. The jump frequency of pyrene, however, is by a factor of two larger in the unsaturated lecithin. (iii) The jump frequencies in phosphatidyl ethanolamines are about equal to those in lecithins. (iv) In phosphatidic acid layers the hopping frequencies depend on the charges of the head groups of both the lipids and the probes. (v) Cholesterol strongly reduces the jump frequency in fluid layers. (vi) The lateral mobility in biological membranes is comparable to that in artificial lipid bilayers. The experimental results are discussed in terms of the free volume model of diffusion in fluids. Good agreement with the predictions made from this model is found. A striking result is the observation of a tilt in dioleyl-lecithin bilayer membranes from the hopping frequencies of pyrene and pyrene lecithin. A tilt angle of phi = 17 degrees is estimated.

[Stimulating effects of alanine and glycine on guinea-pig taenia coli (author's transl)].

Effects of alanine and glycine on the mechanical and electrical activities of guinea-pig taenia coli were investigated. Alanine (0.1--10 mM) and glycine (0.5--10 mM) produced a dose dependent contraction of guinea-pig taenia coli. The stimulating effects of alanine and glycine were not inhibited in the presence of atropine (5 muM), tetrodotoxin (0.1 micrograms/ml), diphenhydramine (4 muM), methysergide (3 muM), strychnine (10 muM), and were not influenced by treatment with indomethacin (3 muM). However, these effects were inhibited in the presence of a Ca antagonist, verapamil (10 muM). When the electrical activities of the taenia coli were recorded by the single sucrose-gap method, alanine and glycine (5--10 mM) produced a reduction of membrane potential and increased spike heights and frequencies of action potential. In LiCl or Choline-Cl and in Na-isethionate solutions, the stimulating effect of alanine was not abolished, but was completely inhibited in KCl-depolarized preparation. From these results, it is considered that both alanine and glycine may directly produce contraction by a depolarization of the cell membrane of guinea-pig taenia coli.

Pulmonary ascariasis.

A case of pulmonary ascariasis is reported for the first time in Australia. Because of increasing immigration from countries which have a high incidence of ascariasis (especially those of South-East Asia), and increasing travel to Asian countries, the awareness of this infestation as a cause of respiratory disease may be of great importance.

Posttranscriptional regulation of glucocorticoid-regulated functions.

Relying heavily on studies of TAT regulation in cultured rat hepatoma cell lines, we have attempted in this brief review to discuss possible mechanisms for posttranscriptional regulation of glucocorticoid-sensitive enzymes and to chronicle the evidence for and against posttranscriptional mechanisms for specific enzyme induction by glucocorticoids. Initially, mechanisms were considered that would reconcile results showing sensitivity of both induction and deinduction of TAT to inhibitors of RNA synthesis with studies demonstrating first that glucocorticoids regulate the rates of specific enzyme synthesis and, then, that glucocorticoids regulate levels of enzyme-specific mRNA. Such reconciliation proved unnecessary when it was demonstrated that inhibitors of RNA synthesis such as actinomycin D were not specific for RNA synthesis, but also had effects on mRNA turnover and protein metabolism. The bulk of evidence to date establishes that glucocorticoids promote the production of enzyme-specific mRNA for the proteins whose synthesis is regulated by thses steroids. Nevertheless, there is still very little direct evidence that steroids can modulate rates of specific gene transcription. The glucocorticoid stimulation of mouse mammary tumor virus RNA production in cultured cell lines is the only example to date where such a mechanism is supported by RNA-DNA hybridization studies. Posttranscriptional actions of steroids on the turnover, processing, or extranuclear transport of specific mRNA precursors remain potential steps at which glucocorticoids might function. The rapid turnover of some glucocorticoid-regulated enzymes and their mRNAs not only ensures a rapid response to steroid addition or withdrawal, but also subjects these proteins to relatively large fluctuations upon alterations in overall protein or mRNA metabolism. Thus many of the inductions and repressions of hepatic TAT and TO by mediators other than the glucocorticoids may be attributable entirely to nonspecific mechanisms.

DNA-damaging and mutagenic effects of 1,2-dimethylhydrazine on Bacillus subtilis repair-deficient mutants.

Mutagenic, DNA-damaging, and in vivo alteration of DNA have been demonstrated for 1,2-dimethylhydrazine (DMH), a potent inducer of adenocarcinomas of the large intestine and colon of rats. These activities are pH-dependent, with 6.5 giving optimum response. There was no requirement for metabolic activation with rat-liver S9 mix when the appropriate Bacillus subtilis mutant strains were used. The Rec- strains recA8 and mc-1 were greater than 300-fold more sensitive to the DNA-damaging activity of DMH than was their isogenic wild-type parent. The DNA isolated from DMH-treated mc-1 had altered spectroscopic characteristics, and gave a greatly reduced transformation efficiency. Treatment of B. subtilis strain TKJ6321 with DMH at pH 6.5 induced His+, Met+ mutations in substantial numbers at low concentrations of this chemical. The use of B. subtilis mutants in these studies has therefore made it possible to demonstrate mutagenic and DNA-damaging activity in bacteria for this potent carcinogenic chemical.

The temperature and pH dependence of conformational transitions of the chromatin subunit.

Hydrodynamic, spectroscopic, and chemical crosslinking studies on monomer chromatin subnits are reported as a function of ionic strength, pH, and temperature. In earlier studies, two salt-dependent conformational transitions were described (Gordon et al., Proceedings of the National Academy of Science, 75, 660, 1978). Transition one occurred between 0.7 and 2.0 mM ionic strength and transition two occurred between 5.0 and 11.0 mM ionic strength. Crosslinking at 11 mM ionic strength with formaldehyde suppressed both transitions. In this communication we report that the second transition was characterized by changes in the circular dichroism spectra in the 260--320 nm region as well as by changes in the hydrodynamic properties. As the ionic strength was increased from 5.0 to 11.0 mM, [theta]282 decreased from 2000 TO 1500 DEG CM2/DMOLE AND [THETA]295 decreased from 0 to -400 deg cm2/dmole. Both transitions occurred in the pH range from pH 6.0 to 9.2. At pH 5.0, the two ionic strength-dependent transitions were no longer observed and the characteristic changes in the circular dichroism spectra were suppressed. The spectra of the monomer subunits at pH 5.0 showed only small changes with ionic strength and resembled the spectra of the subunits at 11 mM ionic strength above pH 6.0. In order to characterize the transitions in thermodynamic terms an ionic strength near the midpoint of each transition was selected. Then, changes in s20,w and D20,w were measured as a function of temperature. These data allow an estimation to be made of the enthalpies and entropies of the transitions.

Urinary acidification in turtle bladder is due to a reversible proton-translocating ATPase.

Adverse proton electrochemical gradients (delta muH) applied across the turtle urinary bladder decrease active H+ transport in this epithelium. A delta muH of 180 mV abolishes both transport and its tightly coupled metabolic reaction. Larger gradients should, in theory, reverse the direction of H+ transport and the metabolic reaction leading to synthesis of ATP if the pump is an ATPase, or cause an increase in the oxidized state of a redox pair if it is a redox pump. To distinguish between these two possibilities, we measured ATP levels in epithelial cells that were poisoned to inhibit cellular mechanisms of ATP synthesis. At delta muH of 120 mV or less no ATP synthesis was found. At delta muH of greater than 120 mV there was a linear increase in ATP synthesis. Dinitrophenol, a H+ carrier, prevented synthesis at delta muH of 310 mV. Dicyclohexylcarbodiimide, an inhibitor of H+ transport that works at the cell surface, prevented ATP synthesis at delta muH of 310 mV. These results demonstrate that a reversible proton-translocating ATPase in the mucosal border of the bladder is the H+ pump responsible for urinary acidification.

Mechanism of toxicity of putrescine in Anacystis nidulans.

Putrescine is lethal to the cyanobacterium Anacystis nidulans at extracellular pH values at which significant concentrations of the nonprotonated diamine rapidly diffuse into the cell and accumulate as the charged form. Although over 98% of the accumulated putrescine is not metabolized, a small fraction is rendered trichloroacetic acid-insoluble, and about 90% of this is bound as putrescinie to proteins and cell structures. Various synthetic functions were studied in the presence of a bacteriostatic (40 microM) and a bacteriocidal (150 microM) concentration of putrescine at pH 9.5. Under lethal conditions, protein synthesis was completely inhibited after 45 min and CO2 fixation after 100 min, whereas nucleic acid synthesis was less affected. Spermidine was lost from the cell and its synthesis was arrested. These functions were much less inhibited at 40 microM putrescine. Ribosomes from putrescine-killed cells were found to be irreversibly dissociated into 30S and 50S subunits. Some putrescine (1-4 molecules) cosedimented with each subunit.

Hazards to wintering geese and other wildlife from the use of dieldrin, chlorfenvinphos and carbophenothion as wheat seed treatments.

Chemical treatments of cereal seeds are used in the United Kingdom to prevent damage by a number of pests including the wheat bulb fly, which is a serious pest of winter wheat. The persistent organochlorine dieldrin was introduced in the 1950s as a seed treatment but caused the death of large numbers of grain eating birds and gave rise to unacceptable environmental contamination. The withdrawal of dieldrin as a seed treatment was made possible by the introduction of two less persistent organophosphate insecticides, chlorfenvinphos and carbophenothion. Although the introduction of these chemicals has been beneficial in reducing environmental contamination, some side-effects on wildlife have still been discernible and carbophenothion has now been withdrawn from use in Scotland owing to the deaths of wintering geese from carbophenothion poisoning. Subsequent laboratory studies have demonstrated that Anser geese are particularly susceptible to carbophenothion poisoning, and the underlying biochemical mechanism has been investigated. The fundamental problem of species variation in toxicity among the organophosphorus and carbamate pesticides which this investigation illustrates presents difficulties for registration authorities when they are considered for clearance for agricultural use. The implications of the environmental problems encountered with dieldrin, chlorfenvinphos and carbophenothion for the pre-clearance testing of new chemicals are discussed and the critical surveillance of the early years of commercial use of a chemical is recommended to support pre-clearance studies aimed at assessing the potential hazard to the environment.

Optimum probabilistic processing in colour perception. II. Colour vision as template matching.

A statistical approach to account for psychophysical phenomena in human colour vision is presented. The central visual processor is viewed as an optimum recognizer of stochastic patterns supplied by the periphery. The processor makes an optimum estimate of the spectral parameters of the stimulus, given the wavelength filter characteristics of the periphery, the stochastic nature of the information and an internal template to which the external stimulus is matched. The estimate is constrained in ways inferred from empirical phenomena. Subjective brightness of monochromatic stimuli and related constant brightness manifolds in the colour space constitute the constraint for brightness estimation. Results analogous and in accord with those of earlier line element theories are obtained. The Bezold-Brücke hue shift constitutes the basic constraint for hue estimation. The hue estimate involves interrelation between the fields in the experiment. Similarities and differences both in basic conceptions and results introduced by the template matching notions are discussed.

Antagonism by some beta-adrenoceptor-blocking agents to cholinergic stimulation of skeletal muscle in vitro.

Antiacetylcholine activity some beta-adrenoceptor-blocking drugs was investigated using isolated guinea pig cremaster muscle and frog fectus abdominis muscle. On the cremaster muscle, the antagonism to acetylcholine was non competitive in K0 1313, (+/-)-INPEA and (--)-INPEA, competitive in (+)-INPEA and functional in practolol; All three INPEA isomers, practolol and propranolol behaved as noncompetitive antagonists of acetylcholine on frog rectus muscle. Caffeine-induced contractions of this muscle were partially inhibited by propranolol but not by the other drugs. It is suggested that the beta-adrenoceptor-blocking drugs produce their antiacetylcholine action by interaction with sites on the muscle which are different from the cholinceptor, and which vary between compounds and species.

Experiments with large enclosed ecosystems.

Three of the major advantages of enclosure experiments are that they ensure (1) that the same populations are sampled over a long period; (2) that populations of at least three trophic levels are initially enclosed in naturally occurring proportions and that they are self sustaining over a long experimental period; and (3) that replicate enclosed populations can be experimentally manipulated. There are two disadvantages which must be mentioned. These are (1) that vertical mixing, which may be reduced by as much as an order of magnitude compared to the open sea, will undoubtedly affect the sinking rates of phytoplankton and may influence the structure of the population; and (2) that as a general rule the larger and therefore more expensive the enclosures become, the more difficult it is to run sufficient replicates. An experiment is described in which 1 microgram Hg/l was added to two 95 m3 bags (3 mdiameter by 17 m deep) and the response of the pelagic population monitored over the following 20 days. A further 10 micrograms Hg/l was then added to each enclosure and the response measured for a further 20 days. The results indicated that: (i) inorganic mercury added to the water column is very rapidly transformed into 'bound' or 'non-reactive' mercury and that about 25% of the mercury added was recovered associated with the organic material settling to the bottom of the bags; (ii) the response of the biological population to 1 microgram Hg/l was very limited and in fact a transient reduction in photosynthetic carbon uptake per unit chlorophyll was the only noticeable effect and there were no changes in population size or structure that could be attributed to mercury; (iii) at 10 micrograms Hg/l the zooplankton population was reduced markedly and this did produce changes in the structure of both the zooplankton and phytoplankton populations. These results are similar to the results of a comparable experiment carried out in Vancouver Island (Cepex) and point to the conclusion that the levels of mercury found in surface waters around the coast of the U.K. (0.001--0.022 microgram Hg/l) are one or two orders of magnitude below the levels at which a response of the biological population can be demonstrated. The usefulness of large scale enclosed ecosystems for further pollution research is discussed and it is concluded that those facilities that provided a link between the water column and the sediments would be most useful since they would (1) enable estimates to be made of the flux rates of pollutants from the water column to the sediments; and (2) allow experiments to be carried out with the pollutant in contact with sediment in its natural form.

12-Hour simultaneous registration of acid reflex and peristaltic activity in the oesophagus. A study in normal subjects.

Twelve-hour simultaneous registration of acid gastro-oesophageal reflux and peristaltic activity in the oesophagus was carried out on 30 healthy subjects. The intensity of the acid gastro-oesophageal reflux was determined by automatic integration of the pH variation. Acid gastro-oesophageal reflux to pH less than or equal to 4 occurs in normal subjects within a range of 0--2.4% of the total registration period. The intravariation was measured in 10 investigations on 1 subject, and lay within the intervariation. To maintain the pressure-measuring system intact, 3 ml H2O/h were fed to the proximal and distal pressure catheters, respectively; it has been shown that this small quantity of water has no influence on the pH variation. Peristaltic activity for the entire measuring period was recorded and related to the individual reflex episodes. The total activity was found to be dependent on the level of consciousness, with little activity occurring during sleep. A positive correlation was found between the lowest pH during a reflux episode and the peristaltic activity in the oesophagus (p less than 0.001), between the lowest pH during a reflux episode and the duration of the reflux episode (p less than 0.001), and between the peristaltic activity and the duration of the reflux episode (p less than 0.001). During long-term registration of oesophageal pH it appears that pH less than or equal to 4 is a usable parameter for distinguishing between pathological and non-pathological acid gastro-oesophageal reflux. Sudden falls in pH to below 4 release increased peristalsis in the oesophagus.

Preferential homing of passively transferred T cells into skin allografts of mice.

An assay which uses two differentially labeled cell populations was used to characterize the preferential localization of passively transferred syngeneic cells immunized to specific alloantigens. Splenocytes cytotoxic to B10.D2 and B10.BR alloantigens were harvested from (C57BL/6 X A/J)F1 (B6AF1) donors bearing acutely rejected skin allografts. One population was labeled in vitro with 3H-thymidine and the other with 14C-thymidine. The labeled cells were pooled and then transferred i.v. into B6AF1 hosts bearing 5-day-old skin grafts from B10.D2 and B10.BR donors. After 48 hr the mice were killed, and the relative amount of cells present in the skin grafts and draining axillary lymph nodes was derived by comparing the 3H:14C ratios of the harvested tissues. The results of these studies indicated that cytotoxic splenocytes harvested from donors bearing acutely rejected skin allografts preferentially localize to the relevant skin allograft after passive systemic transfer. The homing behavior of these splenocytes was augmented by T cell enrichment and significantly diminished by pretreatment with anti-Thy-1.2 serum plus rabbit complement. There was no evidence of preferential homing within the draining axillary lymph nodes. It can be concluded that a T cell population derived from in vivo sensitized splenocytes exhibits preferential homing to relevant skin allografts upon passive transfer.

Metabolism of 8-chloro-6-(o-chlorophenyl)-1-methyl-4H-s-triazolo [4,3-alpha] [1,4] benzodiazepine, triazolam, a new central depressant. I. Absorption, distribution and excretion in rats, dogs and monkeys.

1. Peak radioactivity in the blood was reached at 30 min after i.p. and 1 h after oral dosing of [14C]triazolam to rats. In dogs, peak blood level was observed at 30 min after oral dosing. 2. Daily dosing of triazolam to male rats for 21 days caused a gradual increase in blood level, with peak at 1 h after dosing. 3. The rate of binding of triazolam plus its metabolites to plasma protein of rats was about 30% at 15 min and 6 h. 4. In rats, the majority of the activity of the intra-intestinally administered [14C]triazolam was found in the small intestines in 6 h. 5. About 58% of the oral dose and 77% of the i.p. dose were recovered in the bile of rats in 48 h after dosing. When the bile from one rat was introduced into the duodenum of a second rat, approximately 37% was recovered in the bile of the second animal in 24 h. 6. In male rats, high radioactivity was seen in the liver, kidneys, adrenals and heart, and low in the CNS. By 96 h after dosing, radioactivity in the liver, blood and kidneys was very low, and was undetectable in other tissues and organs. Radioactivity levels in tissues after daily dosing for 7, 14 and 21 days did not differ appreciably from single administration. 7. In monkeys, activity was high in the liver, kidneys and skin following oral administration and low in the CNS. 8. After oral administration of [14C]triazolam to pregnant rats, the activity in the uterus and placenta was higher than that in the maternal blood. The activity in the foetus was low. 9. In rats given [14C]triazolam orally or i.p., 85% and 12% of the oral dose, and 82% and 14% of the i.p. dose were recovered in the faeces and urine, respectively, in 96 h. The rate of cumulative faecal and urinary excretion after repeated dosing was similar to the single dosing with 80% and 14% of the activity recovered, respectively, in faeces and urine in 6 days. In dogs, 50% of the oral dose was found in the faeces and 40% in the urine. 10. Radioactivity in the milk of rats was maximal at 4 h after oral dosing. It declined to 34% of the peak level 48 h later.

Mutagenic activation of tris(2,3-dibromopropyl)phosphate: the role of microsomal oxidative metabolism.

The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) is converted to products which are mutagenic for Salmonella typhimurium TA 100 in the presence of rat liver microsomes, NADPH and oxygen. Other bromopropyl-compounds were also mutagenic; 2,3-dibromopropene and 2,3-dibromopropionic acid were directly mutagenic, whereas 2,3-dibromopropanol and tris(2-bromopropyl)phosphate were weakly mutagenic after addition of liver microsomes and cofactors. Typical in vivo and in vitro inhibitors of cytochrome P-450 inhibited Tris-BP mutagenicity. The effects of inducers of cytochrome P-450 on Tris-BP mutagenicity was dependent on the concentration of mutagen and microsomal protein in the assay, indicating complexity in the kinetics involved when dealing with possible multiple pathways that lead to mutagenicity. Addition of glutathione strongly inhibited Tris-BP mutagenicity. It is suggested that Tris-BP is oxidized to a reactive electrophile, possibly the 2-keto derivative, which could react with nucleophilic groups in DNA and thus lead to mutagenic events.

Role of calcium-dependent regulator protein (CDR) in inhibition of 3',5'-c AMP-phosphodiesterase by influenza virus. II. Kinetic studies on inhibition of CDR-dependent phosphodiesterase by influenza virus.

As revealed by spectrophotometry, native but not heat-inactivated influenza virus in the presence of ATP reduced the activity of calcium-dependent regulator protein-stimulated 3',5'-c AMP-phosphodiesterase (CDR-PDE). ATP could be partially replaced by ADP but not by AMP. The degree of CDR-PDE inhibition increased with increasing virus concentration. But at very high virus concentrations the rate of 3',5'-c AMP hydrolysis by CDR-PDE was not linearly dependent on time. At appropriate virus concentrations the degree of inhibition of CDR-PDE activity remained unchanged for the whole reaction time.

Metabolism of totally ischemic excised dog heart. I. Construction of a computer model.

Construction and fit to the experimental data of a computer model of glycolysis, the Krebs cycle, and related metabolism in an ischemic dog heart preparation, involving 122 metabolites, 65 enzymes, and 406 chemical reactions, is described. The experimental preparation simulated is a dog heart excised from the body, placed in a beaker of Tyrode's solution, and sampled for 100 min; the model required only moderate modification from models representing perfused rat hearts, and little modification from a model of another ischemic dog heart preparation. Common underlying mechanisms for the ischemia are indicated, although this preparation appears to evolve more slowly with time, perhpas owing to heavy sedation and diffusion-limited transport. Lactate is, at first, exported and then accumulates intracellularly; pH falls, but not as much in the mitochondria as the cytoplasm; redox couples go reduced, but with counterintuitive time courses; calcium phosphate is calculated to precipitate, as often observed in cardiac ischemia.

Sexual development and activity of men with disturbances of somatic development.

By means of Questionnaires HTDM and SAM the heterosexual development and sexual activity were investigated in the following groups of males: 1. the control group consists of 345 married men from sterile marriages, who were adequately developed somatosexually, had normozoospermia in the ejaculate and a good potency; 2. in 48 unilateral and 57 bilateral adult cryptorchids; 3. in 101 married men with a distinct testicular hypoplasia, the long axis of both sexual glands being shorter than 30 mm; 4. in 110 patients with a Klinefelter's syndrome; 5. in 14 patients with hypogonadotropic hypogonadism. Whereas the retardation of heterosexual development was found only in two groups (group 4 and 5), a distinctly lowered activity in sex life was ascertained in all four pathological samples.

[Comparative study of postoperative nitrogen balance as a function of carbohydrate intake].

Postoperative nitrogen intake, which limits nitrogen catabolism and improves conditions of healing, classically implies a high calorie intake. The risks and dangers of hypertonic or hypersmotic solutions are such that the provision of nitrogen postoperatively in ordinary surgery is often avoided. The authors studied postoperative nitrogen balance for a given nitrogen intake with different calorie levels in a series of 50 patients undergoing digestive surgery. A first group (28 patients--mean age 54.6 years, mean weight 63.3 kg) received 12.4 g of nitrogen and 1000 calories per day. The second group (22 patients--mean age 52.5 years, mean weight 64.7 kg) received 12.4 g of nitrogen and 2200 calories. Daily nitrogen balance was calculated using the method of approximation described by Apfelbaum on the basis of urinary urea excretion. Statistical study of nitrogen balances for the first four days showed no statistically significant difference between the mean values in the two groups. For group A, the cumulative balance for the first four days was 7.60 g +/- 4.75 g, and for group B 7.85 G +/- 6.64 g. Limitation of postoperative nitrogen catabolism does not necessarily impose the need for high calorie intake, implying the use of a central venous catheter and administration at constant flow. The patient undergoing ordinary digestive surgery may benefit from postoperative nitrogen supplies, associated with a moderate calorie intake and administered via usual venous routes.

Isolation, characterization and the role of rabbit testicular arysulphatase A in fertilization.

Arysulphatase A was purified from rabbit testis. The purification was accomplished by a four-step procedure involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, SP(sulphopropyl)-Sephadex and affinity chromatography on concanavalin A-Sepharose. The specific activity of purified preparation was 135 mumol/min per mg of protein, which represented an increase of 900-fold above that of the crude homogenate. The purified enzyme (20-50 micrograms) was found to move electrophoretically as a single band on polyacrylamide gel at pH 7.2 and 8.4. The homogeneous enzyme was shown to be a glycoprotein with 0.8% (w/w) of N-acetylneuraminic acid and 20% neutral sugar. The treatment of purified enzyme with bacterial neuraminidase had no effect on enzyme activity or kinetic properties, but it changed the elution prolife of rabbit testis arylsulphatase A through DEAE-Sephadex. The purified enzyme was strongly inhibited by Cu2+, Fe3+ and Ag+. It hydrolysed several sulphate esters including cerebroside 3-sulphate, ascorbic acid 2-sulphate and steroid sulphates. Pure arysulphatase was effective in dispersing the cumulus cells of rabbit ova.

Antianginal and haemodynamic effects of a new beta-blocking agent, SD 1601.

Seventeen patients suffering from angina pectoris were submitted to bicycle ergometer test until an ST ischemic segment of typical pain occurred. Before and during the effort the ECG was recorded: before and after the exercise, the systolic intervals were calculated and arterial pressure measured. The T.P. index, namely the product of systolic arterial pressure by the heart rate and ejection time was calculated. The recordings and the effort test were repeated 5 min after treatment with the beta-blocking drug 1-(o-methoxyphenoxy)-3-isopropylamino-2-propanol hydrochloride (SD 1601). After treatment with the blocker, patients were able to prolong the duration of exercise or perform a higher mean total external work. SD 1601 significantly diminished O2 myocardial consumption at rest, expressed as T.P. During physical exercise, work and thus O2 consumption rise; given equal external work, SD 1601 rduces significantly O2 consumption. Acutely given, SD 1601 did not affect systolic intervals nor did it exert any negative inotropic effect.

In vitro binding of various biological substances by two hypocholesterolaemic resins. Cholestyramine and colestipol.

The ability of cholestyramine and colestipol, two hypocholesterolaemic resins, to bind in vitro several compounds such as vitamin B12, vitamin B12-intrinsic factor complex, folic acid, iron citrate and calcium chloride was investigated. Both resins bound to a high extent vitamin B12-intrinsic factor complex, folic acid and iron citrate; in addition, cholestyramine also caused appreciable binding of calcium. Throughout a large range of pH, there was no change in the binding capacity; however, at pH 2, cholestyramine exhibited a marked drop in the binding of tested substances (with exception of folic acid). By increasing the molarity of the solutions, the binding to the resins of vitamin B12-intrinsic factor complex and of calcium chloride was completely inhibited. In human gastric and duodenal juices, the uptake by the resins of the studied compounds depends on the molarity of the physiological medium tested and partly confirms the results obtained with aqueous solutions. These data obtained in vitro emphasize the necessity of regular monitoring these biochemical parameters during chronic treatment of hypercholesterolaemia conducted with these two resins.

Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.

The influence of charge on bilayer membranes. Calorimetric investigations of phosphatidic acid bilayers.

The pH-dependence of the phase transition of dimyristoyl phosphatidic acid and dihexadecyl phosphatidic acid has been investigated using differential scanning calorimetry. Varying the pH induces different degrees of ionization of the polar head group. The changes in transition temperature with pH as observed by calorimetry are in good agreement with those obtained by measuring the changes in light scattering, whereas the transition temperatures reported by the fluorescent probe N-phenylnaphthylamine do not always coincide with those determined from calorimetry [1]. The observed maximum of the transition temperature at pH 3.5 corresponds to a minimum in the transition enthalpy vs. pH diagram. At this pH a particular stable bilayer phase is formed. Full protonation of phosphatidic acids leads to suspensions of mycrocrystals. The transition enthalpy approaches the value of the melting enthalpy of crystalline anhydrous phosphatidic acid. The decrease in the transition enthalpy at high pH values is due to a change in the hydrocarbon chain interactions induced by the doubly charged head groups. The cooperativity of the transition varies with the degree of ionization of the head group, being lower for doubly charged phosphatidic acids.

Studies on rat renal cortical cell kallikrein. II. Identification of kallikrein as an ecto-enzyme.

Suspensions of viable renal cortical cells hydrolyzed a synthetic ester substrate (alpha-N-tosyl-L-arginine methyl ester, Tos-Arg-OMe) and generated kinins from a kininogen substrate. This kallikrein-like esterase activity increased linearly with cell number, or time of exposure to substrate. No radiolabelled substrate or product was found within the cells. Most of the activity appeared to be on cell surfaces as supernatant media had less than 20% of the Tos-Arg-OMe esterase activity on the cell suspensions. Cell surface Tos-Arg-OMe esterase activity was inhibited by aprotinin, benzamidine, pentamidine, and a tris-amidine derivative (alpha,alpha',alpha''-tris(3-amidinophenoxy)mesitylene). Preincubation of cells with phospholipase A2 increased renal cell surface esterase activity up to 76% while only slightly increasing supernatant activity. In contrast, preincubation with deoxycholate caused clearing of suspensions and a marked increase in supernatant esterase activity. Renal cell kininogenase (EC 3.4.21.8) activity was inhibited by preincubation with aprotinin, the tris-amidine derivative, or anti-rat urinary kallikrein antibody. Kallikrein elaborated by renal cells formed a single precipitin line with an antibody to rat urinary kallikrein but the two enzymes were not immunologically identical. We conclude that kallikrein's active sites are facing the external environment of renal cortical cells in suspension with access to substrates, inhibitors, and antibody.

Studies on the possible identity of particulate beta-glucosidase and beta-xylosidase of mouse liver.

Mouse liver beta-glucosidase (beta-D-glucosidase glucohydrolase, EC 3.2.1.21) and beta-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) activities were studied under different conditions of incubation in an attempt to determine whether these two activities are due to a single enzyme or two separate enzymes. The results showed that: (a) Particle-bound beta-glucosidase and beta-xylosidase activities exhibit similar characteristics with different buffers and at various pH values, in the presence or absence of taurocholate. (b) Both activities are inhibited by gluconolactone and conduritol B eposice. beta-Glucosidase activity is inhibited competitively by the two inhibitors, but beta-xylosidase activity is inhibited non-competitively. (c) Xylonolactone was a very poor inhibitor of both activities, but the inhibition of beta-xylosidase activity was more pronounced than that of beta-glucosidase. (d) The presence of glucosides or xylosides simultaneously in the incubation medium suggested the presence of one enzyme with both activities. These results, together with the mode of inhibition produced by gluconolactone and conduritol B epoxide also suggest the presence of two different binding sites for the beta-D-glucoside and beta-D-xyloside, respectively.

Evidence for two distinct types of postsynaptic alpha-adrenoceptor in vascular smooth muscle in vivo.

1 The effects of the highly selective alpha(1)-adrenoceptor antagonist, prazosin, and the relatively selective alpha(2)-adrenoceptor antagonist, yohimbine, on the pressor responses to intravenous injections of phenylephrine and noradrenaline have been examined in anaesthetized cats and pithed rats in an attempt to determine whether alpha(1)- and alpha(2)-adrenoceptors are located postsynaptically on vascular smooth muscle.2 In anaesthetized cats prazosin caused a much greater reduction in the pressor responses to phenylephrine than to noradrenaline or splanchnic nerve stimulation (after adrenalectomy). Yohimbine was of similar potency in reducing the pressor responses to each stimulus.3 A differential blocking activity of prazosin against intra-arterial injections of phenylephrine and noradrenaline was also demonstrated in the blood-perfused cat hind limb. As in the whole animal, prazosin was more potent against phenylephrine than noradrenaline. A similar, though less marked, effect was seen in the mesenteric circulation, but not in the renal circulation, where prazosin was almost equipotent in reducing responses to phenylephrine and noradrenaline.4 In pithed rats prazosin was a potent, competitive antagonist of phenylephrine, but had little effect against noradrenaline; only the responses to high doses of noradrenaline were reduced by prazosin. Yohimbine was approximately equipotent as an antagonist of phenylephrine and noradrenaline. In the anococcygeus muscle, prazosin was as potent an antagonist of noradrenaline as it was of phenylephrine on vascular smooth muscle.5 The results suggest that there are two types of alpha-adrenoceptor in the vasculature of cats and rats. Phenylephrine produces pressor responses by stimulating one type of postsynaptic alpha-adrenoceptor that is blocked by prazosin and yohimbine; these are alpha(1)-adrenoceptors. Noradrenaline exerts some of its effect via these receptors but most of its effect appears to be exerted through prazosin-insensitive receptors. The latter receptors appear to differ from alpha(2)-adrenoceptors.

Evidence for dopaminergic vasodilator innervation of the canine paw pad.

1 In chloralose-anaesthetized dogs pretreated with guanethidine and pancuronium, electrical stimulation (0.2 to 5 Hz) of the peripheral end of the cut tibial nerve caused a frequency-dependent increase in femoral blood flow which was restricted to the paw pads. 2 This neurogenic vasodilatation was not attenuated by atropine, mepyramine plus burimamide, indomethacin or propranolol. It was, however, attenuated in a dose-dependent manner by intra-arterial administration of the dopamine receptor antagonist, ergometrine (0.05 to 0.5 mg). 3 The effect of ergometrine could not be explained by non-specific effects on axonal conduction or transmission or by vasospasm of the blood vessels of the paw-pads. 4 In dogs with intact tibial nerves, a pharmacologically similar dilator response localized to the paw-pads could be elicited by electrical stimulation of loci in the ipsilateral diencephalon and midbrain. This response was not due to inhibition of adrenergic vasomotor tone and was abolished by systemic ganglion blockade or by tibial nerve section as well as by femoral arterial administration of ergometrine. 5 It is suggested that the vasculature of the canine paw pads is innervated by a population of autonomic axons which utilize dopamine or a related substance as a transmitter substance and activation of which causes vasodilation.

A survey of psychotropic drug prescriptions in an oncology population.

The present study examined the prescription practices concerning psychotropic drugs in 5 major oncology centers over a 6 month period. During the survey period 1579 patients were admitted to the collaborating institutions, and 51% of them were prescribed at least one psychotropic medication. Hypnotics were the most frequently prescribed drugs, accounting for 48% of total prescriptions, followed by anti-psychotics at 26% and anti-anxiety agents at 25%. Anti-depressant drugs accounted for only 1% of psychotropic prescriptions. Analysis of prescription rationales revealed that 44% of the psychotropic prescriptions were written for sleep, while 25% were given for nausea and vomiting; approximately 17% were attributed to psychological distress, and 12% were associated with diagnostic medical procedures. The overall rate of prescription was approximately 2 psychotropic drugs per patient per admission, with only 2% of prescriptions resulting in chart-documented side effects. At the level of individual compounds, 3 distinct drugs accounted for 72% of total prescriptions--flurazepam (33%), prochlorperazine (21%), and diazepam (17%).

Microsomal target proteins of metabolically activated aromatic hydrocarbons.

The specificity of binding to microsomal proteins of metabolically activated hydrocarbons has been studied. Radioactively labelled benzene, phenol, chlorobenzene, BP and MC were incubated with liver microsomes from control, phenobarbital- and MC-treated rats in the presence of an NADPH-generating system. The patterns of metabolite binding to microsomal proteins were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Benzene, phenol and chlorobenzene metabolites showed one type of binding pattern dominated by a band at 72 000 Mr. This band was strong both in control and induced microsomes. Additional radioactive bands were seen in the 50 000--60 000 Mr region particularly in MC-induced microsomes. BP and MC metabolites showed a different type of binding pattern with incorporation of radioactivity into several fractions in the 50 000--60 000 Mr region of MC-induced microsomes. Two other strongly labelled fractions occurred at 68 000 and 72 000 Mr. The incorporation was low into control and phenobarbital-induced microsomes. Two labelled bands (Mr 56 000 and 72 000) were common for all hydrocarbons in MC-induced microsomes. The 56 000 Mr band had the same mobility in the gel as an MC-induced protein likely to be cytochrome P-448. The NADPH-generating system was essential for metabolite binding and GSH and UDPGA greatly reduced binding. We suggest that differences in metabolite binding patterns reflect differences in the routes of metabolite formation and that activated hydrocarbons are likely to bind to proteins close to their site of formation.

Hepatic estrone and estradiol glucuronyltransferase activity in pregnancy. Induction by pretreatment with 3-methylcholanthrene and phenobarbital.

Hepatic microsomal estrone and estradiol glucuronyltransferase activity were examined in nonpregnant female and pregnant rats and rabbits. Pregnancy decreased glucuronyltransferase activity towards both substrates by 30% in rats and rabbits when activity was expressed per mg of microsomal protein. Because of the increased size of the liver in pregnancy in the rat, activity was increased in this species when expressed per whole liver. The size of the liver was not increased in pregnancy in the rabbit, so that activity per whole liver was also decreased in this species. Pretreatment of nonpregnant rats with 3-methylcholanthrene had little effect on estradiol glucuronyltransferase activity and increased estrone glucuronyltransferase activity 35%. Similar pretreatment of pregnant rats, however, increased estradiol glucuronyltransferase activity approximately twofold and estrone glucuronyltransferase activity approximately threefold. Phenobarbital was a much less potent inducer of estrone and estradiol glucuronyltransferase activities but did increase activities to a greater extent in pregnant rats than in nonpregnant female controls.

Metabolism of digitoxin in the isolated perfused rat liver. Effect of spironolactone pretreatment.

Livers from either control or spironolactone-treated rats were perfused for a 90-min period with 30% rat blood and 3H-digitoxin. At several time periods throughout perfusation, bile was collected and a sample of blood was removed from the perfusate. Extractions were performed on both blood and bile to determine amount of polar and nonpolar metabolites at 60 min. Polar metabolites were cleaved with beta-glucuronidase and high-pressure liquid chromatography was used to separate the resultant nonpolar metabolites from blood and bile cleaved with beta-glucuronidase. Biliary excretion and perfusate disappearance of 3H-digitoxin were significantly increased in livers taken from spironolactone-pretreated animals. Both polar and nonpolar metabolites in bile were significantly increased in pretreated animals. The majority of polar metabolites produced by livers from both treated and nontreated animals were readily cleaved with beta-glucuronidase. Both biliary excretion and metabolic pattern, obtained from these studies in an isolated perfused rat liver, mimic those seen in the intact rat. Thus, the isolated perfused rat liver can be used as a model for in vivo studies of cardiac glycoside metabolism.

Role of enterohepatic circulation in the analgesic action of 1-alpha-acetylmethadol in the rat.

The enterohepatic circulation of 1-alpha-acetylmethadol (LAAM) was investigated in the rat. Bile containing 3H-LAAM metabolites was collected from a biliary cannulated donor rat after administration of 3H-LAAM (5 mg/kg, 15 muCi/kg, sc) and infused into the intestine of a double biliary-cannulated recipient rat for 1 hr, and tritium excreted into the bile of the recipient rat was monitored. Within 1 hr after the end of infusion significant radioactivity was found in the bile of the recipient rat and by 10 hr 50% of the infused dose of 3H had been re-excreted into bile. The contribution of enterohepatic circulation of LAAM metabolites to the analgesic action of LAAM was also assessed. Pretreatment of rats with neomycin sulfate was used as a method of decreasing enterohepatic circulation of biliary glucuronide conjugates of LAAM metabolites, and such pretreatment had no effect on LAAM analgesia (6 mg/kg) measured by the hot-plate method. In rats with a biliary fistula, a situation in which enterohepatic circulation was completely eliminated, the analgesic response to a dose of LAAM (6 mg/kg, sc) was not different from sham-operated control group. The above findings indicate that enterohepatic circulation of LAAM metabolites does not contribute to the intensity or duration of LAAM analgesia.

A fluorescamine-based sensitive method for the assay of proteinases, capable of detecting the initial cleavage steps of a protein.

The properties of the reaction of fluorescamine with proteins are the basis for the development of a sensitive, general and simple method for the assay of proteolytic activities. More importantly, the assay measures the initial step(s) of proteolytic attack, making it specially suitable for the examination of the controlling factors that regulate proteolytic degradation and/or the detection of 'specific' proteinases. The method allows the simple determination of the general parameters of enzyme action, V and Km, using proteins, i.e. the physiological substrates of the proteinases. The more appropriate proteins to be used as substrates are the N-amino-terminally blocked ones. Many proteins fulfill this requirement. If the particular protein whose degradation has to be studied lacks this modification, three different approaches can be used to study its degradation: (a) the accumulation of N-amino termini in excess over that of the intact substrate; (b) a gel filtration/continuous method and (c) the chemical blockage of its amino groups. The particular advantages of each of these approaches are discussed.

Chronotropic effect of histamine on cultured neonatal rat heart cells.

The positive chronotropic effect (PCE) of histamine in cultured neonatal rat heart cells was monitored using a microscopic method as well as an electro-optically recording device. The action potential frequency was also measured (by means of microelectrodes). An increase in PCE was noted when histamine (from 1 X 10(-6) M to 1 X 10(-5) M) was added to the cells. However, higher concentrations (from 1 X 10(-5) M to 1 X 10(-4) M) were less effective. The PCE of histamine was reduced by pretreating the cells with antihistaminic drugs. H1-blocking agents (promethazine and mepyramine) were more potent than H2-blocking drugs (metiamide and cimetidine). In addition, the PCE of histamine was abolished when the cells were in presence of high K+ medium (26 mEq) but contraction and action potential amplitudes were increased. Our results demonstrate that these cultures respond to histamine and that this response is abolished by antihistaminic drugs thus suggesting the H1 and/or H2 receptors may be present in the neonatal rat heart cell cultures.

[Cerebral angiography in collagen disease and arteritis of different aetiology (author's transl)].

The cerebral angiograms of 11 patients suffering from collagen disease are presented. Panarteriitis nodosa was diagnosed in 4 cases, Lupus erythematodes in 2 cases. With 5 patients immunovasculitis with cerebral affection was found, which was, however, not to be classified in detail. More or less characteristic features are to be expected in the angiogram; they might harden the suspicion of collagen disease, although they are not likely to prove its diagnosis. An interpretation of the radiological findings should--in addition to the morphology--primarily take into account the distribution type of the vessel wall lesions. Clinically as well as by means of angiography it is difficult to differentiate between collagen disease and cerebral arteriitis of different aetiology; this applies particularly to the alterations in cases of embolic circumscribed encephalitis in sepsis lenta. The diagnostic value of angiography in cases of collagen disease with cerebral affection is discussed, the criteria of cerebral arteriitis of different aetiology are dealth with.

Kinetic and electrophoretic studies of human erythrocytes deficient in pyrimidine 5'-nucleotidase.

The mutant enzyme of a patient with hereditary pyrimidine 5'-nucleotidase deficiency was analyzed biochemically. Partially purified by DEAE-Sephadex and concentrated by ultrafiltration, the enzyme had a high Km for the substrate uridine monophosphate. Utilization of the substrate cytidine monophosphate was normal, but utilization of adenosine monophosphate was greatly increased. The enzyme was stable to heat; the pH optimum was acidic. Electrophoresis of the enzyme revealed a very faint, slower than normal band.

Pathogenic species of the genus Haemophilus and Streptococcus pneumoniae produce immunoglobulin A1 protease.

Thirty-seven strains of the genus Haemophilus and five strains of Streptococcus pneumoniae were examined for their ability to produce extracellular enzyme that cleaves immunoglobulin molecules. All strains of H. influenzae, H. aegyptius, and S. pneumoniae elaborated enzyme that selectively cleaved human immunoglobulin A1 (IgA1) myeloma proteins but was inactive against a variety of other proteins including human IgA2, IgG, and IgM, porcine and bovine secretory IgA, human and bovine serum albumins, and ovalbumin. Although susceptible, human secretory IgA remained largely undigested. Two strains of H. pleuropneumoniae isolated from fatally infected pigs cleaved porcine secretory IgA, but had no effect on human IgA proteins. None of 16 strains that belonged to nonpathogenic Haemophilus species produced IgA protease. Analyses of the cleavage products of human IgA1 and secretory IgA proteins by immunochemical methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analytical ultracentrifugation revealed that Fab and Fc fragments were produced. Since the production of IgA1 protease by Neisseria meningitidis has been reported previously, our finding that H. influenzae and S. pneumoniae produce an IgA1 protease indicates that this is a property of all three major etiological agents of bacterial meningitis. This suggests that IgA1 protease production may be an important factor in the pathogenesis of this disease.

Influence of alcohol on the reproductive system of the male rat.

Twelve male rats were treated with alcohol by an oral self administration technique. The testes, epididymides, seminal vesicles, kidneys, spleen, and liver were recovered from each animal. All organs were weighed and prepared for histological studies. Organs from experimental animals, with the exception of the liver, weighed significantly less than the corresponding controls. The testes of alcohol-treated animals revealed the following changes: thickened capsule, atrophic seminiferous tubules and damaged germinal epithelium, in addition to multinucleated giant cells, fragmented spermatozoa, and desquamated spermatocytes in the lumen of the tubules. Atrophic ductules containing very few spermatozoa were observed in the epididymides of these animals. The seminal vesicles were small and lined by degenerated epithelium. The serum testosterone levels were significantly reduced in alcohol-treated animals, compared to the controls. The present investigation indicates that alcohol adversely affects spermatogenesis and testicular function.

Oral contraception, mechanical contraception, and carbohydrate and lipid metabolism: a two-year study.

The carbohydrate and lipid metabolism of 100 women using an oral contraceptive (0.5 mg norgestrel + 0.05 mg ethinyl estradiol) and of 96 women using mechanical contraceptives was monitored over a 2-year period. The women had been screened for factors known to adversely affect carbohydrate and lipid metabolism. Two-hour oral glucose tolerance tests were performed at 6-month intervals during the study; serum insulin was determined at the same intervals in half the women. Triglycerides, total cholesterol, free fatty acids, and body weight were also measured. The study showed no significant differences in lipid metabolism nor in weight gain between women using oral or mechanical contraceptives. After 6 months the fasting glucose of women using oral contraceptives was significantly decreased; at 120 minutes, glucose and insulin levels were significantly increased in comparison to women using mechanical contraceptives. A greater percentage of oral contraceptive users had borderline-abnormal oral glucose tolerance tests but the abnormalities did not persist in the same individuals during the study. The incidence of a pathological oral glucose tolerance with oral contraceptives was 1%.

Distribution of coenzyme F420 and properties of its hydrolytic fragments.

The ability of hydrolytic products of coenzyme F420 to substitute for F420 in the hydrogenase and nicotinamide adenine dinucleotide phosphate-liniked hydrogenase systems of Methanobacterium strain M.o.H. was kinetically determined. The nicotinamide adenine dinucleotide phosphate-linked hydrogenase system was employed to quantitate the levels of F420 in a number of methanogenic bacteria as well as in some nonmethanogens. Methanobacterium ruminantium and Methanosarcina barkeri contained low levels of F420, whereas other methanogens tested contained high levels (100 to 400 mg/kg of cells). F420 from six of the seven methanogens was tested by thin-layer electrophoresis and was found to be electrophoretically identical to that purified from Methanobacterium strain M.o.H. The only exception was M. barkeri, which contained a more electronegative derivative of F420. Acetobacterium woodii, Escherichia coli, and yeast extract contained no compounds able to substitute for F420 in the nicotinamide adenine dinucleotide phosphate-linked hydrogenase system.

Lysis of Escherichia coli mutants by lactose.

Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which as a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K+ content no lysis occurred. Lysis required high levels of beta-galctosidase and lactose transport activity. MM6, the parental strain of MM6-13, has lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, beta-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of MG2+, lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher slat concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2+, lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulafte, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and beta-galactosidase activity, are important in lysis.

Purification and properties of biliverdin reductases from pig spleen and rat liver.

Biliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34,000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxiodoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 microM while that for NADH was 1.5-2 mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 microM whereas that in the NADH system was 1-2 microM. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IX alpha-biliverdin was the most effective substrate among the four biliverdin isomers, and the dimethylester of IX alpha-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34,000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IX alpha-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.

Intramitochondrial phospholipase activity and the effects of Ca2+ plus N-ethylmaleimide on mitochondrial function.

Liver mitochondria treated with N-ethylmaleimide can accumulate Ca2+ but cannot retain it. Ca2+ loss following uptake occurs in parallel with a proton uptake and collapse of the membrane potential. Respiration is not activated during Ca2+ release and cannot be stimulated by uncoupler. After Ca2+ release and accompanying phenomena are nearly complete, the mitochondria undergo a large amplitude swelling. Nupercaine inhibits the premature release of Ca2+, proton uptake, decline in membrane potential, inhibition of uncoupler-stimulated respiration, and large amplitude swelling. Ruthenium red also prevents these effects. Neither Sr2+ or Mn2+ will substitute for Ca2+ to induce these effects in N-ethylmaleimide-treated mitochondria. The effects of N-ethylmaleimide plus Ca2+ on mitochondria are not accompanied by a significant alteration in the content or composition of phospholipids but are accompanied by small increases in the mitochondrial content of free fatty acids. Free fatty acids accumulate more rapidly in response to limited Ca2+ loading in the absence of N-ethylmaleimide than they do in its presence. In the absence of N-ethylmaleimide, polyunsaturated fatty acids and saturated plus monounsaturated fatty acids accumulate at nearly equal rates. In the presence of N-ethylmaleimide, polyunsaturated fatty acids accumulate more rapidly than saturated plus monounsaturated fatty acids. Any condition or agent tested which inhibited swelling and the other effects produced by Ca2+ plus N-ethylmaleimide also prevented the more rapid accumulation of polyunsaturated, compared to saturated plus monounsaturated, fatty acids. In the light of a positional analysis of phospholipid acyl moieties, these data suggest that 1-acyllysophospholipids accumulate in swelling mitochondria but not in response to noraml Ca2+ loading or when swelling is blocked by other agents. The free fatty acid accumulation, per se, is not responsible for swelling, but levels of exogenous palmitic acid as low as 1 nmol/mg of protein dramatically alter the dependence of swelling velocity on Ca2+ concentration, producing a shift from a sigmoidal- to a hyperbolic-like relationship. This same alteration is brought about by aging the mitochondrial preparation at 0 degrees C. Either pyruvate or DL-carnitine prevents the effect of exogenous palmitate and restores the Aa2+ swelling dependence of aged N-ethylmaleimide-treated mitochondria to that of fresh N-ethylmaleimide-treated mitochondria. Intramitochondrial acylcoenzyme A or acylcarnitine, or both, therefore, to be the modulator of Ca2+ sensitivity rather than free fatty acid. The findings are discussed in terms of the role of intramitochondrial phospholipase and other phospholipid metabolizing enzymes in the mechanisms of N-ethylmaleimide plus Ca2+ effects on mitochondria.

Spectral transitions of nitrosyl hemes during ligand binding to hemoglobin.

Human deoxyhemoglobin has been titrated with nitric oxide at several pH values ranging from 6.0 to 9.0, in the presence and absence of the allosteric effector inositol hexaphosphate at 25 degrees C. Samples were frozen for EPR measurements or analyzed optically within 30 s after mixing to ensure a kinetic population of intermediates. Fractions of pentacoordinate alpha-NO heme groups were determined by fitting EPR and absorbance difference spectra in terms of linear combinations of standard signals. Equivalent results were obtained by these techniques. The fraction of alpha-NO heme exhibiting pentacoordinate character in Hb4NO increases from 0.07 to 0.73 in going from pH 9 to 6. The fraction of alpha hemes which are pentacoordinate in fully saturated nitrosyl hemoglobin, Hb4(NO), increases from 0.0 to 0.41 over the same pH range. Only in the presence of bound inositol-P6 are all 4 the alpha-NO hemes pentacoordinate. Thus, the expression of modified NO heme character is not simply a reflection of the formation of low affinity quaternary conformations. Rather, within this conformation the alpha chain iron atoms exhibit an equilibrium between hexa- and pentacoordinate structures which is perturbed markedly by both proton and phosphate binding. No intermediate coordination structure of the type suggested by Chevion et al. (Chevion, M., Stern, A., Peisach, J., Blumberg, W.E., and Simon, S. (1978) Biochemistry 17, 1745-1750) appears to occur since the observed alpha-NO heme spectra can always by represented quantitatively as a linear combination of the normal hexacoordinate and pentacoordinate signals. The formation of pentacoordinate alpha-NO causes this subunit to exhibit a higher affinity for nitric oxide. Thus on standing at low levels of saturation, there is a slow (t1/2 approximately equal to 8 min at pH 7, 25 degrees C) re-equilibration of ligand from beta to alpha subunits. The final ratio of alpha-NO to beta-NO is 2 to 1 in the absence of phosphates and greater than 10 to 1 in the presence of inositol hexaphosphate.

Fatigue in industry.

Physical fatigue is a painful phenomenon which is localised in overstressed muscles. Mental fatigue is a diffuse sensation of weariness; it is a functional state, one of several intermediate conditions between the two extremes of alarm and sleep. A neurophysiological model of fatigue, involving an activating and inhibitory system has been developed. Fatigue in industrial practice has clinical symptoms: psychic instability, fits of depression and increased liability to illness. Indicators of fatigue are work of performance, subjective feelings of fatigue, electroencephalography, flicker-fusion frequency and various psychomotor and mental tests. Several field studies do, to some extent, confirm the above-mentioned concept of fatigue.

A combined alkali extraction--ethidium bromide technique for the measurement of DNA in small pieces of tissue.

Alkaline solutions (0.1--0.5 N NaOH) at elevated temperatures can be used to extract DNA from small pieces of tissue. RNA is destroyed by the treatment. In tissues which have been previously exposed to tritiated thymidine, aliquots from the extracting solution can be used directly for the determination of DNA by ethidium bromide fluorescence and radioactivity in both DNA and the nucleotide pool.

pA2 values of selective beta-adrenoceptor antagonists on isolated atria demonstrate a species difference in the beta-adrenoceptor populations mediating chronotropic responses in cat and guinea-pig.

pA2 values for atenolol (beta 1-selective) and alpha-methylpropranolol (beta 2-selective) have been determined on isolated atria of cat and guinea-pig using noradrenaline (beta 1-selective) and fenoterol (beta 2-selective) as agonists. On guinea-pig atria, the pA2 values did not vary with the agonist used. On cat atria the pA2 for atenolol was greater with noradrenaline than with fenoterol and the pA2 for alpha-methylpropranolol was greater with fenoterol than with noradrenaline. Fenoterol was 20 times more potent on cat than on guinea-pig atria whereas noradrenaline was approximately equipotent in the two species. The results have been intrepreted as suggsesting that both cat and guinea-pig atria contain one receptor type in common (beta 1) but that only cat atria contain beta 2-adrenoceptors as well.

Investigation of narcotics and antitussives using drug discrimination techniques.

Rats learned drug discriminations in a shock-escape T-maze task. They were trained to turn right in the maze following injection of a drug (D) and left when no injection (N) was given. Number of training sessions before criterion performance (STC) was used to indicate degree of discriminability of the training drug. STC decreased monotonically as dosage increased, and reached a minimum of 3 to 26 with various agonists. Most agonists were not highly discriminable. Daily maintenance injections of morphine, 200 to 600 mg/kg, increased the STC of morphine, 15 mg/kg, significantly, but complete tolerance to discriminable drug actions was not observed. After rats discriminated D vs. N, they were tested with novel drugs to determine which would elicit D choices. Most morphine-like agonists substituted for one another during substitution tests; the tested agonists included alphaprodine, codeine, fentanyl, heroin, meperidine, methadone, morphine, piminodine and propoxyphene. In a few instances, one of these agonists failed to substitute for another. Naloxone and naltrexone antagonized the discriminable effects of morphine. Cyclazocine, levallorphan, naltrexone, dextromethorphan, ethoheptazine and the narcotic agonists did not substitute for one another, suggesting that six dissimilar discriminable effects were produced by these drugs.

Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 Dalton range.

Cell-to-cell junctional permeability in mammalian cell cultures was probed with a series of fluorescent tracers ranging 300 to 800 in molecular weight, during treatment with metabolic inhibitors, Ca-transporting ionophore, and carbon dioxide. Treatment with the combination of cyanide and iodoacetic acid (1--2 mM each), but not with either one alone, caused reversible junctional blockade to all tracer molecular species, large and small. (Electrical coupling, however, persisted in a proportion of the junctions tested.) Treatment with the ionophore A23187 (2--10 micrometers) or with CO2 (an atmosphere of 100% CO2 equilibrated with the medium) produced selective junctional blockade: transmission of a 688 and an 817-dalton tracer was generally blocked, while that of a 376-dalton traced and, in certain conditions, that of a 559-dalton one, persisted. The junctional effect of the ionophore required the presence of Ca in the external medium; and effective junctional blockade by CO2 required pretreatment in medium with high Ca concentration or, interchangeably, pretreatment in medium with high CO2 concentration. In one cell type, prolonged exposure to medium with high Ca concentration alone sufficed to block transmission of the 688-dalton tracer. These effects are discussed in terms of the Ca hypothesis of junctional permeability regulation. In comparison with mammalian (or other vertebrate and invertebrate) organized tissues or with insect cell cultures, the mammalian cell cultures are more resistant to junctional blockade. This difference in transmission stability is discussed in terms of the electron-microscopic finding in the mammalian cultures of fine, bilateral cell processes connected by gap junctions.

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. I. Enhanced production of interferon and appearance of cytotoxin stimulated by capsular polysaccharide of Klebsiella pneumoniae or bacterial lipopolysaccharide.

Interferon production stimulated by the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) in BCG-infected mice was compared with that by bacterial lipopolysaccharide (LPS). Prior infection with BCG increased the responsiveness of mice to the lethal effect of neutral CPS-K as well as to that of LPS. Associated with this, BCG-infected mice showed a markedly enhanced ability to produce interferon after stimulation not only by LPS but also by neutral CPS-K. In addition, a cytotoxic factor (cytotoxin) was found to be released in the serum of BCG-infected mice after injection of these inducers. The kinetics of production of interferon and cytotoxin stimulated by neutral CPS-K were very similar to those stimulated by LPS. The time pattern of cytotoxin production was not in parallel with that of interferon production. Interferon reached a peak 2 hr and cytotoxin 3 hr after injection with these inducers. Interferon and cytotoxin produced by neutral CPS-K showed essentially the same stabilities to heating at 56 C and to treatment at pH 2 respectively as those produced by LPS. Interferon was inactivated by heating at 56 C more rapidly than cytotoxin. Cytotoxin was inactivated by treatment at pH 2 for 24 hr, whereas interferon activity was well preserved after this treatment. These results suggest that both activities are the result of different substances.

Development of psychiatric illness in drug abusers. Possible role of drug preference.

The origin of the psychiatric illnesses observed in drug abusers is often unclear. This study examines the causal relation between drug abuse and specific psychiatric disorders. Fifty-one male veterans first seen in 1972, who were admitted at least once per year for six consecutive years for inpatient drug-abuse treatment, underwent psychiatric assessments at each admission. Eleven men mainly used stimulants, 14 depressants, and 26 opiates. Initial psychiatric examinations showed low symptom levels in all groups but no statistically significant differences among them. By the end of six years, five of the stimulant users had psychoses, and eight of the depressant users had serious depression. The narcotics users showed no change in psychopathology. Differences between the groups were significant at the 0.01 level. These changes were not due to acute toxic reactions, but our data suggest that abuse of particular drugs has a major role in the development of specific psychiatric illnesses. The possibility that different preexisting personality disorders lead to different kinds of drug abuse cannot be excluded.

Demonstration of alpha-adrenoceptors in the rabbit heart by [3H]-dihydroergocryptine binding.

For direct identification of alpha-adrenoceptors in a membrane fraction of the rabbit heart the potent alpha-adrenoceptor antagonist [3H]-dihydroergocryptine ([3H]-DHE) was used. 1. The binding of [3H]-DHE was saturable with 80 fmol of [3H]-DHE bound/mg protein and of high affinity with an equilibrium dissociation constant (KD) of 11.5 nM. Binding of [3H]-DHE (6 nM) was rapid (t 1/2 = 2 min) and readily reversible. From the ratio of the rate constants for forward (K1 = 1.97 X 10(7) M-1 min-1) and reverse (K2 = 0.206 min-1) reactions a KD-value of 10 nM was calculated, which is in good agreement with that obtained by equilibrium studies. 2. Adrenergic agonists compete for [3H]-DHE binding in an order to potency: (-)adrenaline greater than (-)phenyleprine greater than (-)isoprenaline and adrenergic antagonists in the order: phentolamine greater than yohimbine greater than (-)propranolol. Binding is stereospecific as indicated by the greater potency of (-)adrenaline than (+/-)adrenaline in displacing [3H]-DHE from the binding sites. 3. For comparison the binding of the potent beta-adrenoceptor antagonist (-)[3H]-dihydroalprenolol ((-)[3H]-DHA) was measured in the same membrane fraction. The number and affinity of beta-adrenoceptors amounted to 115 fmol of (-)[3H]-DHA bound/mg protein at saturation and KD = 7.9 nM. Adrenergic agonists compete for (-)[3H]-DHA binding in an order of potency: (-)isoprenaline greater than (-)adrenaline greater than (-)phenylephrine; and adrenergic antagonists in the order: (-)prapranolol greater than phentolamine. 4. It is concluded that in a membrane fraction of the rabbit heart there exist binding sites for [3H]-DHE which have characteristics indistinguishable from alpha-adrenoceptors. Thus the present results are in agreement with previously reported data on the existence of cardiac alpha-adrenoceptors in the rabbit heart (Schümann et al., 1974; Endoh et al., 1976b).

A case of transient renal tubular acidosis type 1,4 hybrid RTA: a study of the pathophysiologic characteristics of the acidification defect.

The diagnosis of transient renal tubular acidosis was made in a 16 months old boy. Bicarbonate titration studies revealed that the acidification defect consisted of an association of proximal and distal tubular acidosis. The effect of experimentally induced potassium depletion revealed that hyperkalemia contributed to the acidification defect. After correction of the acidification disorder a defect in urinary concentration was still present.

Hydrophilic region of lecithin membranes studied by bromothymol blue and effects of an inhalation anesthetic, enflurane.

A pH-indicator dye, bromothymol blue, was used to probe the hydrophilic surface of dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine bilayer vesicles. The apparent pK of the surface-adsorbed dye was larger than the bulk pK value. The contribution of the choline positive charge on the dissociation constant of the dye adsorbed on the vesicle surface was estimated by screening the charge interaction with 2 M KCl. The effective surface potentials interacting with the dye were thus estimated to be 33.2, 45.6, and 46.8 mV, respectively, for the dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine vesicles. From the differences between the obtained effective potentials and the calculated surface potentials of the charge-determining plane of the choline head, the distances between the prototropic part of the dye and the choline charge-determining plane were estimated to be 10.5, 8.0, and 7.8 A, respectively. These values were obtained at 25 degrees C; the dimyristoylphosphatidylcholine membrane was in the liquid-crystalline phase and the other two were in the solid gel phase. Addition of an inhalation anesthetic, enflurane, decreased the distance in the dimyristoylphosphatidylcholine vesicles and increased the distance in the dipalmitoyl- and distearoylphosphatidylcholine vesicles. The increase of precessional motion of choline head by the inhalation anesthetic is apparently responsible for the changes.

Nitrogen control in Salmonella: regulation by the glnR and glnF gene products.

The product of the glnR gene is required for nitrogen regulation of the synthesis of glutamine synthesis (Gln synthetase) [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] and two periplasmic transport proteins that are subject to nitrogen control in Salmonella. Strains with mutations to loss of function of the glnR product [e.g., a strain with a Tn10 insertion or one with an ICR-induced (frameshift) mutation in glnR] have about 3% as much Gln synthetase as a fully derepressed wild-type strain and are unable to increase synthesis of this enzyme or periplasmic transport proteins in response to nitrogen limitation. The structural gene for Gln synthetase, glnA, and those for the periplasmic transport proteins are unlinked on the chromosome; thus, glnR appears to encode a diffusible positive regulatory element. Consistent with this, the mutant glnR allele is recessive to the wild-type allele with regard to expression of glnA (synthesis of Gln synthetase). Although glnR is closely linked to glnA, strains with mutations to complete loss of function of the glnR product can be distinguished from glnA strains by their ability to produce detectable Gln synthetase and to grow in the absence of glutamine. To demonstrate unequivocally that glnR is distinct from glnA, we have purified and characterized Gln synthetase from a strain with a Tn10 insertion in glnR. Because the properties of Gln synthetase from the insertion mutant, most importantly the carboxyl-terminal sequence of amino acids, are the same as those of synthetase from wild type, the Tn10 insertion cannot be in glnA (if it were, the carboxyl terminus of Gln synthetase would have to be altered); therefore we conclude that the Tn10 insertion is in a regulatory gene, glnR, which is distinct from glnA. A model for the function of the glnR product together with the previously defined glnF product in mediating nitrogen control is discussed.

Is genetically transmitted obesity due to an adipose tissue defect?

1. The aim of this investigation was to ascertain of a variety of obese rodents whether the primary cause of fat cell enlargement lay in the fat cell itself, or in its environment. Rodents studied were the mutant mice 'diabetic' (db/db), 'adipose' (dbad/dbad), and 'yellow obese' (Ay/+), New Zealand obese mice, CBA mice made obese with gold thioglucose, and obese BIO 4.24 hamsters. 2. Gonadal fat of obese or lean genotype was transplanted under the kidney capsule of an obese or lean host. Grafts were left in place for at least one month, then examined histologically to measure fat cell diameters, from which fat cell masses were calculated. 3. Immunological rejection of grafts was avoided either by using mice syngeneic except for the obesity producing mutation (db/db, dbad/dbad or Ay/+) or by transplanting into F1 hybrids (NZO X BALB/c) made by mating the strains acting as donors of obese or lean fat. Transplantation of fat between lean BIO 4.22 hamsters and obese BIO 4.24 hamsters was possible because these had common histocompatibility antigens. 4. In all the forms of murine obesity studied, 'lean' fat cells enlarged in an obese recipient to the size typical of cells in 'obese' fat whilst 'obese' fat cells shrunk in a lean recipient to, at least, the size typical of 'lean' fat. Lean hamster fat cells also enlarged in an 'obese' environment and 'obese' hamster cells shrunk in a 'lean' environment. 5. Environment therefore contributes to the determination of fat cell size in all the rodents studied, and in several rodents (db/db, dbad/dbad, Ay/+, and gold thioglucose obese mice) our results showed that environmental factors are of paramount importance in determining cell size, and factors associated with the fat cell itself make a negligible contribution.

Actions and interactions of amphetamine on self-stimulation in rats.

The dose-response relationship for d-amphetamine (0.125-2 mg/kg, IP) and its l-isomer (0.125-3 mg/kg, IP) was studied in self-stimulation behavior of rats each with an electrode at posterior hypothalamus (PH, mainly monoaminergic) or area ventralis tegmentum (A10, dopaminergic). The drug effects increased with the dose reaching a peak (at 0.5 mg/kg with d-amphetamine and at 1.0 mg/kg with 1-amphetamine) and then decreased. The d-isomer was approximately twice as potent as the l-isomer in enhancing intracranial self-stimulation (ICSS) rate with electrodes at either site. Azaperone (mainly an alpha-adrenergic blocker) and haloperidol (an antidopaminergic neuroleptic) used in small doses (0.05 and 0.008 mg/kg respectively) which did not affect the baseline responding, blocked amphetamine-induced enhancement of ICSS in both groups of rats. Thus, amphetamine-induced facilitation of ICSS at both PH and A10 areas and its blockade by an alpha-adrenergic blocker as well as an antidopaminergic neuroleptic show the involvement of both noradrenergic and dopaminergic mechanisms in self-stimulation behavior.

LSD-induced alterations of investigatory responding in rats.

The effects of lysergic acid diethylamide (LSD) on investigatory responses of rats in a novel hole-board were assessed in a series of experiments. LSD (40-160 micrograms/kg) altered the temporal distribution of "nose-poke" response during a 24-min session; LSD-treated rats responded less than controls initially, yet increased their response rates late in the session. This dose-dependent effect was not related to the time course of the drug's action nor to alterations in general locomotor activity. Only partial tolerance was found after eight daily injections of 100 micrograms/kg LSD. When handling stress was minimized by placing the animals in an anteroom for 10 min before starting the test, the distribution of responding was normal although the overall frequency was still reduced. Conversely, vigorous handling potentiated the LSD effect. These results are interpreted as indicating an increased sensitivity of the LSD-treated rats to the stimuli associated with being handled and placed into the novel hole-board rather than a direct effect on investigatory tendencies. This LSD-induced potentiation of defensive responses appears to compete with the active exploration of the novel environment.

[Clinical study of benorilate. Co-ordinated multi-centre test (author's transl)].

During a co-ordinated test realized by four centres of rheumatology, 91 patients had been treated by benorilate,giving so the possibility to appreciate its efficiency and its tolerance as well in the articulary fits of degeneration as in the inflammatory rheumatisms. The observations had been collected during ambulatory treatments and consequently in the conditions of daily practice. The efficiency of benorilate has been estimated by the two big disadvantages of the rheumatic person: importance of pains and capacity to do principal movements of daily life. The almost totality of patients having been preliminarily treated by another anti-inflammatory and/or antalgesic, the judgement aimed at the amelioration given by the benorilate : 41% of patients affected by arthropathy of degeneration feeled better or much better after taking benorilate. The amelioration reaches 61% for cases of rheumatoid polyarthritis. About the four criterions which could have been numerically quoted, the amelioration that had been observed is statistically significant. No anomaly had been pointed out as for the biological tolerance. The suspension form is well accepted by the rheumatic persons.

CO2-induced kidney calcification.

Light microscopic examination of kidney tissue of guinea pigs exposed to 1.5% CO2, 21% O2, and balance N2 for periods as long as 42 days and of rats exposed to the same CO2 concentrations for up to 91 days showed that the incidence of focal kidney calcification increased with length of exposure. Calcification occurred primarily in the tubules of the renal cortex. Another group of guinea pigs were exposed to 1% CO2, 21% O2, and the balance N2 for periods up to six weeks and were later killed at regular intervals, together with control animals of the same litter. In the exposed animals, arterial PCO2 was elevated by 3-4 mmHg and hydrogen ions by about 4 nmol/liter. The standard bicarbonate level was lowered by 1-1.5 mmol, indicating a lack of renal reabsorption of bicarbonate (HCO3), which in turn placed greater stress on the bone buffer system and apparently caused bone calcium and phosphorus mobilization. Bone calcium and phosphorus levels exhibited a cyclic decrease, which resulted in cyclic hypercalcemia and hyperphosphatemia, after one week and six weeks of exposure to 1% CO2. Kidney calcium content increased significantly after two weeks of exposure (27%) and remained at this elevated level during subsequent exposures between the third and sixth weeks. These findings indicate that once the kidney calcification process has started, kidney mineralization is independent of fluctuations in the blood calcium level. A rise in plasma phosphate level that occurred after one day of exposure could have been a precipitating factor in the calcification process. The small but consistent increases in ionized calcium during a 4-week exposure to 1% CO2 may have stimulated the parathyroid, causing an increased blood calcium level that was independent of the two calcium tides in the blood associated with marked bone calcium loss.

[Analysis of oxalic acid and oxalates].

It is reported on individual methods for the estimation of the oxalic acid in body fluids, particularly in the urine. The case in question is a survey of the oxalate estimation methods, which, however, has no pretensions to completeness. The at present most actualestimation methods are brought somewhat more in detail. The data are not sufficient for the laboratorytechnical performance of the individual methods, this would transgress the possibilities of the work. However, the original papers are cited which contain all the necessary details. Some technical difficulties and disturbances in the individual estimation methods are also entered. Despite excellent work of several teams the problems of standardization, of the absolutely reliable reference methoda as well as of an objective consideration of advantages and disadvantages of individual, often subjectively judged methods is not yet solved. Comparing these methods, one gets the impression that several reliable methods of the same value are established. It seems that this estimation method brings the greatest progress which will reliably establish so small quantities of oxalate as they are in the blood or in the liquor. By this also the oxalate clearance and the renal oxalate treatment becomes more exactly establishable than up to now.

Renal function in hypercalcemic dogs during hydropenia and during saline infusion.

The effects of calcium-gluconate infusions on renal function were studied in unanesthetised dogs. Each dog was studied during hydropenia and saline infusion. Hypercalcemia, mean serum calcium 3.85 mmol/l (hydropenia) and 3.62 mmol/l (saline infusion), increased fractional excretion of sodium (CNa/CIn), calcium (CCa/CIn), and magnesium (CMg/CIn). The increase was significantly higher in saline-expanded dogs than in hydropenic dogs. Fractional excretion of potassium (CK/CIn) was increased in hydropenia but remained unchanged in saline-expanded animals. Fractional excretion of phosphate (Cp/CIn) was not consistently changed by hypercalcemia. Fractional excretion of chloride (CCl/CIn) was markedly increased in saline-expanded dogs but was not changed in hydropenia. Urine osmolality was reduced in hydropenic dogs but unchanged in saline-expanded dogs. In hydropenic as well as in saline-expanded dogs tubular reabsorption of solute-free water (TcH2O/CIn) increased during the first hour of hypercalcemia. In hydropenic dogs hypercalcemia caused a slight but significant decrease in blood pH, standard bicarbonate, and base excess. In hydropenic as well as in saline-expanded dogs glomerular filtration rate (CIn), renal plasma flow (CPAH), and filtration fraction were unaffected.

Inefficacy of digitalis in the control of heart rate in patients with chronic atrial fibrillation: beneficial effect of an added beta adrenergic blocking agent.

The role of digoxin and the new beta adrenergic blocking agent, timolol, in controlling heart rate at rest and during exercise was investigated in 28 patients with chronic atrial fibrillation. Digoxin failed to prevent excessively rapid heart rates during mild to moderate exercise. Increasing digoxin blood levels from a mean of 0.6 to 1.8 ng/ml had no effect on heart rate either at rest or during exercise. The addition of timolol, 20 to 30 mg/day, resulted in a satisfactory and significant attenuation of the rapid heart rates both at rest and during exercise. Heart rates at rest were 91 and 98 beats/min in the patients with low and high digoxin dosage and rose to 135 and 139 beats/min, respectively, during exercise. Timolol reduced the heart rate to 67 at rest and to 92 beats/min during exercise. The effect of beta adrenergic blockade at rest was less pronounced in patients whose initial heart rates were below 90 beats/min. Digoxin alone may not suffice to control excessive heart rate in patients with chronic atrial fibrillation. The additional beta adrenergic blockade actually normalizes the heart rate response in these patients.

[Penicillin biosynthesis and amino acid metabolism in Penicillium chrysogenum in experiments with washed mycelium].

The study of the amino acid metabolism in Penicillium chrysogenum with the use of washed mycelium showed that the amount of the free intracellular amino acids significantly decreased during the process of penicillin production. Still, such a decrease did not cover the nitrogen requirements of the culture for the antibiotic synthesis and mobilization of the protein nitrogen took place. By the end of the process the amount of the protein nitrogen markedly decreased. At the same time alpha-amino nitrogen was absent in the fermentation broth filtrate. About 14 amino acids (including cysteine and valine) which participate in constriuction of the penicillin molecule nucleus were found in the amino acid poll. However, the amounts of cysteine and valine were not high and probably other free intracellular amino acids participated in their synthesis. It was shown that one of the limiting factors in the process of penicillin biosynthesis was synthesis of cysteine, a sulphur-containing amino acid which is one of the precursors of the antibiotic molecule nucleus.

Transport of glutamine by rat kidney brush-border membrane vesicles.

Transport of glutamine by brush-border vesicles prepared from the renal cortex was studied. The transport system had both Na+-dependent and Na+-independent components. The presence of Na+ in the incubation resulted in an 'overshoot' at 30s at which time the rates of transport were approx. 8 times the values obtained in the absence of Na+. Variation of the glutamine concentration showed that the system obeyed Michaelis-Menten kinetics with Km and Vmax. values for the Na+-dependent system of 0.86 mM and 9.6 nmol/min per mg of protein respectively. Vesicles obtained from chronically acidotic rats showed similar kinetic characteristics. The Km and Vmax. values for the Na+-dependent system were 0.76 mM and 9.6 nmol/min per mg of protein respectively. There was increased uptake of glutamine by vesicles from acidotic rats and this increase was associated with increased activity of gamma-glutamyltransferase in these preparations. Vesicles from acidotic rats, however, showed no increase in glucose transport and no increase in the activity of maltase, another brush-border enzyme.

Effects of temazepam, flurazepam and quinalbarbitone on sleep: psychomotor and cognitive function.

1 The effect of temazepam 15 and 30 mg, flurazepam 15 and 30 mg, quinalbarbitone 100 and 200 mg and placebo were studied in 14 healthy male volunteers according to a Latin-square design. At 14-d intervals subjects received capsules 30 min before bedtime on 2 consecutive nights and were evaluated for objective sleep characteristics, for morning estimates of sleep characteristics, and for cognitive and psychomotive performance and subjective state at 3.5, 10.0 and 22.5 h after ingestion. 2 Changes in sleep induction and sleep maintenance were observed with temazepam 30 mg and flurazepam 30 mg had the greater effect on cognitive performance, whereas quinalbarbitone 20 mg had the greater effect on psychomotive performance. Subjective assessments of alertness were most affected by flurazepam, and by quinalbarbitone 200 mg. 4 The results suggest that temazepam produces less residual effects and is shorter acting than quinalbarbitone and flurazepam.

Favism: erythrocyte metabolism during haemolysis and reticulocytosis.

The reduced activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate; NADP+ 1-oxidoreductase; G6PF) in Mediterranean erythrocytes explains the precarious equilibrium of the hexose monophosphate pathway (HMP) and the susceptibility of these cells to haemolytic agents. G6PD-deficient erythrocytes, in steady-state conditions, have a low NADPH/NADP+ ratio, thus allowing the HMP to operate at its maximal intracellular rate and to compensate the intrinsic erythrocyte enzyme deficiency. Studies started soon after accidental intake of fava beans by sensitive G6PD-deficient subjects demonstrate a decrease of both NADPH/NADP+ ratio and reduced glutathione. The metabolic effects induced by fava beans may be attributed to oxidative stress probably associated with an inhibitor effect of some unknown metabolite on the HMP. The availability of erythrocytes from subjects recovering from haemolysis with high reticulocyte counts and increased G6PD activity, provides new information on the rate of synthesis as well as on the in vivo decay of the mutant enzyme. Correlation of G6PD activity to reticulocyte count and extrapolation to an ideally homogenous population of reticulocytes reveal that the mutant enzyme is synthesized at a nearly normal rate. Furthermore, the present correlation allows an estimate of the in vivo half-life of Mediterranean G6PD. The rate of decline of about 8 d observed in this study well correlates to the intracellular metabolic aspects of G6PD Mediterranean erythrocytes.

Electron acceptors of bacterial photosynthetic reaction centers. II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex.

The photoreduction of ubiquinone in the electron acceptor complex (QIQII) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H+ in the reduction was revealed by the pH dependence of the electron transfer events and by net H+ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash QII receives an electron via QI to form a stable ubisemiquinone anion (QII-); the second flash generates QI-. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H+, to produce QIIH2. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H+ uptake. Above pH 6 there is a progressive increase in H+ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H+ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to QII-. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H+ binding electron transfer following the second flash.

A ribonuclease from yeast associated with the 40 S ribosomal subunit.

1. Autodegradation of yeast ribosomes is due to a 'latent' ribonuclease which is associated with the 40 S ribosomal subunit. 2. The ribonuclease was extracted in the presence of EDTA from ribosomes and purified 118-rold by protamine sulphate precipitation, (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. 3. The optimum pH for this enzyme is 5 to 6.5 while the optimum temperature is 45 to 50 degrees C. Incubation for 10 min at 60 degrees C caused a reduction in enzyme activity of 70%. 4. The ribonuclease has an endonucleolytic activity against rRNA, tRNA, poly(A), poly(U) and poly(C) but does not degrade poly(G) or DNA. It hydrolyzes the homopolymers to nucleoside 3'-phosphates. 5. Zn2+, Mn2+, heparin, glutathione and p-chloromercuribenzoate inhibit the ribonuclease, while Na+, K+, EDTA and sermidine have only little or no effect. 6. It binds tightly to yeast ribosomes but only loosely to ribonuclease-free wheat germ ribosomes. 7. Polyribosomes possess less autodegradation activity than monoribosomes, isolated from the same homogenate.

Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue.

CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM.

Studies on synaptic vesicles in mammalian brain characterization of highly purified synaptic vesicles from bovine cerebral cortex.

Synaptic vesicles have been isolated from bovine cerebral cortex by sequential differential and density gradient centrifugations followed by chromatography on a Sepharose 6B column. We have studied the morphology, enzymatic markers, neurotransmitter and ATP contents and protein composition of the vesicles. The specific contents of acetylcholine, gamma-aminobutyric acid, aspartate, glutamate and catecholamines were 4--8-fold higher in the vesicle fraction compared to the crude synaptosomal pellet. Electron micrographs of the vesicle preparation showed enrichment of vesicular material with an average diameter of 50 nm. The purity of the preparation was assessed by the very low activities of enzymatic markers of cellular membranes and cytosol components. Some Ca--Mg-activated ATPase activity was detected in the vesicle preparations, but its content relative to the neurotransmitters fell on chromatography, suggesting that this activity may be partially contributed by non-synaptic vesicle components, such as small microsomes. The isolated synaptic vesicles were solubilized with 1% sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. The major Coomassie blue stained bands observed with apparent molecular weights of 160,000 and 55,000 were enriched in parallel to the increase in purity of the preparation.

Novel 125I-labeled nortriptyline derivatives and their use in liquid-phase or magnetizable solid-phase second-antibody radioimmunoassays.

Nortriptyline derivatives prepared by reaction with fluorescein isothiocyanate or conjugation to N-acetyl-L-histidine were radioiodinated and the products purified with Sephadex LH-20 columns to obtain two novel nortriptyline radioligands. Antisera were raised in rabbits by immunization with nortriptyline conjugated to succinylated ovine albumin. By use of the iodinated fluorescein derivative we developed a liquid-phase second-antibody radioimmunoassay that gives results correlating closely (r = 0.98) with those by an established radioimmunoassay of similar specificity in the assay of apparent total amitriptyline and its metabolite nortriptyline in serum or plasma from patients being treated with these drugs. With the iodinated N-acetyl-L-histidine derivative we developed a magnetizable solid-phase second-antibody radioimmunoassay. The cross reactivities of amitriptyline and nortriptyline could be made equal by performing the assay at pH 9.0, which makes it possible to measure true total active drug concentrations in patients receiving amitriptyline.

The adenylate cyclase system in human liver: characterization, subcellular distribution and hormonal sensitivity in normal or cirrhotic adult, and in foetal liver.

1. Adenylate cyclase (EC 4.6.1.1) activity was characterized in human liver, and its subcellular distribution compared with that of three other potential enzyme markers of the pericellular membrane: leucine aminopeptidase (EC 3.4.11.1), gamma-glutamyltransferase (EC 2.3.2.2) and 5'-nucleotidase (EC 3.1.3.5). Although these three enzyme activities were detected in each of the subcellular fractions studied, 85% of the total adenylate cyclase activity was found in the 1000 g pellet ('nuclear' fraction) with a threefold increase in specific activity as compared with the homogenate. No adenylate cyclase activity existed in the 150 000 g supernatant fraction. 2. In the 'nuclear' fraction, adenylate cyclase activity was increased in a dose-dependent fashion by glucagon with a half-maximal stimulation at 10 nmol/l and a maximal four- to seven-fold increase at 1 mumol/l. Catecholamines activated adenylate cyclase 2.5- to three-fold, with an order of potency (protokylol greater than isoprenaline greater than adrenaline greater than noradrenaline) typical of a beta 2-adrenoreceptor. Prostaglandin E1 and NaF also stimulated cyclase two- and four-fold respectively. Insulin, serotonin, dopamine, thyroid-stimulating hormone and ACTH had no effect. Adenosine provoked a weak inhibition at 0.1 mmol/l. Finally guanosine triphosphate and 5'-guanylyl imidodiphosphate induced a marked increase in basal activity, four- and eight-fold respectively, but both reduced the relative increase in enzyme activity due to glucagon or adrenaline. 3. Cyclase from foetal liver (12--16 weeks old) and cirrhotic adult liver appeared to behave similarly to that from normal liver; however, foetal cyclase was more active, and cirrhotic enzyme less active than normal adult liver. Both systems responded to catecholamines via a beta 2-adrenoreceptor. 4. These results validate the use of rat liver adenylate cyclase as a tool for pharmacological and physiological studies.

Hormonal regulation of acetyl-CoA carboxylase activity in the liver cell.

Chick liver cell monolayers synthesize fatty acids at in vivo rates and are responsive to insulin and glucagon. High rates of fatty acid synthesis are maintained with insulin present and lost slowly without insulin. Glucagon or 3',5'-cyclic AMP cause immediate cessation of fatty acid synthesis. The site of inhibition appears to be cytoplasmic acetyl-CoA carboxylase which catalyzes the first committed step of fatty acid synthesis. Liver carboxylase exists either as catalytically inactive protomers or active filamentous polymers. Citrate, an allosteric activator of the enzyme, is required for both catalysis and polymerization. Glucagon and cAMP cause an immediate decrease in the cytoplasmic citrate concentration of chick liver cells apparently by inhibiting the conversion of glucose to citrate at the phosphofructokinase reaction. Since fatty acid synthesis and citrate level are closely correlated, citrate appears to be a feed-forward activator of the carboxylase in vivo. Compelling evidence indicates that carboxylase filaments are present in the intact cell when citrate levels are high and depolymerize when citrate levels fall. Hence, carboxylase activity and fatty acid synthetic rate appear to be determined by cytoplasmic citrate level.

[Gonadotropin secretion following pernasal stimulation with synthetic gonadotropin releasing hormone (GnRH) and a highly effective GnRH-analog in healthy men and prepubertal boys].

Potent long acting analogs of GnRH are of great interest especially in view of pernasal (p.n.) treatmen of hypogonadism of hypothalamic origin and of cryptorchidism. To find the necessary p.n. dosage of such a substance, serum LH and FSH were measured in 6 normal adult human males after p.n. application of various doses of D Leu6-des-Gly10-GnRH ethylamide. 50 microgram of the GnRH analog were necessary to obtain increased serum gonadotropins over a period of at least 8 hours. By repeated p.n. application of 200 microgram of synthetic GnRH every 2 hours in 6 normal adult males a considerable increase of serum gonadotropins could be demonstrated as well. Pernasal application of 200 microgram GnRH repeated at an interval of 1 hour in 3 cryptorchid boys produced a distinct increase of the serum gonadotropins. The intraindividual comparison of 200 microgram GnRH and 20 microgram of the GnRH analog in one boy showed equivalent net increases of the gonadotropins. With the analog the gonadotropin increase lasted for about 6 hours.

Role of the renin-angiotensin system in the blood pressure rebound to sodium nitroprusside in the conscious rat.

Intravenous infusions of sodium nitroprusside (SNP) at doses of 20, 40 or 80 micrograms/kg min-1 for 30 min produced dose-related decrements in blood pressure in conscious rats fitted with indwelling aortic and vena caval catheters. Immediately upon termination of SNP infusions, blood pressure rebounded to levels which were significantly above pre-SNP control values. The following evidence indicates that the rebound increase in blood pressure was due to increased activity of the renin-angiotensin system: (1) plasma renin activity was increased approximately four-fold by SNP, (2) rebound did not occur in nephrectomized rats, (3) rebound was markedly attenuated in animals treated with an angiotensin converting enzyme inhibitor, SQ14225, (D-3-mercapto-2-methylpropanoyl-L-proline) and (4) beta-adrenergic receptor blockade with propranolol reduced the rebound response. In addition, the magnitude of the rebound following SNP infusions was directly related to the dose of SNP infused. These results are consistent with the hypothesis that renin accumulates during SNP infusion more rapidly than it is metabolized. Consequently, the accumulated renin elicits a hypertensive response when SNP treatment is withdrawn.

The Royal College of Midwives: past, present and future.

The initial vision of Miss Louise Hubbard and 7 midwives provided the impetus for the founding of the Midwives Institute in 1881. The British Midwives Act was passed in 1902. It set up a statutory body, the Central Midwives Board, which trained and controlled the practice of midwives in England and Wales, and made it illegal for unqualified persons to practice midwifery. From its early days the Midwives Institute was interested in the continued education of the midwife. Lectures were regularly held by eminent obstetricians and were attended by midwives who paid for the lectures. The Institute grew and formed an organization with local groups of midwives throughout England and Wales. The Royal College of Midwives is now a membership organization whose headquarters are in West End, London. Full membership is open to all midwives on the Roll of the appropriate Statutory Body. As a voluntary organization, the College is supported by members' subscriptions and donations and fees for postcertificate educational courses. Changing maternity care patterns, increased use of technology, high standards of clinical ability, and changes in society have shaped the Education Department of the College. Recently, the College worked toward the passage of the Nurses, Midwives and Health Visitors Bill through Parliament.

Reversal of vasectomy.

Vasovasostomy to reverse a previous vasectomy for sterilization was attempted for 27 men, the procedure being technically impossible in only one case. A testicular biopsy was performed at the time of operation and a number were investigated for cell-mediated immunity to sperm and for the presence of circulating sperm-agglutinating and cytotoxic antibodies. The first 17 cases have been studied and of these there have been 11 pregnancies, ten of which have already come to term with the birth of normal infants, including one set of twins. Of the rest, two are known to have oligozoospermia and four have been lost to follow-up, although two of them were euspermic when last examined. In spite of these encouraging results, it is considered that there are no grounds for alterning the present basis of vasectomy counseling which is that the operation is likely to be irreversible.

Anaerobic isolates in chronic recurrent suppurative otitis media. Treatment with carbenicillin alone and in combination with gentamicin.

Tympanocentesis was performed in 32 pediatric patients with chronic recurrent suppurative otitis media. The aspirate was cultured aerobically and anaerobically. Aerobes were isolated from ten patients (31.2%); anaerobes from one patient; and both aerobes and anaerobes from 21 patients (65.6%). There were 46 aerobic isolates. The aerobes commonly recovered were Pseudomonas aeruginosa (24 isolates) Proteus sp. (5) and Staphylococcus aureus (3). There were 32 anaerobes isolated including anaerobic gram-positive cocci (19 isolates) and Bacteroides sp., the latter of which included Bacteroides fragilis group and Bacteroides melaninogenicus (9). The patients were treated by parenteral carbenicillin 300 to 400 mg per kg per day given in four dosages administered for a period of 12 to 21 days (average 17 days). An aminoglycoside (gentamicin) was added in 15 patients. The clinical response was good in 17 patients and poor in 15. There were no side effects or adverse reactions noted during therapy. The above findings demonstrate the polymicrobial etiology of chronic recurrent suppurative otitis media and suggest that treatment directed against the aerobic and anaerobic isolates is efficacious in more than half of the cases.

Effect of cerebral extracellular fluid acidity on total and regional cerebral blood flow.

Total and regional cerebral blood flow (CBF), and cerebrospinal fluid (CSF), and arterial blood acid-base status were measured in 26 chloralose-urethan-anesthetized dogs before and after 30 and 60 min of ventriculocisternal perfusion with artificial CSF equilibrated with 7% CO2 and containing either low (8.7 or 9.1 meq/l), normal (19.6 meq/l), or high (34.7 meq/l) bicarbonate ion concentration ([HCO3-]). An inverse linear relationship was observed between the CSF pH and total CBF. Regional blood flow changes were greater in structures that were closest to the ventricular system. In addition, regional blood flow changes were greater in all tissues studied after 60 min of perfusion than after 30. Perfusion with the control [HCO3-] caused no significant changes in either acid-base status or CBF. We believe that the regional cerebral blood flow changes are the result of changes in the H+ concentration gradient across the cerebral extracellular fluid (ECF) space due to the diffusional exchange of HCO3- between CSF and ECF. It is concluded that cerebral ECF acidity is important in the local regulation of cerebral blood flow.

Tracheal fluid in fetal lambs: spontaneous decrease prior to birth.

We studied tracheal fluid (TF) production in 14 fetal lambs: 6 controls, 6 receiving atropine on 1 or more of the last 7 days before birth, and 2 with bilateral section of the cervical vagosympathetic trunk. A cannula diverted all TF into an intrauterine bag; we collected TF intermittently and measured its volume. All ewes delivered spontaneously at 128-150 days' gestation. TF production decreased before birth in all fetuses except one control. TF production decreased before birth in all fetuses except one control. TF production did not correlate with fetal arterial blood gas tensions, hematocrit, or plasma proteins. In controls only, TF production correlated with fetal arterial pH (P less than 0.02); however, the pH range was small and the correlation has questionable physiological significance. For all fetuses, TF production during the 7 days before birth correlated inversely with the plasma cortisol concentration of 48 h previously (n = 36; r = -0.603; P less than 0.001). We conclude a) TF production in fetal lambs decreases before spontaneous term or preterm labor; b) this decrease is not affected by atropine or by section of the cervical vagosympathetic trunk; and c) the decrease in TF production may be related to increased secretion of cortisol.

Occurrence of old yellow enzyme in Gluconobacter suboxydans, and the cyclic regeneration of NADP.

Old yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88,000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.

Fluorescent antibody test kit for rapid detection and identification of members of the Bacteroides fragilis and Bacteroides melaninogenicus groups in clinical specimens.

The Fluoretec fluorescent antibody test kit (Pfizer Inc., New York, N.Y.), developed for the rapid detection of members of the Bacteroides fragilis and B. melaninogenicus groups, was evaluated by testing 58 stock cultures and 76 clinical specimens. The test reagents detected 100% of 40 B. fragilis and B. thetaiotaomicron stock culture strains, although only 22% of 18 B. vulgatus, B. distasonis, and B. ovatus strains showed positive fluorescence. The 76 clinical specimens were evaluated by examining fluorescent antibody-stained smears of 49 specimens of purulent material and smears of 27 blood cultures which were positive for gram-negative bacilli by Gram stain or subculture. The fluoretec reagent detected members of the B. fragilis group in 28 (97%) of the 29 specimens of purulent material and all (100%) of the 16 blood cultures in which these anaerobes were demonstrated by culture. Overall, the Fluoretec reagent detected members of B. fragilis group in 44 (98%) of the 45 clinical specimens which were shown by culture to contain these anaerobes. Two of the 76 clinical specimens gave positive fluorescence for members of the B. fragilis group but failed to yield these organisms by culture. Members of the B. melaninogenicus group were detected by culture in 15 specimens and in each case their presence was demonstrated by the Fluoretec reagent. No members of the B. melaninogenicus group were isolated from five clinical specimens that gave positive fluorescence with the B. melaninogenicus reagent.

Discontinuous density gradient separation of human mononuclear leucocytes using Percoll as gradient medium.

The use of a new commerically available medium (Percoll) for fractionation of human mononuclear leucocytes is described. Cells can be fractionated on the basis of their densities with high reproducibility. The separated cells were characterized by morphological and functional criteria. Monocytes can be obtained in the low density fractions with a purity of 70%--90%. Lymphocytes were found in high density fractions with a purity up to 99%. No separation between E-rosette forming (E-RFC) and surface immunoglobulin-bearing lymphocytes was obtained. However, a reduced number of high avidity E-rosette forming lymphocytes (HAE-RFC) was found within low density lymphocytes. Best spontaneous DNA synthesis and reaction in mixed leucocyte culture (MLC) were obtained with cells isolated from the 1.066/1.068 density interface, whereas the stimulation with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) had a peak response with cells from the 1.064/1.066 density interface. Colony forming myelopoietic stem cells and colony forming T-lymphocytes were detected in fractions of low density.