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Linoleic acid absorption in the unanesthetized rat: mechanism of transport and influence of luminal factors on absorption.

Linoleic acid intestinal absorption was studied in the unanesthetized rat. At low (21-1260 micrometer) intraluminal concentrations, absorption took place by facilitated diffusion; while at high (1.26-2.5 mM) concentrations, simple diffusion was the predominant mechanism of transport. At low concentrations (840 micrometer), the equimolar additions of oleic, linolenic, and arachidonic acids or lecithin inhibited the absorption of linoleic acid. Substitution of potassium for sodium in the buffer solution, substitution of Tween 80 for sodium taurocholate, or decrease in the hydrogen ion concentration all resulted in decreased rate of linoleic acid absorption. Increase in sodium taurocholate concentration, or perfusate flow rate increased linoleic acid's absorption. These experiments demonstrate that linoleic acid is absorbed by a concentration-dependent dual mechanism of transport. The absorption rate is modified by the pH, surfactant type and concentration, the simultaneous presence of other polyunsaturated fatty acids, and the thickness of the unstirred water layer.

Acid-base, calcium, potassium and aldosterone metabolism in renal tubular acidosis.

Classic renal tubular acidosis is characterized by a primary defect in establishment of a large hydrogen ion gradient across the distal renal tubule. Thus the development of hyperchlorenic metabolic acidosis follows. In addition, hypokalemia results from renal potassium wasting secondary hyperaldosteronism from sodium wasting and contraction of the extracellular fluid. The presenting signs and symptoms are growth retardation, fatigue, periodic paralysis, polyuria, polydipsia, vomiting and constipation as well as nephrocalcinosis and nephrolithiasis. It is suggested that effective treatment with alkali therapy requires markedly higher doses than formerly recommended, and may related to a higher rate of endogenous acid production from (1) intermediary metabolism of sulfur amino acids and organic acids, (2) impaired tubular reabsorption of bicarbonate and (3) hydrogen ion release from hydroxyapatite formation. It is also suggested that acidosis may interfere with vitamin D metabolism and thus play an important role in the pathoetiology of the growth failure in children with this disorder.

Dark field imaging of biological macromolecules with the scanning transmission electron microscope.

A scanning transmission electron microscope (STEM) equipped with a field emission gun has been employed for the examination of biological macromolecules at high resolution. The quality of micrographs obtained with the STEM is dependent upon the quality of the substrate used to support biological objects because the image contrast in dark field is proportional to the mass density of the specimen. In order to reduce deleterious effects of the substrates on the image quality, we have developed a method of fabricating substrates consisting of very thin, very clean carbon films supported on very clean fenestrated plastic films. These films are approximately 15 A thick. Well-known biological macromolecules such as glutamine synthetase and tobacco mosaic virus (both stained) and low-density lipoprotein and ferritin (both unstained were placed on these substrates and examined with the STEM by using various modes of contrast. The micrographs obtained by using the dark field mode of contrast employing an annular detector were free from phase contrast, as expected. Using this contrast mode, we have been able to directly observe (in-focus) 2.5- to 4.4-A lattice spacings in the ferritin core. The effect of electron radiation damage on the helical structure of tobacco mosaic virus was also examined. Micrographs as well as corresponding optical diffraction patterns obtained with moderately low doses showed very clear helical structure from both sides of the virus. In addition, the (11.5 A)(-1) layer lines indicated the effective resolution attained on these particles.

A new analytical procedure for two-dimensional electrophoresis of cellular proteins: comparison of protein compositions of parent strain and a K+-accumulation mutant of E. coli.

An improved two-dimensional analytical electrophoretic technique fractionates according to molecular weight in the presence of dedecyl sulfate in the first dimension, then fractionates according to isoelectric point in a perpendicular dimension. Electrofocusing in the second dimension achieves nearly complete removal of most protein components while providing true estimates of their isoelectric points. Because not all proteins penetrate isoelectric focusing gels, some proteins may go unrecognized using conventional two-dimensional systems where isoelectric focusing precedes electrophoresis. However, such components do enter dodecyl sulfate gels; hence the presence and molecular weight of those components can be established by the new procedure. A concurrent finding was that, in general, penetration of isoelectric focusing gels by discrete protein subunits dissolved in 9 M urea is an all-or-none phenomenon depending on the solubility of the specific subunit. The procedure was applied to comparison of the protein compositions of a parental strain (CBH) of Escherichia coli and a derived mutant strain (RD-2) deficient in ability to accumulate K+. The strains showed similar two-dimensional patterns except for one discrete isoelectric component absent in the parent strain but present in the mutant.

[The gamma-glutamyltransferase (GGT) isoenzymes in human serum (author's transl)].

With this report we communicate the results of a method for the fractionation of the isoenzymes of gamma-GT in human serum based on a modified technique of Hetland et al., using cellulosa acetate. With this method we observed the appearance of a complex of four band representing: gamma-GT1 = prealbumin-albumin, gamma-GT3 = alpha 1-globulin, gamma-GT3 = alpha 2-globulin, gamma-GT4 = beta-globulin. It has been noted that the gamma-GT2 is the only fraction which constantly appears. This isoenzyme also corresponds to the form, fisiologically present in serum and is presumably preduced in the cells of the bileduct. In the results we observed the appearance of gamma-GT1 only in the course of processes involving certainly cholestasis. This fraction was once reported by other AA., but it has an unknown origin. It was not possible to correlate the appearance of the fractions gamma-GT3 and gamma-GT4 with any specific pathology alterations even though Hetland et al. have affirmed that the gamma-GT3 band derives from hepatic parenchyma.

[The present treatment of headaches (author's transl)].

Considered from the point of view of clinical practice, the treatment of chronic headache may be either symptomatic and etiological or physiopathological. Progress in symptomatic treatment depends first on the reasonable and graduated use of pure analgesics, looking out for the toxic side effects of the usual drugs and then the fairly definite efficacy of certain psychotropic drugs. The discovery of an etiology gives a specific dimension to the treatment: either anti-cerebral oedema drugs with above all tetracosactide, a diagnostic test of cerebral tumours, or antidepressor or tranquillizer drugs, depending on the variety of disturbance to be corrected. An attack of migraine always benefits from ergotamine used occasionally and in limited dosage (not more than 6 mg daily or 10 mg per week). For the basic treatment the drugs act mainly peripherally and fairly regularly in the following order: methysergide, beta-blockaders, pizotifene, cyproheptadine, oxetorone. Other drugs have a central effect, Tiapridal, MAO inhibitors which are too often neglected, and clonazepam which is not very easy to use.

Neurologic and cardiovascular effects of hypotension in the monkey.

Thirty monkeys were exposed to controlled systemic hypotension of different magnitudes and duration to determine factors leading to brain injury or cardiovascular failure. Fourteen monkeys developed brain injury. Of these, 6 survived indifinitely and 8 were sacrificed or died within 12-62 hours due to neurologic deterioration accompanied by respiratory failure. Sixteen animals did not develop brain injury, but 9 of these died within 24 hours from documented cardiovascular failure with the remaining 7 survived indefinitely. A highly reproducible threshold for the development of brain injury was found at a mean arterial blood pressure (MABP) of 25 mm Hg. Maintenance MABP was less than or equal to 25 mm Hg in 13 of 14 lesioned monkeys and greater than 25 mm Hg in 15 of 16 non-lesioned monkeys. Maintenance MABP averaged 20.1 +/- 1.1 mm /g in lesioned and 32.1 +/- 1.7 mm Hg in non-lesioned animals (p less than 0.001). Among the non-lesioned animals, death from delayed cardiovascular failure ensued when MABP was maintained between 27 and 35 mm Hg for 90 min or longer. Animals exposed to this range of hypotension for less than 90 min or to MABP exceeding 35 mm Hg for as long as 3 h survived intact. EEG changes occurring during hypotension most accurately predicted neurologic outcome. The threshold MABP required to produce cerebral electric silence was 21-22 mm Hg. Monkeys developing marked brain injury had greater than 25 minutes of EEG flattening, while slightly injured animals had it for 5-15 minutes and those without injury for less than 5 min. Changes in acid-base state, common carotid artery blood flow, and cerebral uptake of glucose and oxygen during hypotension also correlated with neurologic and cardiovascular outcome. Hypoxemia and hypercarbia were not contributory factors in the production of brain injury in this study.

Effect of non-carbonic acidosis on total splanchnic perfusion and cardiac output during anaesthesia with O2-N2O-halothane.

Six dogs, premedicated with pethidine 10 mg kg-1 b.w, were anaesthetized with mebumal natrium (NFN) 25 mg kg-1 b.w. and 80 mg gallamoni jodidum (NFN). Anaesthesia was continued with O2-N2O-halothane and artificial ventilation. Non-carbonic acidosis was induced by i.v. infusion of hydrochloric acid, during which the related values of pulse, blood pressure, cardiac output, total splanchnic prefusion and portal pressure were measured. The pulse remained unchanged down to pH 7.0. At this pH, arrhythmia suddenly occured and developed into ventricular fibrillation. Before this occured falling cardiac output was observed (cardiac output 1 min-1 = -21.49+3.21 x pH, N = 23, r = 0.75, P less than 0.001) and rising oxygen consumption (O2 ml min-1 kg-1 = 25.79--2.96 x pH, N =28, r = 0.52, P less than 0.01), rising oxygen extraction and rising peripheral resistance, while the mean pressure in the aorta was almost unaltered. During this course towards circulatory failure, an unchanged to slightly rising total splanchnic perfusion (Qsp1) was demonstrated, which with the lowest pH, represented up to 40% of the cardiac output (Qtot): Qsp1/Qtot = 3.11--0.39 x pH (N = 28, r = 0.52, P less than 0.01). Portal pressure rises slightly during acidosis, and oxygen saturation in the portal vein is high. It is probable that the retained splanchnic blood flow is caused by retention of the portal flow. This is quite different from observations during anaesthesia with barbiturates. It is concluded that halothane modifies considerably the circulatory response in the systemic circulation and the splanchnic region during non-carbonic acidosis.

[Contribution of biology to nosology of depressive states. Neurochemical, endocrine and genetic factors (author's transl)].

Genetic factors have been evidenced in the etiology of manic-depressive syndromes through twins, morbidity risk studies, linkage studies with genetic markers such as color blindness and the Xga blood group, as well as through adoption studies. Most genetic studies indicate that there is a genetic and biological heterogeneity in manic-depressive illness. Among these manic-depressive syndromes, one group is consistent with a dominant X-linked transmission of the disease. From the neurochemical point of view, most investigators emphasize the importance of cerebral neurotransmitter substances such as catecholamines and indolamines in the pathogenesis of bipolar depressive states. According with this hypothesis, depression is associated with a functional deficit in brain monoamines while mania may be due to an hyperproduction of monoamines. These neuropharmacological studies are of importance because they also have neuroendocrine implications. Some pituitary hormones are secreted under the control of brain monoamines, and they are also implicated in the pathogenesis of depressive states.

A communication on health and development in the Kainji Lake area of Nigeria.

A study of the health care delivery facilities in the Kainji Lake area of Nigeria (an artificial lake created in 1968) showed that hospitals, a health centre, maternal and child health centres, public health units, dispensaries and leper institutions, controlled by various organizations, are available. Dispensaries and leper settlements/clinics form the most numerous health providers in the rural areas. Analysis of 1973 data from eight dispensaries around Lake Kainji showed that malaria, gastroenteritis, chest and skin infections, venereal diseases and shistosomiasis constitute the major health problems. Observations of the environmental sanitation in the study area by the author support the idea that the diseases emanate particularly from the low standard of environmental health. A suggestion is made for the establishment of a central organization charged with the responsibilities for health planning and development. The evaluation of the impact of hte dispensaries as health providers is needed for future health planning. A health care delivery system supported by operational research should be initiated at the village level.

Role of blood platelets in coronary artery disease.

Over the past decade, research in blood platelet physiology has led to the suggestion that platelets play an important part in the pathogenesis and complications of coronary artery disease. Occlusive intravascular platelet aggregates have been shown to cause ischemic myocardial damage in the experimental animal and to be present in some patients who die suddenly. The interplay between endothelial damage and platelet aggregation has been implicated in the etiology of atherosclerosis. Products released from platelets during aggregation may cause arterial spasm. Patients with overt ischemic heart disease and with the risk factors associated with coronary artery disease have been found to have abnormally reactive platelets. Clinical studies of drugs that inhibit platelet aggregation have been reported to show a beneficial effect in preventing cardiac deaths or myocardial infarction; other studies have been negative or shown only a trend toward benefit. This report reviews the theoretical and experimental basis for the platelet hypothesis and the current data on the use of antiplatelet drugs in patients with coronary disease.

Plasma secretin concentrations in fasting and postprandial states in dog.

In four dogs with a modified Herrara pancreatic fistula and gastric cannula and three dogs with two duodenal cannulas, ingestion of a meat meal resulted in a significant and sustained increase in the mean plasma immunoreactive secretin concentrations, from mean fasting levels of less than 10 pg/ml to 25--55 pg/ml. This increase in the plasma secretin concentration coincided with a marked increase in pancreatic bicarbonate output and frequent decreases in the mean proximal duodenal pH to less than 4.5 from the range of 6.5 in the fasting state. Intravenous administration of cimetidine, 150 mg, produced a marked suppression of postprandial increases in both pancreatic bicarbonate output and plasma secretin concentration. Moreover, the postprandial duodenal pH rarely reached below 5.0 after cimetidine administration. These studies indicate that plasma secretin concentration does increase significantly after a meal. The postprandial increase in plasma secretin concentration appears to depend on the gastric acid delivered in the proximal duodenum. A possible physiological role of secretin in the pancreatic secretion after a meal is indicated by these findings.

Sputum counterimmunoelectrophoresis in the diagnosis of pneumococcal pneumonia.

Fifty-six patients with pneumonia were grouped according to degree of clinical certainty that the etiologic agent was Streptococcus pneumoniae. Of 14 patients with definite or probable pneumococcal pneumonia, 12 had pneumococcal antigens detected in sputum by counterimmunoelectrophoresis (CIE), 13 had a positive sputum culture, and 12 had a Gram-stained smear of sputum suggestive of the diagnosis. Of 9 patients with definite nonpneumococcal pneumonia, none had pneumococcal antigens detected by CIE, but one had pneumococci isolated from sputum culture, and one had a Gram stain of sputum suggestive of pneumococci. Of 34 control patients without pneumonia, five had a positive CIE, 11 had a positive culture, and 15 had a positive Gram stain. When used to differentiate pneumococcal from other types of pneumonia, CIE of sputum appears to be a sensitive and specific test. Among patients without pneumonia, however, CIE lacks specificity. Additionally, sputum Gram stain may correlate as well as CIE with pneumococcal pneumonia, but further substantiation of this observation is necessary.

Purification and some properties of two extracellular proteolytic enzymes produced by Vibrio SA1.

The purification and characterisation of an extracellular endo and amino-peptidase of the marine Vibrio SA1 is described. The endopeptidase was purified by ammonium sulphate precipitation, gel filtration and affinity chromatography. It had a molecular weight of approximately 31,000, a pH optimum at 7.8 and a temperature optimum at 50 C. The enzyme was rapidly inactivated at 65 C. The aminopeptidase was purified by ammonium sulphate precipitation, gel filtration and preparative polyacrylamide gel electrophoresis. This enzyme had a molecular weight of approximately 21,000, a pH optimum at 8.6 and a temperature optimum at 60 C. Both proteases were inactivated by EDTA while reactivation occurred by Ca2+, Zn2+ and Mg2+ ions. The endopeptidase hydrolysed several peptide bonds in the oxidized B-chain of insulin, particularly those involving amino groups of hydrophobic amino acid residues with bulky side chains. It was unable to hydrolyse synthetic dipeptides, but a number of tripeptides were hydrolysed at a low rate. The aminopeptidase hydrolysed leucinamide and di- and tripeptides containing hydrophobic bulky amino acids as the N-terminal residue. It was concluded that the endopeptidase and the aminopeptidase of Vibrio SA1 possess complementary specificities.

Human tear buffering capacity.

With the use of a closed chamber microelectrode system, we measured the relative buffering capacities of 490 human tear samples from young healthy adults. The buffering capacities of the 457 waking-hour samples did show small but regular oscillations that were similar to those previously reported for blood and tear pH, but only rarely did the buffering capacity of one sample approach double that of another. The buffering capacities of 33 tear samples, associated with periods of prolonged eye closure (sleep), were not significantly different (P less than 0.5) from those of the open eye.

Prevention and treatment of space sickness in shuttle-orbiter missions.

Today it is impossible accurately to predict susceptibility to space sickness of crew members making their first transition into orbit, for want of a ground-based validated model of free fall. Even assuming that space sickness is simply a specific designation for motion sickness that may be experienced in orbital flight (and here agreement is not general), preventive therapy poses difficult problems because, for a priori reasons, either all crew members or none should receive treatment. If all receive preventive therapy, everyone should execute head movements in a programmed manner to ensure rapid adaptation to the environment; at least a large minority will not benefit but rather will experience whatever sideeffects inevitably accompany administration of a drug. If none receive preventive therapy prelaunch, at least a large minority will pose two problems--treatment for acute motion sickness and rapid acquisition of adaptation. Trade-offs will involve the identification of long-acting antimotion sickness drugs for use prelaunch that will be efficacious for at least 90% of those going aloft for the first time and the effectiveness of combining rapid adaptation with treatment of motion sickness. The following report describes recent experiments dealing with these problems.

Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points.

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.

The efficacy of four respiratory analeptics on healthy young men in a CO2 rebreathing experiment.

Four respiratory analeptics were examined in a CO2 rebreathing experiment with increase from 0--6% in the inspiratory CO2 concentration during 30 min on 24 healthy young men. The respiratory response curves (VE/pCO2) showed a parabolic shape. They were examined on parallel shift and change of slope. The "excitability ratio" is the first derivative of the response parabola and as such becomes a linear function of the actual arterial CO2 partial pressure. The following effects have been shown: 1. 150 mg amiphenazole (2.07 mg/kg) cause a small rise in the ventilation by accelerating the frequency at increased CO2 partial pressures above 45 mmHg. 2. 240 mg theophylline ethylenediamine (3.32 mg/kg) produce a nearly parallel upward-shift of the respiratory response curve, i.e., the respiratory minute volume increases independently by a deepening of the respiration. 3. 450 mg prethcamide (6.22 mg/kg) cause a slight increase of the "excitability ratio" at CO2 partial pressures above 45 mmHg. The CO2-dependent respiratory minute volume is not changed significantly but the pattern of breathing changes by accelerating the frequency and decreasing the tidal volume. 4. 40 mg fominobene (0.55 mg/kg) raise the "excitability ratio" convincingly at CO2 partial pressures above 40 mmHg. However, the total volume ventilated during the experiment does not increase because of a diminished ventilation at rest.

Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation.

Resonance Raman spectra of the heme protein chloroperoxidase in its native and reduced forms and complexed with various small ions are obtained by using laser excitation in the Soret region (350-450 nm). Additionally, Raman spectra of horseradish peroxidase, cytochrome P-450cam, and cytochrome c, taken with Soret excitation, are presented and discussed. The data support previous findings that indicate a strong analogy between the active site environments of chloroperoxidase and cytochrome P-450cam. The Raman spectra of native chloroperoxidase are found to be sensitive to temperature and imply that a high leads to low spin transition of the heme iron atom takes place as the temperature is lowered. Unusual peak positions are also found for native and reduced chloroperoxidase and indicate a weakening of porphyrin ring bond strengths due to the presence of a strongly electron-donating axial ligand. Enormous selective enhancements of vibrational modes at 1360 and 674 cm-1 are also observed in some low-spin ferrous forms of the enzyme. These vibrational frequencies are assigned to primary normal modes of expansion of the prophyrin macrocycle upon electronic excitation.

Kinetics and mechanism of the reduction of horse heart ferricytochrome c by glutathione.

A detailed investigation of the reduction of cytochrome c by glutathione has shown that the reaction proceeds through several steps. A rapid combination of the reducing agent with the cytochrome leads to the formation of a glutathione-cytochrome intermediate in which the glutathione most likely interacts with the edge of the heme moiety. The electron transfer takes place in a subsequent slower step. Since cytochrome c(III) exists in two conformational forms at neutral pH [Kujundzic, N., & Everse, J. (1978) Biochem. Biophys. Res. Commun. 82, 1211], the reduction of cytochrome c by glutathione may be represented by cyt c(III) + GS- reversible K1 cyt c(III) ... GS- reversible k1 products cyt c*(III) + GS- reversible K2 cyt c*(III) ... GS- reversible k2 products At 25 degrees C, pH 7.5, and an ionic strength of 1.0 (NaCl), k1 = 1.2 X 10(-3) S-1, k2 = 2.0 X 10(-3) S-1, k1 = 2.9 X 10(3) M-1, and K2 = 5.3 X 10(3) M-1. The reaction is catalyzed by trisulfides, and second-order rate constants of 4.55 X 10(3) and 7.14 X 10(3) M-1 S-1 were obtained for methyl trisulfide and cysteine trisulfide, respectively.

D-Gluconate transport in Arthrobacter pyridinolis. Metabolic trapping of a protonated solute.

D-Gluconate uptake was studied in whole cells of Arthrobacter pyridinolis; the uptake activity was inducible, mutable and showed saturation kinetics (Km = 5 micrometer). Uptake of D-gluconate was not mediated by a phosphoenol-pyruvate : hexose phosphotransferase system, nor was it directly energized by ATP. A transmembrane pH gradient, delta pH, of --63 mV was generated by A. pyridinolis cells at pH 6.5, while at pH 7.5, delta pH = 0. Addition of 8 micrometer D-gluconate significantly reduced the delta pH. The transmembrane electrical potential, delta psi, which was --87 mV over a range of pH from 5.5 to 7.5, was unaffected by the presence of substrate. D-Gluconate accumulated at the same rate and as the protonated solute, at both pH 6.5 and 7.5. Experiments in which a diffusion potential was generated in cyanide-treated cells, indicated that the delta psi did not energize transport. Rather, the rate of D-gluconate uptake metabolism: (a) treatment of cells with valinomycin or nigericin, under conditions in which there was a loss of intracellular potassium, inhibited both D-gluconate uptake and the metabolism of pre-accumulated D-gluconate; (b) the effects of cyanide and azide on D-gluconate uptake were much more severe at pH 6.5 than pH 7.5, a pattern which paralleled the effects of these inhibitors on D-gluconate metabolism; (c) extraction and chromatography of intracellular label from D-gluconate uptake revealed that accumulation of unaltered D-gluconate was negligible; (d) a series of mutant strains with lower D-gluconate kinase activities also exhibited low rates of D-gluconate uptake; (e) spontaneous revertants of these mutant strains consistently regained both D-gluconate kinase activity and wild type levels of uptake.

Mechanism of uricase action.

Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) exposes a positional and steric specificity in the enzymic conversion of urate to allantoin. C-2 of urate was recovered as C-2 of allantoin. By the consecutive oxidation and hydrolysis reactions a levorotatory intermediate was formed, presumably (-)-2-oxo-4-hydroxy-4-carbohydroxy-5-ureido-imidazoline. The absorption and optical rotation dispersion spectra of the intermediate were established. In the presence of borate buffer, the intermediate was transformed to (+)-alloxanate. The decay of the former compound depends on general base and acid catalysis. RS-(+/-)-allantoin was formed by chemical decarboxylation and S-(+)-allantoin by enzymic decarboxylation.

Effects of ATP on the intermediary steps of the reaction of the (Na+ + K+)-ATPase. IV. Effect of ATP on K0.5 for Na+ and on hydrolysis at different pH and temperature.

The pH optimum for (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) depends on the combination of monovalent cations, on the ATP concentration and on temperature. ATP decreases the Na+ concentration necessary for half maximum activation, K0.5 for Na+ (Na+ + K+ = 150 mM), and the effect is pH and temperature dependent. At a low ATP concentration a decrease in pH leads to an increase in K0.5 for Na+, while at the high ATP concentration it leads to a decrease. K0.5 for ATP for hydrolysis decreases with an increase in pH. The fractional stimulation by K+ in the presence of Na+ decreases with the ATP concentration, and at a low ATP concentration K+ becomes inhibitory, this being most pronounced at 0 degrees C. The results suggest that (a) ATP at a given pH has two different effects: it increases the Na+ relative to K+ affinity on the internal site (K0.5 for ATP at pH 7.4, 37 degrees C, is less than 10 microM); it increases the molar activity in the presence of Na+ + K+ (K0.5 for ATP at pH 7.4, 37 degrees , is 127 microM), (b) binding of the cations to the external as well as the internal sites leads to pK changes (Bohr effect) which are different for Na+ and for K+, i.e. the selectivity for Na+ relative to K+ depends both on ATP and on the degree of protonation of certain groups on the system, (c) ATP involves an extra dissociable group in the determination of the selectivity of the internal site, and thereby changes the effect of an increase in protonation of the system from a decrease to an increase in selectivity for Na+ relative to K+.

The acyl-CoA desaturases of microsomes from rat liver and the Morris 7777 hepatoma.

We have investigated the role of the microsomal oxidative desaturase in defining the aberrant phosphoglyceride fatty acid composition of hepatomas. The microsomal delta 9-stearoyl-CoA, delta 6-oleoyl(linolenoyl)-CoA, and delta 5-eicosatrienoyl-CA desaturase activities were studied in control and host liver and in the poorly differentiated Morris 7777 hepatoma. The delta 9-stearoyl-CoA desaturase of the hepatoma was significantly decreased (42%) relative to control liver, yet the hepatoma specific activity was twice that of host liver. Additionally, the specific activity of the delta 9-stearoyl-CoA desaturase of the tumor was found to decrease with increasing tumor weight. Also this desaturase was inactivated by freezing and thawing. The delta 6-oleoyl(linolenoyl)-CoA and delta 5-eicosatrienoyl-CoA desaturases of the hepatoma were 39% and 4% of control, respectively. The electron transport components involved in the desaturase system were reduced, although this did not appear to be rate-limiting. In addition, two competing metabolic reactions which could lower the observed desaturase activities, hydrolysis of the thioester and incorporation of substrate acyl-CoA molecules into glycerides, did not appear to be responsible for the lowered desaturase activities of the tumor. Thus, it appears that reduced levels of the desaturases themselves may be responsible for the observed activities. These results indicate that the capacity of the hepatoma to biosynthesize polyunsaturated fatty acids is greatly reduced and this is consistent with the decreased polyene content observed in many neoplasms.

Effects of cholesterol, hydroxycholesterols and calcium on pregnenolone production rates in mitochondrial fractions from rat testes.

The in vitro regulation of the mitochondrial conversion of cholesterol to pregnenolone in rat testis tissue has been further investigated. Pregnenolone production rates by isolated mitochondrial fractions could be stimulated by the addition of cholesterol. The stimulation was always highest in mitochondria isolated from lutropin-treated testes relative to control and cycloheximide treated testes. Addition 20- or 25-hydroxycholesterol resulted in a greater stimulation of pregnenolone production rates and these rates were unaffected by prior treatment with cycloheximide. When both cholesterol and 20- or 25-hydroxycholesterol were present in the incubation medium, pregnenolone production rates were mainly influenced by the hydroxycholesterol, even in the presence of a ten-fold excess of cholesterol. Ca2+ in vitro stimulated pregnenolone production rates from endogenous cholesterol as well as from added cholesterol. However, pregnenolone production rates in the presence of hydroxycholesterol were not influence by the addition of Ca2+ in vitro.

Studies on neurotransmitter-stimulated phospholipid metabolism with cerebral tissue suspensions: a possible biochemical correlate of synaptogenesis in normal and undernourished rats.

The phenomenon of neurotransmitter-stimulated incorporation of 32Pi into phosphatidic acid and inositol phosphatides (neurotransmitter effect) in developing brain was studied in vitro as a possible measure of synaptogenesis. While the neurotransmitter effect was not observed with brain homogenates, highly consistent and significant effects were noted with brain tissue suspensions obtained by passing the tissue through nylon bolting cloth. The magnitude of the effect decreased with the increase in mesh number. Maximum stimulations obtained with the 33 mesh adult brain cortex preparations (mean +/- S.E.M. of 6 experiments) were 203 +/- 8%, 316 +/- 17% and 150 +/- 8% with 10(-3) M acetylcholine (ACh) + 10(-3) M eserine; 10(-2) M norepinephrine (NE) and 10(-2) M serotonin (5-HT), respectively. Experiments with developing rat brain at 7, 14 and 21 days of age showed that the neurotransmitter effects due to ACh, NE and 5-HT increase progressively in different regions of the brain but that there are marked regional differences. It is suggested that the neurotransmitter effect is a valid biochemical correlate of synaptogenesis. In rats undernourished from birth to 21 days of age, by increasing the litter size, the neurotransmitter effect with ACh, NE or 5-HT was not altered in the cortex but was significantly reduced in the brain stem. In cerebellum the effects due to ACh and NE were significantly altered, while that with 5-HT was unaffected. It is concluded that cholinergic, adrenergic and serotonergic synapses are relatively unaffected in the cortex but are significantly affected in the brain stem by undernutrition. In the cerebellum of undernourished rats the adrenergic and cholinergic, but not serotonergic systems, are altered.

Some bases of differences in vascular response to sympathetic activity.

Basic patterns of neuroeffector organization vary widely in the vasculature, in general, with vessel diameter and type, and confer distinctive properties. The smaller the vessel, the more intimate the neuroeffector relationship, the more localized the action of the released transmitter, and the more important myogenic conduction compared to transmitter diffusion for the coordination of vascular effector response. Seemingly superimposed upon these basic general patterns are other variable features, conferring upon vessels of similar size and type diversity of function. These variables include sensitivity and magnitude and possible location of alpha- and beta-receptors and their subtypes, presence and nature of intrinsic vascular tone, and the density and pattern of adrenergic innervation to mention the more important. Functional diversity in neuroeffector characteristics can, to some extent, be understood in relation to embryological development, neurotrophic influences, effector regulation of innervation, and the mural response to an increase in intravascular pressure.

Evaluation of urinary enzyme patterns in patients with kidney diseases and primary benign hypertension.

The urinary excretion of lactate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, arylsulphatase A, alpha-glucosidase, beta-galactosidase, trehalase, N-acetyl-beta-glucosaminidase, beta-glucuronidase, and leucine arylamidase was studied in 68 patients with biopsy-proved glomerular, 54 with interstitial renal disease and in 97 patients suffering from primary hypertension. The enzyme output of these 219 patients was compared to that of a reference population of 100 thoroughly selected healthy subjects. The highest incidence of elevated enzyme excretion was observed for N-acetyl-beta-glucosaminidase with 88% in glomerulopathies and 78% in interstitial disease, followed by beta-galactosidase. 94% of the patients with glomerular kidney disease, 90% of those with interstitial disease and about 60% of the subjects with primary benign hypertension revealed an output of at least one enzyme above upper reference limit. The highest average enzymuria occured in glomerulopathies, particularly high values in patients with the nephrotic syndrome. Application of discriminant analysis to the urinary enzyme pattern of glomerular and interstitial renal diseases resulted in an overall correct classification into the appropriate group of 89% of all patients. The discrimination between glomerular and interstitial disease was better in patients with normal renal function than in those with reduced function. Results show, that the analysis of urinary enzyme patterns may be a helpful adjunct for differential diagnosis of kidney diseases.

Multiple factors regulating the release of norepinephrine consequent to nerve stimulation.

Whereas extracellular calcium is absolutely required for neurotransmitter release consequent to stimulation of adrenergic and other neurons, a large number of substances are known to modify the amount of norepinephrine released per nerve impulse. In general, cyclic nucleotides, phosphodiesterase inhibitors, beta-adrenoceptor agonists, cholinergic nicotinic agonists, and angiotensin are able to enhance neurally mediated norepinephrine release, whereas alpha-adrenoreceptor agonists, cholinergic muscarinic agonists, prostaglandins of the E series, opiates, enkephalins, dopamine, and adenosine inhibit neurally mediated norepinephrine release. Although it has been proposed that cyclic AMP may enhance, and endogenous cyclic GMP may inhibit, neurotransmitter release, no consistent relationship between the effects of the several modulators of neurally mediated norepinephrine release and their effects on adenylate and guanylate cyclase is as yet apparent. The demonstration of whether such a relationship exists must await the development of techniques that will allow the measurement of cyclic nucleotide levels in the presynaptic adrenergic nerve terminal after exposure to the putative modulators of release and consequent to nerve stimulation.

Effect of acute and chronic ethanol administration on the content of coenzymes linked to energy transfer in the liver of rats fed standard or low-protein diet.

1) In rats fed a standard diet or a protein restricted diet the effect of acute and chronic ethanol administration on liver content of adenine nucleotides was studied. In the long-term experiments the total liver content of NAD and NADP was additionally determined. 2) a single oral ethanol load does not significantly influence the total adenine nucleotide content. Liver AMP content increases immediately following ethanol ingestion about 2-fold and remains elevated for 12 hours. ATP content and ATP/ADP ratio are significantly reduced within 30 minutes after ethanol administration. Both return to initial values after 2 hours adn decrease again thereafter. 3) The increase in the AMP content is dose dependent, i.e. it is more pronounced after small doses of ethanol and is not observed when blood ethanol concentrations are very high. The elevation of the AMP levels during ethanol oxidation is interpreted as a consequence of increased ATP consumption and of inhibition of citric acid cycle. 4) In animals fed nearly protein-free diet, total adenine nucleotide content and ATP content are distinctly reduced. An increase in AMP concentration is not observed in these rats where ethanol oxidation is markedly inhibited. 5) Chronic ethanol application does neither in rats kept on a standard diet nor in those fed a protein restricted regimen affect the liver content of total adenine nucleotides or ATP. Similarly the total content of NAD and NADP shows no major changes. 6) It is concluded that the relatively small alterations in total liver adenine nucleotide content and in the different adenine nucleotide fractions are not important for ethanol-induced fat accumulation or other disturbances in the liver.

Clinical studies of thyrotropin-releasing hormone tartrate (TRH-T) as a direct stimulant to the central nervous system.

Thyrotropin-releasing hormone tartrate (TRH-T) was administered in 17 cases of organic brain lesions and 2 cases of disturbed mental activity (psychical depression), and its effect, mainly on the level of consciousness and electroencephalogram, was examined. Ten consecutive administrations of 0.5--1.0 mg/day TRH-T, as TRH, resulted in improvement of disturbance of consciousness in 8 of 16 cases. This effect was not necessarily correlated with the degree of disturbance or the site of the lesion. Improvement was seen even in those cases where disturbance of consciousness had been fixed over a long period. The effect on the electroencephalogram was small and did not parallel the degree of improvement of the level of consciousness. Abnormal TSH and thyroid hormone values were not seen despite the continued administration of TRH-T. These results would appear to indicate that the continuous administration of TRH-T has a mild activating effect directly on the central nervous system, and not through the endocrine mechanism, and exerts no damage on the internal environment in vivo.

Studies on regulatory functions of malic enzymes. VI. Purification and molecular properties of NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme [EC 1.1.1.40] was highly purified from Escherichia coli W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471,000, 438,000, and 495,000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6 M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76,000 and 82,000, respectively. The sedimentation coefficient (S(0)20, W) was 13.8S, and the molecular activity was 44,700 min-1 at 30 degrees C. The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g. The Km value for L-malate was 2.3 mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and alpha-keto acid reductase activities. Based on the above results, the molecular properties of the enzymatic reaction are discussed.

Influence of hydrogen and chloride ions on the interaction between sulfaethidole and bovine serum albumin studied by microcalorimetric and acid-base titrimetric methods.

From earlier studies it is known that bovine serum albumin has one high affinity binding site and several lower affinity sites for the sulfa drug N1-(5-ethyl-1,3,4-thiadiazol-2-yl)sulfanilamide (sulfaethidole) (Kostenbauder, H.B., Jawad, M.J., Perrin, J.H., and Averhart, V. (1971) J. Pharm. Sci. 60, 1658-1660). This binding has been further studied using equilibrium dialysis, microcalorimetry, and pH titration technique. Results of these studies show that the binding of sulfaethidole to the first (high affinity) site may be accompanied by an uptake of protons. Proton uptake is found to be zero at pH 7.4 and approximately 0.6 at pH 8.5 for each sulfaethidole molecule bound. The other binding sites for sulfaethidole are not proton linked. The first, and probably the other binding sites, are also Cl- ion linked; for example, the binding of sulfaethidole to the first binding site is accompanied by the displacement of (on average) one Cl- ion at pH 7.4 in 0.1 M NaCl. This explains the observation that the heat of binding of sulfaethidole to the high affinity site is -33.0 kJ.mol-1 in the absence of chloride ions, but only -22.8 kJ.mol-1 in the presence of 0.1 M Cl- (at pH 7.4).

Quaternary structure of methemoglobin. Pulse radiolysis study of the binding of oxygen to the valence hybrid.

The pulse radiolysis of solutions of adult human methemogolbin was used in order to reduce a single heme iron within the protein tetramers. The valence hybrids thus formed were reacted with oxygen. Kinetics of the reactions were studied. The effects of pH and inositol hexaphosphate were examined. The kinetics of the ligation of oxygen to stripped valence hybrids showed a single phase behavior at the pH range 6.5 to 9. As the pH was lowered below 6.5, a second, slower phase became apparent. In the presence of inositol hexaphosphate, above pH 8, the kinetics of oxygen binding was of a single phase. As the pH was lowered, a transition to a second, slower phase was noticed. Below pH 7, the slower phase was the only detectable one. The analysis of the relative contribution of the faster phase to the total reaction as a function of the pH showed a typical transition curve characterized by a pK = 7.5 and a Hill parameter n = 2.9. On this basis, it is concluded that human adult stripped methemoglobin resides in an R quarternary structure, while the presence of IHP stabilizes the T structure at pH below 7.5. This transition between the quaternary structures of methemoglobin cannot be accounted for by the switch between the high spin and the low spin states of the ferric iron. This switch of spin state takes place at pH greater than 8.2.

Kinetic studies on the reaction of p-hydroxybenzoate hydroxylase. Agreement of steady state and rapid reaction data.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is a NADPH-dependent, FAD-containing monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate in the presence of NADPH and molecular oxygen. The mechanism of this three-substrate reaction was investigated in detail at pH 6.6, 4 degrees C, by steady state kinetics, stopped flow spectrophotometry, and equilibrium binding experiments. The initial velocity patterns are consistent with a ping-pong type mechanism which involves two ternary complexes between the enzyme and substrates. The first ternary complex is formed by random addition of p-hydroxybenzoate and NADPH to the enzyme, followed by the release of the first product (NADP+). The reduced enzyme . p-hydroxybenzoate complex now reacts with oxygen, the third substrate, to form the second ternary complex. The enzyme-bound p-hydroxybenzoate then reacts with the activated oxygen to give 3,4-dihydroxybenzoate which is released regenerating the oxidized enzyme for the next cycle. The binding of p-hydroxybenzoate to the oxidized enzyme to form a 1:1 complex causes large, characteristic spectral perturbations and fluorescence quenching. The dissociation constant for the enzyme . substrate complex was obtained by titrations in which absorbance and/or fluorescence quenching was measured. The binding constants of NADPH to the enzyme with and without p-hydroxybenzoate were determined kinetically by measuring the rate of reduction of the enzyme at different concentrations of NADPH. The reduction of the enzyme proceeds extremely slowly in the absence of p-hydroxybenzoate. The presence of the substrate causes a dramatic stimulation (140,000-fold) in the rate of enzyme reduction. The anaerobic reduction of the enzyme by NADPH in the presence of p-hydroxybenzoate produces a transient charge-transfer intermediate. On the basis of the proposed mechanism, the dissociation constants for p-hydroxybenzoate and NADPH as well as the Michaelis constants for all the three substrates were calculated from the initial velocity data. The agreement obtained between various kinetic parameters from the initial rate measurements and those calculated from the individual rate constants determined in rapid reactions, strongly supports the proposed mechanism for the p-hydroxybenzoate hydroxylase reaction.

Purification and characterization of a human neutrophil neutral protease. The neutral peptide-generating protease.

A human neutrophil neutral protease which generates a low molecular weight peptide from a plasma protein substrate and cleaves the basic amino acid ester substrates alpha-N-p-tosyl-l-arginine methyl ester HCl, alpha-N-benzoyl-l-arginine-methyl ester HCl, and alpha-N-carbobenzoxy-l-lysine-p-nitrophenyl ester has been purified to homogeneity and distinguished from the known lysosomal neutrophil proteases. The starting activity was obtained from purified human neutrophils by homogenization, sedimentation by low-speed centrifugation, and high salt elution of the insoluble material. Purification was achieved by aprotinin-affinity chromatography, precipitation at low ionic strength, and gel filtration. The overall recovery, relative to the activity in the starting eluate of the neutrophil fraction, was congruent with50% with a 200- to 400-fold increase in specific activity. After treatment with diisopropylfluorophosphate to eliminate autodegradation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced and unreduced protein gave a single protein band of 29,000-30,000 mol wt. The isoelectric point determined in sucrose gradients ranged from pH 7.8 to 8.3 with a peak at pH 8.0. This neutrophil protease, like cathepsin G and elastase, is composed of a single polypeptide chain of congruent with30,000 mol wt, but differs from cathepsin G and elastase in its less cationic isoelectric point and its failure to cleave synthetic substrates presenting an aromatic amino acid ester linkage and alanyl peptide bonds, respectively.

Role of cerebrospinal fluid [H+] in ventilatory deacclimatization from chronic hypoxia.

Once ventilatory acclimatization begins in sea level residents sojourning at high altitude, abrupt restoration of normal oxygen tensions will not restore ventilation to normal. We have investigated the role of cerebrospinal fluid (CSF) [H(+)] in this sustained hyperventilation by measuring CSF acid-base status in seven men (lumbar) and five ponies (cisternal) in normoxia, first at sea level and then periodically over 13-24 h of "deacclimatization" after 3-5 d in hypoxia (P(B) = 440 mm Hg). After 1 h deacclimatization, hyperventilation continued at a level only slightly less than that obtained in chronic hypoxia (+1-2 mm Hg Pa(CO2)), whereas CSF pH was either equal (in man) or alkaline (in pony, +0.02, P < 0.01) to sea level values. Between 1 and 12-13 h deacclimatization in all humans and ponies Va fell progressively (Pa(CO2) increased 4-7 mm Hg) and CSF pH became increasingly more acid (-0.02 to -0.05, P < 0.01). Between 12 and 24 h of normoxic deacclimatization in ponies, Pa(CO2) rose further toward normal, coincident with an increasing acidity in CSF (-0.02 pH). Similar negative correlations were found between changes in arterial pH and Va throughout normoxic deacclimatization. We conclude that [H(+)] in the lumbar or cisternal CSF is not the mediator of the continued hyperventilation and its gradual dissipation with time during normoxic deacclimatization from chronic hypoxia. These negative relationships of Va to CSF [H(+)] in normoxia are analogous to those previously shown during acclimatization to hypoxia.

Guanylate cyclase in neuroblastoma N1E 115 cells: presence of endogenous activator.

Guanylate cyclase in cultured neuroblastoma N1E 115 cells was readily solubilized. MgCl2 as well as MnCl2 served as a metal cofactor of the guanylate cyclase. The maximal guanylate cyclase activity obtained with MgC12 was 80% of that with MnCl2. When the supernatant of cell homogenate was adjusted to pH 5.2, all of enzyme activity was precipitated. The guanylate cyclase activity recovered in the pH 5.2 precipitate was reduced to about 10% of the original supernatant. Combination of the pH 5.2 supernatant and precipitate fractions, however, restored guanylate cyclase activity, indicating that the pH 5.2 supernatant contains an endogenous activator for guanylate cyclase. The activating factor in the pH 5.2 supernatant remained in the aqueous phase after proteins were removed by perchloric acid. The factor was filterable through Diaflo ultrafilter membranes UM 2 and UM 10 indicating that the factor is a small molecule. The activation by the endogenous activator was prevented by N-methylhydroxylamine and lysolecithin.

The fate of IgE bound to rat basophilic leukemia cells.

The present study investigates the fate of the cell-bound IgE by using a well-characterized rat basophilic leukemia cell line and a purifed IgE myeloma protein. Both histamine-releasing and nonreleasing cell lines were examined. In both cases, no evidence for cell-mediated IgE catabolism could be elicited. Both the dissociated IgE and the receptors remained intact for prolonged periods of time, as demonstrated by binding assays. Internalization and/or recycling of membrane-bound IgE could not be demonstrated by E. M. autoradiography. We found only limited time-dependent changes in accessibility to anti-IgE antibody, trypsin, or elution at low pH (2.9 to 3.1). A biphasic dissociation of cell-bound 125I-IgE during incubation in the presence of excess unlabeled IgE was reproducibly observed; the more slowly dissociated IgE was also less readily dissociated at pH 3.4. These studies lead us to conclude that, in vitro, IgE resides in a functional orientation on the surface of RBL-1 cells, for prolonged periods of time.

A dual, concentration-dependent absorption mechanism of linoleic acid by rat jejunum in vitro.

Linoleic acid absorption was studied using everted rat jejunal sacs. At low concentrations (42-1260 microM), the relationship between linoleic acid concentration and its absorption rate fitted best to a rectangular hyperbola. At high concentrations (2.5-4.2 mM) the relationship between the two parameters was linear. The separate additions of 2,4-dinitrophenol, cyanide, or azide, or decrease in the incubation temperature from 37 to 20 degrees C did not change the absorption rate of linoleic acid. Absorption rate of linoleic acid at low concentrations increased as the hydrogen ion and taurocholate concentrations were increased or as the unstirred water layer thickness was decreased. Linoleic acid absorption rate was decreased after the additions of lecithin, oleic, linolenic, and arachidonic acids or the substitution of taurocholate with the nonionic surfactant Pluronic F 68. These observations indicate that a concentration-dependent, dual mechanism of transport is operative in linoleic acid absorption. Facilitated diffusion is the predominant mechanism of absorption at low concentrations, while at high concentrations, simple diffusion is predominant. At low concentrations, the absorption rate of linoleic acid is influenced by the pH, surfactant type and concentration, the simultaneous presence of other polyunsaturated fatty acids, and the thickness of the unstirred water layer.

Persistent changes in central catecholaminergic system after recovery of perinatally undernourished rats.

Pregnant rats from day 14 of pregnancy or pups were fed a control diet (24% casein) or a deprived diet (8% casein) in order to obtain the following groups: 1) Control (C-C group); 2) Prenatal deprived (D-C group); 3) Postnatal deprived (C-D group); 4) Pre and postnatal deprived (D-D group). From 50 days of age on, all groups were fed a balanced commercial stock diet until 140 days of age. A significant reduction in corporal and brain weights was observed in C-D and D-D groups during deprivation and after nutritional recovery. Twenty-four day old deprived rats showed a decrease in brain noradrenaline (NA) content but no significant change in NA concentration. By the end of the deprivation period (50 days), brain NA levels tended to be reduced or increased in postnatal deprived rats, depending upon the method of expressing the results (microgram NA/whole brain or microgram NA/g fresh tissue, respectively). At 140 days of age, i.e., after 90 days of nutritional recovery, no differences were detected between deprived and control rats. However, conversion rate of [14C]tyrosine to brain catecholamines and tyrosine-hydoxylase activity were higher in D-D rats as compared with controls at this age. These results suggest that perinatal undernutrition produces, even after a prolonged period of nutritional recovery, a permanent activation of the central catecholaminergic system in adulthood. This fact may explain the different behavioral alterations described as a consequence of protein deprivation in early life.

The non-specific membrane binding properties of delta9-tetrahydrocannabinol and the effects of various solubilizers.

The binding of [3H]delta9-tetrahydrocannabinol to crude and purified synaptosomal membrane suspended in either Krebs solution or 10 mM sodium phosphate buffer was examined. The membrane/buffer partition coefficient was found to be 12,500, and was constant over a free concentration range of 10(-8) to 10(-6)M. Binding was similar in both suspending media, and to both crude and purified synaptosomal membrane. The solubilizing agent Cremophor E.L. (8 microgram ml-1) decreased the partition coefficient by one-half, and by greater than 99% at 0.4 mg ml-1. Similar effects were observed with Tween 80, while ethanol caused a maximum decrease of 60%. Membrane concentrations of THC were calculated at various effective concentrations reported in the literature, and were within the range predicted by the Meyer-Overton rule of anaesthesia. An apparent non-specific interaction with neuronal membranes and effective membrane concentrations of the order 2 x 10(-4) to 1 x 10(-2) mol kg-1 suggests THC may exert some of its effects by a mechanism analogous to the general anaesthetics, and thus may be classified as a partial anaesthetic.

Experimental central hypertension produced by chemical degeneration of the locus coeruleus in the rat.

The role of noradrenergic neurons originating from the locus coeruleus (LC) in blood pressure regulation was studied by stereotaxic administration of 6-hydroxydopamine (6-OHDA) into the LC in Wistar-Kyoto rats. The administration of 6-OHDA (12 micrograms/6 microliters) into the bilateral LC resulted in hypertension and tachycardia, which lasted for 12 days and gradually returned to the levels observed before administration. The systolic blood pressure and heart rate were 172 mmHg and 460 beats/min on the mean respectively, one day after administration. The induced hypertension and tachycardia were closely correlated with the depletion of norepinephrine (NE) content in the cortex and the medulla-pons in rats in a hypertensive state. The correlation between the NE content of the cortex and blood pressure was particularly marked (r = -0.793, p less than 0.02). Furthermore, destructive change of the LC was observed histologically in the hypertensive rats. They hypertension was completely prevented by pretreatment with desipramine before 6-OHDA administration. These findings suggest that 6-OHDA induced degeneration, probably mainly in the dorsal bundle originating from the LC and in the LC itself. It is suggested, therefore, that localized chemical degeneration of the bilateral LC causes hypertension and tachycardia as a consequence of denervation of the dorsal bundle of noradrenergic neurons originating from the LC.

Insulin release: the fuel hypothesis.

The immediate and direct regulation of insulin release by circulating nutrients, especially glucose, is thought to be mediated in the pancreatic B-cell by a sequence of metabolic, ionic, and motile events. On the basis of previous work, it is assumed that the process by which glucose is recognized as an insulinotropic agent entirely depends on the metabolic changes evoked by the sugar in the islet cells. Several factors are considered as possible candidates for the coupling between these metabolic changes and subsequent ionic events such as altered phosphate, chloride, sodium, potassium, and calcium handling. It is acknowledged that changes in the concentrations of glycolytic intermediates and cyclic nucleotides (adenosine- or guanosine-3', 5'-cyclic monophosphate), or both, could play a modulatory role upon stimulated insulin release. However, the initiation of insulin release seems to depend on the generation of two essential coupling factors: H+ and reduced pyridine nucleotides. The changes in H+ fluxes may account for the glucose-induced decrease in K+ and Ca2+ fractional outflow rate, all three parameters displaying hyperbolic-like dose-response curves with half-maximal values at noninsulinotropic glucose concentrations. The changes in NAD(P)H concentration may account for a glucose-induced Ca2+--Ca2+ exchange process due to a change in affinity of a native ionophoretic system. The dose-response curves for these parameters yield a sigmoidal pattern analogous to that which depicts the rate of insulin release at increasing glucose concentrations. It is proposed that such a coupling between metabolic and cationic events is operative in response to other insulinotropic nutrients and that its time course may be relevant to the phasic aspect of insulin release. Thus, the nutrient-induced release of insulin (and possibly other pancreatic hormones), which is essential for the regulation of fuel homeostasis, would depend on the capacity of circulating nutrients to act as a fuel in the islet cells. This concept raises a question as to the existence and nature of feedback mechanisms regulating the metabolic fluxes in the islet cells as a function of their energy expenditure.

[Photooxidation of aspartate transaminase from chicken heart cytosol].

Rose-bengal-sensitized photooxidation of aspartate transaminase from chicken heart cytosol results in a loss of enzymatic activity which follow first order kinetics down to 70--75% inactivation. 0.9 Histidine, 0.9 tryptophane residues and 1.5 SH groups per enzyme subunit were found to be modified in the photooxidized transaminase, which retained 26% residual activity. Photodestruction of the coenzyme was about 16%. The rate of enzyme photoinactivation is constant in the pH range 6--8, and drastically decreases with lowering pH from 6 to 4. alpha-Ketoglutarate partially protects the holoenzyme from inactivation. The apoenzyme undergoes photoinactivation at a rate almost twice as rapid as the holoenzyme. Photooxidized apotransaminase retains affinity to pyridoxal phosphate and binds as much coenzyme as the native apoenzyme. Photooxidation induces no significant alterations in the circular dichroism pattern of the enzyme in the 200 to 240 nm range. However, positive circular dichroism is markedly increased in the absorption bands of aromatic amino acids (260--300 nm). The affinity of photooxidized holoenzyme for glutarate and alpha-methyl aspartate is greatly decreased. On the other hand, photooxidized enzyme retains its ability to bind alpha-alanine and to catalize the transamination half-reaction between alpha-alanine and the bound coenzyme. These findings imply that photooxidation disturbs the binding of the distal carboxyl group of dicarboxylic substrates. This may be due to a localized conformational change induced by destruction of a photoreactive histidine residue at the active site. A role of the histidine residue in transamination reaction is discussed.

Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis.

A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-K1, the uridine-requiring auxotroph Urd(-)A, and selected Urd(-)A revertants. The enzymes of Urd(-)A and the Urd(-)A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-K1. The results presented permit the conclusion that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (ii) the aspartate transcarbamoyltransferase activity is located on a portion ( approximately 20,000 daltons) at one end of the polypeptide; (iii) this portion may also be required for monomers to aggregate into the multimeric from present in mammalian cells; (iv) the mutations in Urd(-)A and the Urd(-)A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.

Cellular energy metabolism, trans-plasma and trans-mitochondrial membrane potentials, and pH gradients in mouse neuroblastoma.

A method for quantitative evaluation of transmembrane electrical potential and pH gradients across a subcellular compartment in an intact cell is presented. This approach has been applied in studies of mouse neuroblastoma C-1300 clone NB41A3, in which the transmembrane electrical potential and pH gradients and the mitochondrial volume percent have been determined. Membrane potentials and pH gradients were measured by two different methods. Equilibrium distributions of [(3)H]triphenylmethyl phosphonium and [(14)C]-thiocyanate ions gave calculated apparent membrane potentials of -77.0 and -29.6 mV, respectively, at 20-25 degrees C; a value of -60.8 mV was obtained from microelectrode measurements. Equilibrium distributions of weak acids ([(14)C]trimethylacetic acid and 5,5-di[(14)C]methyl-2,4-oxazolidine-dione) and of weak bases ([(14)C]dimethylamine and [(14)C]trimethylamine) gave calculated upper and lower limits of the pH gradient (Delta pH = pH(e) - pH(i)) of -0.14 and -0.21 pH unit, respectively. The microelectrode measurements showed that the intracellular pH is within 0.1 of a pH unit or less of the extracellular pH over the extracellular pH range of 7.35-6.85. The mitochondrial volume percent calculated on the basis of the measured cytochrome c content is 5.6 +/- 1.2% and compares well with estimates of 5.4 +/- 1.1% obtained from 25 electron micrographs. Measurements of the cellular energetic parameters gave values within the range found in other cells and perfused organs. Comparison of the results of the microelectrode and equilibrium measurements permits estimates of the electrical potential and pH gradients across the mitochondrial membrane (mitochondria-to-cytoplasm gradients) to be made and suggests that the trans-mitochondrial membrane protonmotive force in the intact cell cannot be greater than -143 mV.

The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms.

Intracellular genera are found in all the major groups of Protista, but are particularly common among the dinoflagellates, trypanosomatid zooflagellates and suctorian ciliates; the Sporozoa are nearly all intracellular at some stage of their life, and the Microspora entirely so. Intracellular forms can dwell in the nucleus, within phagosomal or other vacuoles or may lie free in the hyaloplasm of their host cells. Organisms tend to select their hosts from a restricted taxonomic range although there are some notable exceptions. There is also great variation in the types of host cell inhabited. There are various reasons for both host and cell selectivity including recognition phenomena at the cell surfaces. Invasion of host cells is usually preceded by surface interactions with the invader. Some organisms depend upon phagocytosis for entry, but others induce host cells to engulf them by non-phagocytic means or invade by microinjection through the host plasma membrane. Protista avoid lysosomal destruction by their resistance to enzyme attack, by surrounding themselves with lysosome-inhibiting vacuoles, by escaping from the phagosomal system into the hyaloplasm and by choosing host cells which lack lysosomes. Nutrition of intracellular heterotrophic organisms involves some degree of competition with the host cell's metabolism as well as erosion of host cell cytoplasm. In Plasmodium infections, red cells are made more permeable to required nutrients by the action of the parasite on the host cell membrane. The parasite is often dependent upon the host cell for complex nutrients which it cannot synthesize for itself. Intracellular forms often profoundly modify the structure and metabolism of the host cell or interfere with its growth and multiplication. This may result in the final lysis of the host cell at the end of the intracellular phase or before the infection of other cells. Certain types of intracellular organisms may have arisen initially as forms attached to the cell surface of digestive or other organs, but the intracellular habit appears to have arisen independently in several groups of Protista.

Interactions between Bdellovibrio and its host cell.

The bdellovibrios are extremely small bacteria with the unique property of being parasites of other (gram-negative) bacteria. In the presence of viable and susceptible bacteria a Bdellovibrio cell physically 'attacks' an individual host cell, attaches to its surface, penetrates the cell wall, and multiples within the periplasmic (intramural) space of its prey. The invading Bdellovibrio and its progeny degrade and consume the cellular constituents of the invaded host bacterium. This process finally results in complete lysis of the host cell and release of the Bdellovibrio progeny. From a population of parasitic bdellovibrios, derivatives can be selected that grow on complex nutrient media. Currently, none of the different nutritional types can be propagated in a fully defined synthetic medium. By degradation of the cellular constituents of the host the Bdellovibrio cell in its periplasmic space has available all the monomeric subunits needed to synthesis of the macromolecules. Peculiarities of Bdellovibrio metabolism with respect to uptake of preformed molecules and energy efficiency are discussed.

Effect of desglycinamide(9)-lysine(8)-vasopressin and prolyl-leucyl-glycinamide on oral ethanol intake in the rat.

Rats given ethanol in their drinking water at a concentration that permitted adequate fluid intake gradually accepted higher concentrations and consumed larger amounts of ethanol. These increases were augmented when daily subcutaneous injections of 1 microgram of desglycinamide9-lysine8-vasopressin (DGLVP) or 10 microgram of prolyl-leucyl-glycinamide (PLG) were given concomitantly. Nonsignificant changes in ethanol consumption were seen with injections of 1 microgram PLG, or 0.42 or 42 microgram of lysine8-vasopressin (LVP). In a second experiment 4 microgram DGLVP given every second day as a long-acting zinc phosphate complex, commencing after the increases in ethanol intake had taken place, failed to produce any change in ethanol consumption subsequently. In both Experiments 1 and 2, the rats were switched from forced ethanol intake to a choice between ethanol and tap water. On these tests there was only marginal evidence of peptide-produced changes in ethanol intake.

Inhibition of cardiac protein synthesis by prolonged ethanol administration.

Effects of prolonged ethanol consumption have been studied on the rates of cardiac protein synthesis. Prolonged ethanol consumption resulted in a significant decrease in cardiac contents of total protiens and RNA. Chronic exposure to ethanol did not result in an alteration in cardiac DNA content. The rates of protein synthesis measured by determining the rates of (U-14C)-leucine incorporation into cardiac proteins showed that chronic ethanol-feeding leads to a significant inhibition of protein synthesis. Studies with ribosomes and pH 5 enzyme fractions of heart showed that prolonged ethanol consumption inhibits the capacity of both these fractions to synthesize proteins. Acute administration of ethanol or in vitro addition of ethanol does not affect the cardiac protein synthesis in the heart. The acetaldehyde-mediated inhibition of cardiac protein synthesis can be partially prevented by antabuse. These observations suggest that, at least some of the deleterious effects of chronic ethanol consumption on the heart may be exerted through the inhibition of protein synthesis in the cardiac muscle.

Steroid specificity of human placental 5-ene-3 beta-hydroxysteroid oxidoreductase.

The properties of 5-ene-3 beta hydroxysteroid oxidoreductase (3 beta-HSD) from human placental homogenates were studied in vitro. The apparent Michaelis constants for 3 beta-HSD with the substrates pregnenolone (delta 5P) and dehydroepiandrosterone (DHA) were 170 nM and nM respectively. The optimal pH for both these substrates was between 10 and 12. With NAD as the substrate, the Km for the pregnenolone was 20 microM and for DHA, 17 microM. The activity of 3 beta-HSD was inhibited by various steroids. Competitive inhibitors (pregnenolone substrate) included: ethynylestradiol (inhibition constant Ki=7.3 nM), DHA (Ki=46 nM), estradiol-17 beta (Ki=46 nM), cholesterol (Ki=0.68 microM) and 16 alpha-hydroxydehydroepiandrosterone (16 alphaOHDHA) (Ki=2.2 microM). When the substrate was DHA, competitive inhibition occurred with the following steroids: ethynylestradiol (Ki=6.4 nM), estradiol-17 beta (Ki=69 nM), pregnenolone (Ki=91 nM), cholesterol (Ki=1.3 microM) and 16 alphaOHDHA (Ki=1.9 microM). 4-Ene-3-ketosteroids such as androstenedione, progesterone (delta 4P), norethindrone and chlormadinone acetate acted as noncompetitive inhibitors towards both substrates.

On the deacetylase activity of Vi bacteriophage III particles.

1. Using the complete phage particles as an enzyme, O-acetyl (1 leads to 4)-alpha-D-galacturonan (acetylated pectic acid) as a substrate, and gas-liquid-chromatography for the determination of the acid liberated, the virus-catalysed deacetylation of the polymer was studied. The activity was found to be stable up to about 50 degrees C, and from pH 4.5 to 9, with an optimum at pH 7.8; it was not affected by EDTA, or by 1,10-phenanthroline. The initial reaction velocity (at 37 degrees C) exhibited a simple hyperbolical dependence on the substrate concentration, with Km = 10.5 mM for O-acetyl (independent of virus concentration), and Vmax = 15 nmoles/min and 10(10) plaque forming units. The reaction was, however, rapidly inhibited by a partially deacetylated product (but neither by acetate, nor by pectic acid itself). 2. Using the natural substrate, acetylated (1 leads to 4)-2 amino-2-deoxy-alpha-D-galacturonan (Vi polysaccharide, Vi antigen), and a variety of structural analogues, the following conclusions about the substrate specificity of the Vi phage III deacetylase (acetyl-alpha-1,4-galacturonan acylhydrolase) were reached: (a) acetylated galacturonan is as good a substrate as acetylated aminogalacturonan; (b) of the two substrate diastereomers, acetylated alpha-L-guluronan (also 1 ax leads to 4 ax-linked units, but with axial acetyl residues at C-3), and beta-D-mannuronan (1 eq leads to 4 eq-linkages, and axial acetyl groups at C-2), only the former was acted upon, possibly indicating a specificity for the conformation of the polymer rather than for the configuration of the single residues; (c) all acyl analogues tested, O-monofluoroacetyl, O-propionyl, and O-butyryl galacturonan, were inert, showing a high degree of specificity for O-acetyl; (d) the oligomers, acetylated tri- and digalacturonic acid, as well as methyl-alpha-D-galacturonide, were still deacetylated, although more slowly, demonstrating tolerance of the enzyme of substrate size.

Alpha1-antitrypsin quantitation and Pi typing of stored postmortem blood.

Correlation of necropsy findings with protease inhibitor levels and phenotype is sometimes desirable. This study was designed to determine the feasibility of postmortem protease inhibitor assessment. One hundred fifteen consecutive postmortem samples, stored at -20 C for 24 to 53 months, were analyzed. The time from death to necropsy, storage time, and the pH values of the sera were correlated with alpha1-antitrypsin levels, trypsin inhibitory capacity, and Pi typability. The alpha1-antrypsin level and trypsin inhibitory capacity were not significantly correlated with morgue time, serum storage time, or pH, and mean values were within the expected ranges. A significant decrease in Pi typability occurred when pH was less than 7.0. Moreover, while most (86%) of the sera stored for 2 to 2 1/2 years were typable, only 30% of those stored for more than four years were typable. Determination of alpha1-antitrypsin and trypsin inhibitory capacity are possible with the use of stored postmortem blood. Pi typing is usually possible, provided sera are not acidic and are examined within 2 1/2 years.

Efficacy of antiparkinson agents in preventing antipsychotic-induced extrapyramidal symptoms.

The types of extrapyramidal syndrome (EPS) reactions produced by antipsychotic agents and the prophylactic use of antiparkinson agents in preventing EPS are reviewed. EPS are classified as akathisias, dystonias, parkinson-like symptoms and tardive dyskinesia, and have a varied incidence reported to range from 10.6 to 100%. Incidence may vary with age, gender, drug and dosage. The prophylactic use of antiparkinson agents to prevent EPS is controversial. Many psychiatrists believe the effect of EPS on patients is more harmful than the side effects of anticholinergics, whereas others believe that because of side effects, increased cost, greater risk to tardive dyskinesia and improper use, the use of antiparkinson agents cannot be justified. Most studies of prophylactic use of antiparkinson agents have lacked adequate control groups, adequate blinding procedures for investigators rating EPS, uniform definitions of EPS, random sampling and careful reporting of group characteristics such as dosage and drugs received. There is a lack of definitive studies of the value of antiparkinson agents in preventing the occurrence of EPS in patients receiving antipsychotics. A large, multicenter study should be undertaken to resolve the issue.

Relation between transmucosal potential difference, ionic flux, and the intraluminal supply of H+ in the ferret stomach.

To investigate the relation between gastric transmucosal potential difference, ionic flux, and the intraluminal concentration of H+, test solutions containing various concentrations of HCl were instilled into ferret stomachs both before and after exposure to various doses of acetic acid. The changes in ionic composition of the test solution were determined and the transmucosal potential difference was recorded throughout each experiment. The results showed that after exposure to the organic acid (1) ionic flux was proportional to the concentration of acetic acid used and was a direct function of the concentration of H+ within the lumen, and (2) the decrease in transmucosal potential difference was proportional to the concentration of acetic acid used but did not appear to be related to the subsequent magnitude of ionic flux. It is suggested that the decrease in gastric transmucosal potential difference that follows exposure of the gastric mucosa to an organic acid is a measure of the increase in mucosal permeability, whereas the subsequent ionic movements are governed by the concentration of H+ within the gastric lumen.

Properties of beta-glucan synthetase from Saccharomyces cerevisiae.

Properties of beta-glucan synthetase from S. cerevisiae were studied. The enzyme exhibited optimal activity at pH 6.7 and 24 C. Km for UDP-glucose was 0.12 mM. Addition of Mg++ or Mn++ stimulated its activity by 60% and 21% respectively. High concentrations of EDTA and hydroxyquinoline were inhibitory. Glucan synthetase was fully active in cell-free extracts. Small concentrations of trypsin or subtilopeptidase A from Bacillus subtilis, caused only a slight increase in glucosyl transferase activity, but larger concentrations destroyed beta-glucan synthetase. Acid proteases were neither stimulatory nor destructive. Thus it seems unlikely that beta-glucan synthetase exists in a zymogen form. Glucan synthetase was unstable. It was inactivated more rapidly at 28 C than at 0 C. The presence of substrate, beta-glucan or the protease inhibitors PMSF, Antipain or Pepstatin A did not protect beta-glucan synthetase from inactivation. Glucan synthetase was not stimulated by addition of cellobiose or beta-glucans. The synthesis of beta-glucans was competitively inhibited by UDP (Ki = 0.45 mM). Glucono-delta-lactone, a known inhibitor of beta-glucosidases was a strong non-competitive inhibitor of beta-glucan synthetase.

Determinants in microbial colonization of the murine gastrointestinal tract: pH, temperature, and energy-yielding metabolism of Torulopsis pintolopesii.

Torulopsis pintolopesii is an indigenous yeast that colonizes the secreting epithelia in the stomachs of mice and rats. A wild-type strain of this microbe was isolated and identified. To attempt to learn characteristics of the yeast that are advantageous to it in colonizing its natural habitat in vivo, we examined some aspects of its nutrition and energy-yielding metabolism and some environmental conditions that influence its growth in vitro. The yeast appeared to be limited in the compounds it can utilize as carbon and nitrogen sources. It grew best at 37 degrees C and did not grow at 23 or 43 degrees C. It grew optimally at neutral pH but could grow aerobically at pH values as low as 2.0 and anaerobically at pH values as low as 3.4. As assessed by measurements of growth rates and yield coefficients, it grew better aerobically than anaerobically. When grown aerobically, it had a cyanide-sensitive system for taking up O(2) and tested positively for cytochrome c oxidase activity. A petite mutant strain isolated from the wild-type strain had a growth rate and yield coefficient when incubated aerobically that were essentially the same as those of the wild-type parent grown anaerobically. Likewise similar to the wild-type parent grown anaerobically, the petite strain, though incubated aerobically, did not take up O(2). Yeast-free mice associated with either the wild-type or the petite mutant strain were colonized at essentially the same rates and to similar final population levels by both strains. The yeast's capacity to respire may be of little advantage to it in its natural environment. By contrast, its abilities to grow best at 37 degrees C and to grow at low pH values are undoubtedly advantageous characteristics in this respect. The limitations in its carbon and nitrogen nutrition are difficult to evaluate as ecological factors in its colonization of the natural habitat.

Respiratory acidosis with the small Storz-Hopkins bronchoscopes: occurrence and management.

Carbon dioxide retention in the Storz rigid ventilating bronchoscope with the Hopkins lens system was investigated in the laboratory. The 3.5, 4.0, and 5.0 30-cm Storz bronchoscopes with a 3.95-mm (outside diameter) telescope lens were used in 10 mongrel dogs weighing between 8 and 15 kg. Significant (p less than 0.01) accumulation of arterial carbon dioxide tension (PaCO2) (respiratory acidosis) was observed after 5 and 10 minutes of ventilation through the 3.5 and 4.0 bronchoscopes, but no significant increase in PaCO2 was noted with the 5.0 bronchoscope. There was no significant change in arterial oxygen tension under the same conditions. Manual compression of the upper anterior abdominal wall during expiration was applied during bronchoscopy in 6 children. Arterial blood samples were taken before insertion of the bronchoscope and 5 minutes later with and without abdominal compression during expiration. A significant increase (p less than 0.05) in PaCO2 and a decrease in pH were observed after 5 minutes of the bronchoscopic procedure without manual compression of the abdominal wall, while no significant changes in PaCO2 were observed with abdominal compression.

Clinical usefulness of sodium amobarbital interviewing.

We report a double-blind, randomized, placebo-controlled study utilizing a within-subjects design on 20 hospitalized, psychiatric patients who participated in sodium amobarbital interviews to determine if the drug has a specific effect in eliciting clinically useful information. The patients selected had difficulty communicating with their primary therapists during the postadmission, diagnostic interviews. Two raters completed a Hamilton Depression Scale, a New Haven Schizophrenia Index, and a Brief Psychiatric Rating Scale after each interview. Although both the amobarbital and saline interviews were moderately useful in obtaining new information, we found no significant difference in the primary therapists' assessments of clinical usefulness. In addition, the drug interview did not uncover material that would aid in the differential diagnosis between depression and schizophrenia. There was, however, a significant negative correlation between the assessment of general usefulness and the time interval between admission and interviewing. We report our only exception, a case of catatonic schizophrenia, in which the patient responded specifically to the drug.

Properties of ATP-driven reverse electron flow in chloroplasts.

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (deltapH). ATP-induced reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in deltapH formation. The rapid first phase and formation of a deltapH occur also in the absence of the electron transfer mediator phenazine methosulfate. 2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30 degrees C. The rate of formation of delta pH and of reverse electron flow were faster at high temperatures, but the maximal extent of delta pH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15 degrees C was found. Light activation of the ATPase occurred throughout the range studied. At 0 degrees C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more than 10 min. 3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.

The plastoquinone pool as possible hydrogen pump in photosynthesis.

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H+ + e-) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H+ uptake and the internal H+ release led to the following conclusions: (1) A stoichiometric coupling of 1 : 1 : 1 between the external H+ uptake, the electron translocation through the plastoquinone pool and the internal H+ release (corrected for H+ release due to H2O oxidation) is valid (pHout = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation. These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H+ + e-) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.

Identification, partial purification and biochemical characterization of gamma-glutamyltranspeptidase present as a membrane component in skimmed milk and milk fat-globule membranes, and in mammary-tumour virus from the milk of infected mice.

The enzyme gamma-glutamyltranspeptidase was reproducibly found to be associated with mouse milk particles; it is present in milk fat-globule membranes and mouse mammary-tumour virus of infected Swiss mice, also in particles from the milk of uninfected mice. The enzymatic activities observed range among the highest reported for mammalian tissues. The enzyme was partially purified from mouse mammary-tumour virus, and from milk fat-globule membranes. The molecule requires the presence of detergents to remain soluble, behaves as a high molecular weight component, properties characterizing integral membrane proteins. Kinetics, and the effect of competitors as well as of specific inhibitors show this enzyme to be identical to the well-known kidney gamma-glutamyltranspeptidase ((gamma-glutamyl)-peptide:amino-acid gamma-glutamyltransferase, EC 2.3.2.2). Other oncornaviruses budding from cultured cells originally expressing the enzyme in their plasma membrane also incorporate the enzyme in their structure.

Action of histamine receptor agonists and antagonists on the rat uterus.

1 Histamine and a series of compounds acting selectively on H1- and H2-receptors were tested on the isolated oestrous uterus of the rat. 2 Histamine had a dose-dependent inhibitory effect on the contractions elicited by acetylcholine. This action was unaffected by H1-blockers but was competitively inhibited by H2-blockers. The H1-selective agonist, 2-(2-aminoethyl)thiazole was ineffective at doses 100 times greater than those of histamine. Conversely, all the H2-agonists showed activity in the order of potency: N'-methylhistamine greater than histamine greater than N'-N'-dimethylhistamine greater than 5-methylhistamine greater than 5-methyl-N'-methylhistamine. Among the non-imidazole compounds, dimaprit had an activity identical to that of histamine, but all the dimaprit-like compounds showed negligible activity. 3 The data obtained suggest that in the rat uterus, (a) the activation of H2-receptors is responsible for the inhibitory effect of histamine and its analogues; (b) the integrity of the histamine molecule seems to be less crucial than that of the dimaprit molecule for the maintenance of the H2-activity, since changes in its structure modify but do not abolish the biological activity as they do in the case of dimaprit; (c) the order of activity of the various H2-receptor agonists is different from that observed in other tissues.

Effects of dietary vitamin B6 on the in vitro inactivation of rat tyrosine aminotransferase in host liver and Morris hepatomas.

Control rats or rats bearing Morris hepatoma 5123C (intact), 5123C (adrenalectomized), 7794A, 7800, 8999, 9121, or 9618A were fed a purified diet either deficient or adequate for vitamin B6. The concentration of pyridoxal phosphate in the plasma, host livers, and hepatomas was determined, as well as the in vitro rate of inactivation of induced tyrosine aminotransferase in homogenates of host livers and hepatomas. The results demonstrated the presence of a cysteine-independent inactivating system for tyrosine aminotransferase in hepatomas 5123C (adrenalectomized), 7800, 8999, and 9121. Only in hepatoma 9121 was there a dramatic influence of the dietary vitamin B6 on the rate of cysteine-independent inactivation. A cysteine-dependent inactivating system for the enzyme was present in all host livers and hepatomas. The rate of this in vitro inactivation for both host livers and hepatomas apparently was a function of the concentration of pyridoxal phosphate, but inactivation of tyrosine aminotransferase occurred at a significantly lower concentration of pyridoxal phosphate in the hepatomas than in the host livers.

Effects of equiblocking doses of nadolol and propranolol on left ventricular performance.

Nadolol, a recently developed noncardioselective beta-adrenergic blocker, has the potential advantages of a longer oral half-life (t 1/2) than propranolol and, in animal studies, markedly fewer direct myocardial depressant effects. Neither the relative intravenous potency of nadolol and propranolol nor the comparative effects of the 2 drugs on left ventricular performance has been studied in man. We compared equiblocking intravenous doses of nadolol and propranolol in 10 subjects with ischemic wall-motion disorders. Nadolol was on the average 6.2 times as potent on a milligram-for-milligram basis. Both drugs decreased resting heart rate (p less than 0.02) and produced small rises in both mean pulmonary artery (p less than 0.03) and mean pulmonary artery wedge (p less than 0.03) pressures without significantly reducing the cardiac output. Both drugs also produced depression of the radionuclide ejection fraction (p less than 0.002). There were no significant differences between the effects of the 2 drugs on any of the aforementioned variables. Thus, the effects of nadolol on left ventricular performances are similar to those of propranolol. Because of its long oral t 1/2, nadolol may prove to be a clinically useful drug.

Oral contraceptives and depressive symptomatology: biologic mechanisms.

The biological mechanisms through which oral contraceptives influence the central nervous system and produce depression were examined. Oral contraceptives reduce the level of serotonin and norepinephrine available at the central adrenergic receptor sites, alter folate and B12 levels, and perhaps influence hypothalamic releasing hormone levels. The level of serotonin is influenced in the following manner. The estrogens in oral contraceptives increase tryptophan available for the brain to convert to serotonin and tryptamine. Depression is associated with lower levels of serotonin, tryptamine, and perhaps tryptophan in the brain. Estrogens in oral contraceptives may also alter pryridoxal phosphate which in turn affects the production of serotonin. Oral contraceptives possibly lower norepinephrine levels by 1) decreasing tyrosine; 2) influencing coenzymes necessary to norepinephrine production; and 3) increasing monoamine oxidase levels. Oral contraceptives apparently inhibit the metabolism of folate and B12, and lower levels of these substances are associated with depressive symptoms. Decreased norepinephrine and serotonin levels may inhibit the release of gonadotrophin-releasing hormones, and these hormones may in turn influence behavior. Recommendations to clinicians were: 1) patients should be screened for a history of depression prior to prescribing oral contraceptives; 2) pill users should be monitored for depression; and 3) 25 mg daily of pyxidoxine should be administered if a patient taking oral contraceptives is deficient in B6.

Hormonal regulation of perinatal enzyme differentiation in the mammalian liver.

The adaptation of newborn mannals to extrauterine life depends in large part on the maturation of biochemical and physiological functions during perinatal development. Hormones such as glucocorticoids, catecholamines and glucagon can stimulate enzyme induction during development; on the other hand, insulin has been shown to antagonize these stimulatory effects. Only the surface of the problem of hormonal regulation of enzyme differentiation during the perinatal period has been reached, especially as regards human development. Each enzyme presents unique problems of chemical regulation; the functional consequences of these factors are not exactly the same in each tissue and perhaps not in each species. The possibility of using inducing agents such as hormones, drugs and substrates to promote biochemical enzyme differentiation is a new and exciting aspect which needs to be explored further as a means of facilitating survival and ensuring optimal extrauterine development of the immaturely born human infant or the full-term infant with delayed-enzymic development. However, any intervention in the carefully programmed interplay of different hormones which regulate normal enzymic adaptation and development during the perinatal period should be undertaken only after careful consideration. The possibilities of long-term harm must be weighed against short-term benefits.

Drugs, alcohol and driving.

Driving a car is a complex psychomotor and perceptual task which is subject to impairment by many factors. Several workers have studied the potential effects of drugs and alchol in crash production by epidemiological and laboratory studies. Both types of studies have yielded useful data but their limitations must be borne in mind when applying the results in pratice. Alcohol is obviously the most common single cause of traffic accidents. A progessively increased risk with increasing blood alcohol levels is well documented; fatigue and/or drugs increase this risk. Drugs are related much more infrequently to traffic accidents although on the basis of statistics, there is a potential risk with drug use. However, drugs alone are not as important as alcohol. The most significant drugs as regards driving risk are obviously certain antianxiety agents, hypnotics, stimulants, hallucinogens, marihuana, lithium and narcotic analgesics, as well as ganglionic blocking agents, insulin and sulphonylurea derivates. Patients should not drive after taking these drug until they are objectively fully alert and capable. Anticholinergics, antihistamines, antidepressants, antipsychotics, phenybutazone, indomethacin, alpha-methyldopa, and beta-blockers may in some cases cause central side effects (e.g. drowsiness) strong enough to affect driving performance. After starting therapy with these drugs, or after a significant change in dose, driving should be avoided until it is known that unwanted effects do not occur. Psychotropic drugs may enhance the deleterious effect of alcohol, and with most hypnotics there is still an effect the next morning. Some drugs (e.g. anticonvulsants or antiparkinsonian drugs) may make driving safer, but the disease (epilepsy, Parkinsonism, cardiovascular diseases, psychic disorders, etc.) ofter precludes driving. Clinicians should warn their patients about an impairment of driving skills if this is likely to occur due to the drug or the illness concerned.

1H nuclear-magnetic-resonance studies of porcine lutropin and its alpha and beta subunits.

The titration curves of the histidine residues of porcine lutropin and its isolated alpha and beta subunits have been determined by following the pH-dependence of the imidazole C-2 proton resonances. The isolated alpha subunit contains a buried histidine, whose C-2 proton does not exchange with solvent, and which has the unusually low pK of 3.3. In the native hormone all the histidine residues have relatively normal pK values (between 5.7 and 6.2). The four histidine C-2 proton resonances have been assigned to specific residues in the amino-acid sequence, by means of deuterium and tritium exchange experiments on the alpha subunit and its des(92-96) derivative. The histidine with a pK of 3.3 is identified as His-alpha87. The effects of pH on tyrosine and methyl proton resonances show that the titration of His-87 in the isolated alpha subunit is accompanied by a significant conformational change which involves loosening of the protein structure but which is not a normal unfolding transition. The role of conformational changes in the generation of biological activity by subunit association in the glycoprotein hormones is discussed.

The effect of rifampicin on norethisterone pharmacokinetics.

The pharmacokinetics of norethisterone have been studied in 8 women during and one month after treatment with rifampicin (450--600 mg/day). Rifampicin caused a significant reduction in the A.U.C. of a single dose of 1 mg norethisterone from 37.8 +/- 13.1 to 21.9 +/- 5.9 ng/ml X h (p less than 0.01). The plasma norethisterone half life (beta-phase) was also reduced from 6.2 +/- 1.7 to 3.2 +/- 1.0 h (p less than 0.0025). In one additional woman on long term oral contraceptive therapy the 12 hour plasma norethisterone concentration was reduced by rifampicin from 12.3 ng/ml to 2.3 ng/ml. Rifampicin caused a significant increase in antipyrine clearance, 6 beta-hydroxycortisol excretion and plasma gamma-glutamyltranspeptidase activity but there was no significant correlations between changes in these indices of liver microsomal enzyme induction. There was a significant correlation between the percentage increase in antipyrine clearance and the percentage decrease in norethisterone A.U.C. during rifampicin. The changes in norethisterone pharmacokinetics during rifampicin therapy are compatible with the known enzyme inducing effect of rifampicin.

Plasma corticosterone response to serotonin altering drugs in the 3-day-old rat.

Three-day-old rats were injected with various neurotransmitter altering agents to demonstrate a functional relationship between these drugs and plasma corticoid levels. Plasma corticosterone levels were increased after injection of methiothepin, methysergide, and 5-hydroxytryptophan (5-HTP), but were not changed by cholinergic, adrenergic, and dopaminergic compounds. Imipramine alone had no effects on plasma corticoids but in combination with 5-HTP resulted in a more sustained response than 5-HTP alone. Afunctional relationship between plasma corticosterone and serotonin receptors has been demonstrated in the 3-day-old rat. The presence of this relationship just after birth suggests the possibility that serotonin may be a mediator of early experience effects on later adrenocortical function.

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum.

Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a KM for putrescine of 2.58 x 10(-6) M and a Vmax of 0.53 nmol per hr per 50 microliter serum. Aminoguanidine competitively inhibited the enzyme with a KI of 1.8 x 10(-8) M. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The Vmax for the ABS putrescine oxidase was 2.10 nmol per hr per 50 microliter serum, and the KM for putrescine, 50.3 x 10(-6) M. The K1 of the ABS putrescine oxidase for aminoguanidine was 41 x 10(-6) M. On the basis of both the KM and KI values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56 degrees C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed.

Superoxide production in pulmonary alveolar macrophages and killing of BCG by the superoxide-generating system with or without catalase.

The superoxide production of BCG-infected and noninfected alveolar macrophages was measured by superoxide dismutase-inhibitable nitro blue tetrazolium reduction. The cells were incubated with or without cell-free bronchial lavage fluid (pulmonary washings). When control alveolar macrophages were infected by BCG, superoxide production was decreased markedly, probably due to bacterial cytotoxic factors. In contrast, the production of superoxide in alveolar macrophages exposed to pulmonary washings was increased and not appreciably influenced by BCG infection. Superoxide production by alveolar macrophages was dependent on time and on the protein concentration in the pulmonary washings. In controls, it was inversely proportional to the infecting dose of BCG. We observed previously that alveolar macrophages activated by pulmonary washings inhibited intracellular growth of BCG. We now present evidence that enhanced production of superoxide contributes to such inhibition, especially in the presence of catalase at acid pH. These findings are pertinent to the defense of inflamed lungs, where serum and serum immunoglobulin G transuded from blood into alveolar spaces probably induce such activation on alveolar macrophages.

Isolation of human spermatozoa membrane antigens binding sperm-immobilizing and sperm-agglutinating antibodies.

Peptides of human spermatozoa were dissolved with Hyamine 2389 and Triton X-100 and separated by chromatography on Biogel P4 columns into seven fractions. The antigenic activities of the separated sperm-membrane fractions were tested according to their capacity to inhibit sperm agglutination and sperm immobilization (immune inhibition test) in human sera of sterile patients. Sperm-agglutinating and sperm-immobilizing activity was tested by the microtray agglutination and microtray immobilization test. A titer reduction was achieved only in sperm-immobilizing sera. Four sperm antigenic fractions revealed in the majority of the repeatedly tested sperm-immobilizing sera an inhibition of the antigen-antibody reaction. No reaction was observed after exhaustive absorption of the tested seven antigen fractions with sperm-agglutinating sera. Therefore the conclusion can be drawn that sperm-agglutinating and sperm-immobilizing antibodies react with different sperm antigens. Normal human sera without sperm antibodies served as control. As no sperm agglutination or sperm immobilization was obtained after absorption of these control sera our antigen fractions do not produce sperm agglutination or sperm immobilization.

Adaptations in skeletal muscle following strength training.

Five men were studied before and after 7 wk of isokinetic strength training to determine its effects on muscle enzyme activities and fiber composition. One of the subject's legs was trained using 10 repeated 6-s maximal work bouts, while the other leg performed repeated 30-s maximal knee extension exercise. The total work accomplished by each leg was constant. Training 4 times/wk achieved similar gains in peak torque for both legs at the training velocity (3.14 rad/s) and at slower speeds. Fatigability of the knee extensor muscles, as measured by a 60-s exercise test, was similar in both legs after training. Biopsy specimens showed significant changes in the % of the muscle area composed of type I and IIa fibers as a result of both strength training programs. In terms of muscle enzymes, only the 30 s exercise program resulted in elevated glycolytic, ATP-CP and mitochondrial activities. Despite these changes, none of the parameters measured were found to be related to the gains in either muscle strength or fatigability during maximal isokinetic contractions.

Effect of cyclic guanosine 3',5'-monophosphate on nitrogen fixation in Rhizobium japonicum.

The addition of exogenous cyclic guanosine 3',5'-monophosphate (cGMP) at a concentration of 0.1 mM to a free-living culture of Rhizobium japonicum 3I1b110 was found to completely inhibit the expression of nitrogenase activity and markedly inhibit the expression of hydrogenase and nitrate reductase activities. The effect was specific for cGMP. Experiments on the in vivo incorporation of radioactive methionine and subsequent analysis of the labeled proteins on polyacrylamide gels showed that the biosynthesis of nitrogenase polypeptides was inhibited. It appears that the time of addition of cGMP is important since the effect was only seen during the early stages of nif gene expression. The intracellular level of cGMP was found to respond to physiological changes in the cell, and there was a fall in cGMP concentrations when nitrogenase was induced. Microaerophilic-aerobic shift experiments showed that intracellular levels increased from 0.25 pmol/mg of cell protein under microaerophilic conditions to 2.6 pmol/mg of cell protein under aerobic conditions, suggesting that the cellular pool size of cGMP may be under redox control.

Effect of NADP+ and its analogs on the Rose Bengal-sensitized photoinactivation of D-erythrulose reductase from beef liver.

Upon addition of NADP+, the rose bengal-sensitized photoinactivation of D-erythrulose reductase from beef liver is prevented to a remarkable extent. Adenosine 2',5'-diphosphate (2',5'-ADP) also has a protective effect, but to a lesser extent. On the other hand, 2'-AMP markedly enhances the photoinactivation. Other nucleotides which have no 2'-phosphoryl group, such as NAD+, 3'-AMP, 5'-AMP, ADP, and NMN, are ineffective. Further, only 2'-AMP derivatives (NADP+, 2',5'-ADP, and 2'-AMP) among these nucleotides were found to be potent competitive inhibitors of the enzyme with small Ki's (6--13 muM). Photooxidation of some methionine residues in the enzyme is prevented by the addition of NADP+ and accelerated in the presence of 2'-AMP. Photooxidation products(s) of 2'-AMP derivatives have no effect upon the enzymatic activity. Although NADP+ and 2'-AMP induce detectable conformational changes of the enzyme, the changes are not characteristic to the compounds. Based on these observations, we present a possible action mechanism of 2'-AMP derivatives on the photoinactivation of D-erythrulose reductase.

Anaphylactic release of a basophil kallikrein-like activity. I. Purification and characterization.

These studies describe the IgE-mediated relase of a basophil kallikrein-like enzyme that is an arginine esterase and is inhibited by plasma, diisopropylphosphofluoridate, and Trasylol. The substrate specificity for the synthetic amino acid ester substrates p-toluenesulfonyl-L-arginien methyl ester, benzoyl-arginine methyl ester, and acetyl-tyrosine methyl ester is similar for the basophil enzyme and plasma kallikrein. The interaction of arginine esterase-active fractions from ion-exchange (DEAE-Sephacel) and gel filtration (Sepharose 6B) chromatography, with human plasma kininogen, generates immunoreactive kinin. The basophil arginine esterase and kinin-generating activities co-chromatograph on Sepharose 6B and the quantity of kinin generated is, in general, proportional to the arginine esterase activity of the column fractions, suggesting that these two activities are subserved by the same protease. The ability of this protease to generate kinin equally well from heat- and acid-treated plasma, as from fresh human plasma, suggests that this protease has kallikrein-like activity. These data suggest that kallikrein-like activity can be generated from human basophils as a direct result of a primary IgE-mediated immune reaction, thus providing a potential link between reactions of immediate hypersensitivity and the plasma and(or) tissue kinin-generating systems.

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.

The reversal by pyridostigmine of neuromuscular block produced by soman.

The effect of pyridostigmine on neuromuscular block produced by soman was studied in the isolated phrenic nerve-diaphragm preparation. In the rat, soman produced an irreversible reduction in tetanic tension and functional acetylcholinesterase (AChE) activity. Pretreatment with pyridostigmine before exposure of the diaphragm to soman, followed by removal of the anticholinesterase from the organ bath, produced a return of tetanic tension and an increase of 5% in functional AChE activity. Similar results were obtained in the guinea-pig. The changes in synaptic AChE activity were verified pharmacologically by showing a decrease in the blocking activity of acetylcholine in preparations pretreated with pyridostigmine in comparison to those given soman alone following removal of the anti-cholinesterase. The blocking dose of carbachol did not change in these two groups indicating that desensitization was not a component of the protective action. A comparison was also made of the results obtained by measuring inhibition of AChE in situ with those obtained from muscle homogenates. The implications of these results are discussed.

Metabolic conversion of fluorenone oxime to phenanthridinone by hepatic enzymes.

Fluorenone oxime is converted to phenanthridinone by enzymes present in rat liver homogenates. The reaction is analogous to the chemical Beckman rearrangement. The oxime-amide rearrangement enzyme is localized primarily in the microsomes, with some activity in the cytosol. The reaction requires reduced nicotinamide adenine dinucleotide phosphate and observes Michaelis-Menten kinetics. The reaction is relatively slow (Vmax = 7.75 +/- 2.01 nmoles of phenanthridinone formed/100 mg of liver/15 min), but the enzyme reaches maximum velocity at relatively low substrate concentrations (Km = 3.90 +/- 1.85 x 10(-5) M). The reaction is strongly competitively inhibited by 1-decylimidazole (KI = 3.75 +/- 1.77 X 10(-7) M) and inhibited to a lesser extent by the chelating agents bipyridyl (KI = 1.33 +/- 0.21 X 10(-3) M) and ethylenediamine tetraacetate (KI = 1.00 +/- 0.28 X 10(-3) M) and the sulfhydryl binding agent p-chloromercuribenzoate (KI = 2.71 +/- 0.07 X 10(-4) M). Studies also suggest that the reaction mechanism does not involve initial enzymatic substrate esterification through acetylation, glucuronidation, phosphorylation, or sulfation.

Hemorrhagic cystitis and ureteritis, and interstitial nephritis associated with administration of penicillin G.

Hemorrhagic cystitis and ureteritis, and interstitial nephritis developed in a patient receiving penicillin G and streptomycin as therapy for bacterial endocarditis. After therapy was changed to vancomycin there was prompt resolution of these abnormalities.

Proliferative capacity of erythropoietic stem cell lines and aging: an overview.

The earliest bone marrow precursor cell types, often called stem CELLS, have a very large capacity for self renewal. This makes them a useful model system in which to test the hypothesis that normal somatic cells have a limited proliferative capacity. Marrow precursor cells differentiate and multiply to replenish the supply of various blood cell types that constantly turn over. Especially with erythrocyte production, this function is well difined and can be tested rigorously to determine whether a significant amount of the stem cell proliferative capacity is exhausted. Functional tests generally show that marrow stem cell lines are exhausted after three to six serial transplantations into successive recipients; the few exceptions are cases in which functioning by cells from the irradiated recipients has not been ruled out. Genetic markers unambiguously identifying marrow stem cell lines from the original donor are necessary for clear cut interpretations of transplantation experiments. No significant differences are found when comparing erythrocyte production by marrow stem cell lines from old and young adult donors. This suggests that little or none of the erythropoietic stem cell's proliferative capacity is exhausted by a lifespan of normal functioning.

On the mechanism of film neoplastogenesis.

Since the inception of experimental cancer research, all theories concerning the mechanism of transformation of normal cells into neoplastic cells were constructed on the basis of the postulate that interaction of an agent and cell constituents is a condition indispensable for the occurrence of the neoplastic transformation. In the course of the past three decades, however, all attempts to uncover agents responsible for the transformations induced by chemically inert films implanted in living tissues or for the "spontaneous" transformations in vitro in cell cultures devoid of added neoplastogens, yielded consistently negative results. In regard to the neoplastogenic effect of imbedded inert films, the present article takes into consideration that films implanted in living tissues hinder the circulation of interstitial fluid in their vicinity and as a result elicit anoxia and protracted low pH gradients in the environment of the cells adjacent to the films. The effects of such protracted low pH on the metabolic activities of the surviving cells are examined and a working hypothesis is offered concerning the role of protracted environmental low pH in the mechanism of film neoplastogenesis.

[Temperature dependence of conductance of isoionic DNA solutions. Determination of dissociation constants of primary phosphoryl groups].

Concentration dependence of the equivalent conductance of isoionic DNA solutions has been studied at different temperatures. The limiting equivalent conductance (lambda infinity) at every temperature investigated has been obtained by extrapolation to the infinite dilution in Kohlraush's plots. At the same time in plots c lambda c versus 1/lambda c (lambda c is equivalent to conductance at the concentration c), corresponding to the linear form of Ostwald's dilution law, the straight lines were obtained. Both lambda infinity and acidity constants (K) have been determined from these plots. The values of lambda infinity by two methods are in well agreement. The average values of lambda infinity were used for energy activation of conductivity calculation, equal to 2,40 +/- 0,05 kcal/mole. The acidity constant of primary phosphoryl groups passes through a maximum near 33 degrees. Equivalent conductance of hydrogen ions calculated by neglecting of macroion's mobility and by using of potentiometric determined concentration (cH+) has been shown to increase with cH+. Unusual behavior of DNA in isoionic solutions is discussed.

Reactions of cysteamine and other amine metabolites with glyoxylate and oxygen catalyzed by mammalian D-amino acid oxidase.

Pig kidney D-amino acid oxidase [D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3] catalyzes a rapid uptake of oxygen when high concentrations (50-100 mM) of glyoxylate and the following amines are present under usual assay conditions (pH 8.3): cysteamine, 2-aminoethanol, putrescine, D,L-1-amino-2-propanol, D,L-2-amino-1-propanol, 3-amino-1-propanol, D,L-octopamine, ethylenediamine, and L-cysteine ethyl ester. Notable physiological amines that do not support a rapid O2 reaction under the above conditions include histamine, serotonin, epinephrine, norepinephrine, spermidine, spermine, and cadaverine. A more detailed kinetic investigation of the reactions involving the first four reactive amines listed above indicated that the cysteamine reaction proceeds at a rapid rate even when cysteamine and glyoxylate are present at less than millimolar concentrations, but greater than millimolar concentrations are needed in the other amine reactions in order to observe a reasonable rate. At low concentrations and pH 7.4, the cysteamine-glyoxylate substrate (presumably thiazolidine-2-carboxylic acid) reacts an order of magnitude faster than any other known D-amino acid oxidase substrate. Considerable circumstantial evidence suggests that the reaction involving cysteamine is occurring physiologically, but the reactions of other amines would be occurring in the cell at a very low rate, if at all. It is proposed that the product of the enzymic reaction may be a metabolic effector that can modify the reactivity of proteins or nucleic acids by covalent attachment.

Structure and histogenesis of tooth plates in Sagenodus inaequalis Owen considered in relation to the phylogeny of post-Devonian dipnoans.

The histology of tooth plates of Sagenodus inaequalis has been investigated to obtain information on the histogenesis of the tissue. The histological mechanisms of growth and replacement of the tooth plate are described in terms of an increase in area of the tooth plate by addition of denticles to the lateral margins of the ridges, replacement of worn dentine at the tritoral surface by basal growth of dentine and invasive growth of dentine into the surrounding bone at the anterior and medial margins. The histogenesis of a specialized character for dipnoans is described, namely hypermineralized dentine, or petrodentine, within the tooth plates. This has placed an advanced character further back in the phylogenetic organization of dipnoans than was previously recognized. The implications of these observations are discussed in relation to proposed phylogenies and previous observations on tooth plates of other dipnoans. It is concluded that Sagenodus inaequalis shared a common ancestor with both the ceratondontids and the lepidosirenids. A sequence is proposed for the phyletic relationship of tooth plate-bearing dipnoans. From a consideration of the features of histogenesis of this specialized dentine, the alternative terminologies are reviewed and petrodentine (Lison 1941) chosen for the hypermineralized dentine and syndentine (Thomasset 1928) as the collective term for the entire mass of dentine in the tooth plate.

Further observations on Calliacantha Leadbeater Choanoflagellata), with special reference to C. simplex sp. nov. from many parts of the world.

C. simplex is a new species with an unusually wide geographical range, occurring at sea temperatures as varied as 0 degrees C under ice at Pt Barrow or -1 degrees C in arctic Canada, to 22 degrees C on the equator at the Galapagos Islands. The morphology and range of variation are illustrated by means of light microscopy, transmission electron microscopy and scanning electron microscopy applied to dry whole mounts prepared in situ from freshly-gathered wild material. Among the diagnostic structural features, special interest attaches to the position of the anterior transverse costa which is located unusually far back in comparison with other members of the genus; it is also shown to be within and not outside the ring of four longitudinal costae building up the lorica chamber, although this feature can only be ascertained by scanning. The differences between this species and C. natans (type species of the genus) are shown to be greater and to involve more characters than could previously have been recognized, and some possible functional implications are discussed in a preliminary way.

Distribution of glutamate dehydrogenase and glutamine synthetase activity in the sheep and chicken digestive tract.

Glutamate dehydrogenase (GLDH, EC 1.4.1.3) and glutamine synthetase (GS, EC 6.3.1.2) activity were determined in the contents and tissues of the various parts of the sheep and chicken digestive tract, GLDH activity in the tissues of the sheep omasum, duodenum, rumen, reticulum, colon, caecum, jejunum and ileum ranged from 3.25+/-0.7 U (mumol/g dry weight . min) to 5.94+/-2.28 U; in the abomasum it was 9.67+/-1.27 U. GLDH activity in the contents of the ileum, abomasum, jejunum and duodenum varied from 0.85+/-0.19 U to 3.29+/-0.53 U and in the colon, caecum, reticulum, omasum and rumen from 6.34+/-2.64 U to 16.96+/-3.83 U. GS activity in the tissues of these parts of the digestive tract varied from 2.8+/-0.59 U to 8.6+/-1.4 U and their contents from 2.49+/-0.85 U to 10.76+/-2 U. GS activity in the contents of the colon was very low (0.26+/-0.07 U). In the tissues of the chicken duodenum, caecum, jejunum and ileum we found GLDH activity of 4.68+/-1.64 U to 7.96+/-1.73 U; in their contents it was 3.31+/-1.06 U to 3.8+/-0.73, but in the caecum it attained up to 66.7+/-24.3 U. GS activity was high from 57.6+/-2.0 U to 231+/-84 U in the tissues and 357+/-53 U to 383+/-76 U in the contents (in the caecum up to 2,500+/-233 U). The results show that conditions for the utilization of ammonia are present in the tissues and the contents in the whole of the sheep and chicken digestive apparatus. The hypothesis is confirmed that the different ability of ruminants and fowls to utilize ammonia formed from urea added to their feed, including ammonia formed by hydrolysis of blood urea, is due to the different GLDH and GS activity in their digestive tract as well as in their liver.

GABAergic and glycinergic mechanisms within the substantia nigra: pharmacological specificity of dopamine-independent contralateral turning behavior and interactions with other neurotransmitters.

The pharmacological specificity of the GABA agonist muscimol-induced contralateral turning behavior after unilateral injection into substantia nigra pars reticulata (SNR) has been studied. Muscimol-induced turning was antagonized by intranigral bicuculline methochloride (BMC) and picrotoxin, whereas antagonists of glycine, morphine, dopamine, noradrenaline, and serotonin were ineffective. Glycine induced a qualitatively similar turning behavior which was strychnine-sensitive but relatively BMC and picrotoxin-insensitive. Other drugs, including substance P, kainic acid, clonidine, oxymetazoline, serotonin, and carbachol, induced turning that could be dissociated from the effect of muscimol. Muscimol-induced turning was dopamine-independent, indicated by resistance to haloperidol (1 mg/kg), to pretreatment with reserpine (7.5 mg/kg) plus alpha-methyl-p-tyrosine (200 mg/kg), to haloperidol injections into the SNR, striatum and nucleus accumbens, and finally to kainic acid lesions of the striatum. 6-Hydroxydopamine lesions increased the efficacy of intranigral muscimol, while kainic acid lesions of the SNR antagonized muscimol. Muscimol-induced turning was inhibited by oxotremorine (0.25 mg/kg), by intranigral carbachol, and by apomorphine (0.1--0.5 mg/kg), but only moderately by intranigrally injected apomorphine. These data suggest specificity of GABA-agonist-induced contralateral turning and indicate an interaction between nigral GABA and other neurotransmitters, particularly dopamine and acetylcholine.

Anaerobic pulmonary infections.

The main cause of anaerobic pulmonary infections is aspiration of saliva, upper airway secretions or gastric content. Predisposing conditions include prominent dental disease, chronic upper respiratory tract infections and reduced consciousness. Fusobacterium nucleatum, Bacteroides melaninogenicus and anaerobic Gram-positive cocci are the most frequently encountered organisms. The clinical presentations are lung abscess, lung gangrene and empyema, which all tend to take a slow and indolent course. Preferred localization are dependent lung segments, most often on the right side. For bacteriological examination in these infections, only transtracheal aspirate and aspirate from the lung or pleural space are considered adequate. In 26 cases of empyema treated in our hospital during the last 3 years, adequate specimens had been taken in 19. Fifteen had been adequately examined, and anaerobes were cultured in 6. Among 29 abscesses treated during the same period, adequate specimens had been taken in only 14, and 11 had been properly examined. Seven specimens grew anaerobes on culture. In prospective studies of transtracheal aspirate in 15 chronic bronchitics without emphysema, anaerobes were not demonstrated. In 11 patients with bronchiectasis, anaerobic bacteria were cultured in 3. Finally, no anaerobic bacteria could be demonstrated in the transtracheal aspirate from 76 patients with acute exacerbation of chronic bronchitis. Anaerobic, pulmonary infections do not represent an intriguing medical problem in our region. However, knowledge of these infections is necessary to secure adequacy in collection of specimens and in their bacteriological examination.

In search of treatment for tardive dyskinesia: review of the literature.

Studies on the treatments for neuroleptic-induced tardive dyskinesia published in the English literature until August 1978 are reviewed. There is a yet no single satisfactory method of treatment for tardive dyskinesia. Withdrawal of neuroleptics results in a remission of symptoms in younger and non-brain-damaged patients. Paradoxically, the most effective treatment for suppressing dyskinesia is administration of neuroleptics. The possibility that continued use of neuroleptics in dyskinesia patients produces irreversible brain damage remains to be validated (or invalidated). Anticholinergic and dopaminergic drugs are of no value in the treatment of tardive dyskinesia. Cholinergic drugs have not lived up to their initial promising results in this condition. About one third of the dyskinetic subjects seem to respond to various nonspecific measures. Tardive dyskinesia probably consists of at least two subtypes-- reversible and persistent. Methodological aspects of earlier studies and possibilities for future research in this field are discussed. Suggestions for treatment of individual cases are also outlined.

Studies on the adrenergic systems of the eye at the Wilmer Institute.

The study of the ocular adrenergic systems at the Wilmer Institute over the last 40 years began with the examination of the formation and outflow of aqueous humor by Jonas Friedenwald and his co-workers in the early 1940's. At that time, researchers were attempting to understand the mechanisms of fluid dynamics in the eye, with the idea of altering these mechanisms to alleviate glaucoma only a distant goal. In the late 1950's, Langham and others reported on the effect of superior cervical ganglionectomy on intraocular pressure, following this work with a succession of papers on the relationship between alpha and beta adrenergic substances and aqueous humor dynamics. Sears, in collaboration with Bárány, described the effects of alpha and beta adrenergic substances on outflow resistance. Eakins, in 1963, suggested a mechanism of action for isoproterenol in the lowering of intraocular pressure. Work on timolol at Wilmer began with a report from Radius, Diamond, Pollack and Langham covering experimental studies with rabbits and clinical studies of the drug's ocular hypotensive effects on glaucoma patients. Richter and others also studied a small number of patients on maximal therapy and found further intraocular pressure reduction with added timolol.

Specific suppression of graft-versus-host responsiveness by incubation of donor lymphoid cells with a solubilized membrane fraction of host lymphoid cells.

BALB/c spleen cells were incubated with a solubilized membrane fraction (SMF) prepared from spleen and thymus cells of (BALB/c x C3H/He)F1 or (BALB/c x A/J)F1 hybrid mice. Cells incubated with (BALB/c x C3H/He)F1 SMF produced less graft-versus-host (GVH) splenomegaly in (BALB/c x C3H/He)F1 hosts than did untreated BALB/c cells. The reduction of GVH splenomegaly was specific, inasmuch as the GVH activity of (BALB/c x C3H/He)F1 SMF-treated and untreated cells was similar in (BALB/c x C57BL)F1 hosts, and BALB/c cells treated with (BALB/c x A/J)F1 SMF showed no alteration of GVH activity in either (BALB/c x C3H/He)F1 hosts or (BALB/c x C57BL) F1 hosts. The time course of splenomegaly did not differ for SMF-treated and untreated cells. Donor cells that were labeled with tritiated adenosine and treated with (BALB/c x C3H/He) F1 SMF produced a reduction in the amount of label appearing in (BALB/c x C3H/He)F1 host spleens but not in (BALB/c x C57BL)F1 host spleens. Mechanisms which could account for the ability of SMF to cause specific reductions in both GVH activity and host spleen labeling are discussed.

[Dynamics of acid-base indices in the blood of lambs from the 7th day of postnatal life to adult age].

Under the conditions of a specialized farm equipped with amodern technology or rearing, sixteen ewe lambs of the Improved Wallachian breed were studied for the dynamics of acid-base indices in the blood from the seventh day of post-natal life unitl the age of 19 months. The acid-base parameters were measured by the Astrup BMS 2 apparatus. After weaning at an age of 45 days, the lambs were fed mixture COJ 1 for ten days, then mixture COJ 2, followed by the administration of a VJ mixture and granular feed in a 1 : 1 ratio. The mentioned feeds were given to the lambs ad libitum. At the beginning of July the animals were driven to pasture where they were kept with no supplementary feeding. In the winter season they were kept indoors and were fed briquettes of forage at a ration of 2.3 kg per head per day. In the second year of age the sheep were driven to pasture in early. May. Throughout the period of study, the values of actual pH in the blood ranged ranged from 7.29 to 7.39 and pCO2 from 4.33 to 6.25 kPa, on an average; BE ranged from -8.2 to -1.8 mmol 1(-1), BB from 35.7 to 49.3 mmol 1(-1), and SB had limiting means of 18.0 and 22.7 mmol 1(-1), AB 16.7 and 23.5 mmol 1(-1), and tCO2 ranged from 17.7 to 24.8 mmol 1(-1), on an average. The highest numerical fluctuations in the values of the indices under study were recorded in the period of transition to the different kinds of solid feeds and to pasture.

[The unaffected primary rejection of xenogeneic kidney transplants in the closely related fox-dog species system].

The present study reports of three kinds of experiments of unaffected primary rejection of xenogenous kidney transplanats in the close-related fox-dog species system. The issue is whether there is a relation between the amount of grafted parenchyma and the immune induced potency, that is whether the course of rejection of transplanted single kidneys (group I a) differs from the course after en-bloc transplantation of both kidneys (group I b). In group II alterations of blood chemism and behavior of humoral antibodies are followed in dogs to which a fox kidney was transplanted, while keeping their own functioning kidneys. This experiment is to give information whether the uremic syndrome influences the development of humoral immunity, and what changes of blood chemism may primarily be related to destruction of the graft, under the condition of absent uremia. Untreated graft recipients survived for 5,4 +/- 0,49 days (n = 5) when single kidneys were transplanted (group I a), and 5,2 +/- 0,75 days (n = 5) when both kidneys were grafted en-bloc (group I b). As to the rejecting reactions, both groups are almost equal: the increasing functional failure causes a fast increase of creatinine and urea nitrogen; alkaline phosphatase and LDH show distinct alterations, related to the progress of the graft's destruction. Decrease of albumin level and loss of cholinesterase activity indicate an impaired hepatic function as reaction to uremic intoxication. Gamma-globulins and leucocytes show alterations that can be related to non-specific inflammatory reactions. The immunologically specific initial lymphopenia suggests that after revascularization these cells migrate to the graft, and later react with antigenic structures of vascular endothelium and still later with those of the organ cells. Cytotoxic antibodies appear on the 4th postoperative day in increasing amount. Post mortem histologic examination shows round cell infiltrates in the vastly necrotic renal parenchyma. When the recipient's kidneys are kept in situ and a fox kidney is transplanted (group II) uremia is avoided and the animals survive. During the 30-days period of observation, that is longer than the term of rejection, the titer of cytotoxic antibodies remains stable or tends to increase. LDH and alkaline phosphatase show characteristic changes that are considered sequels from destructed transplantate. The experiments show, aside from certain reservations, that the donor-host combination fox-dog is suitable to serve as preclinic model for human transplantation using xenogenous donors of organs, i. e. anthropoid primates.

Fetal heart rate variability: an approach to automated assessment.

Three hundred seventy-five hours of fetal heart rate (FHR) data derived from the direct fetal electrocardiogram (ECG) were studied. This data had been stored on magnetic tape from 83 intrapartum patients. By means of a computerized technique, the FHR variability was assessed quantitatively. The degree of variability was then related to: (1) state of labor, (2) fetal scalp pH values, and (3) the 1-minute Apgar score. FHR variability was computed from differences between consecutive R-R intervals measured from the R wave of each fetal ECG. A trend of increasing variability was seen with advancing labor, defined by either time prior to delivery or cervical dilatation, but values were not statistically significant. Significantly less FHR variability was encountered when fetal scalp pH values below 7.20 were compared to higher values. FHR variability assessed during the 20 minutes immediately preceding delivery was significantly lower in infants with 1-minute Apgar scores less than 7. Machine assessment of FHR variability thus could be correlated with fetal condition as determined by scalp pH and neonatal outcome determined by Apgar score.

The endodermal origin of digestive and respiratory tract APUD cells. Histopathologic evidence and a review of the literature.

Twenty-seven small cell carcinomas of the lung and three tumors of the large intestine with combined adenocarcinomatous and small cell and/or anaplastic carcinoid-type histologic features were studied by light and electron microscopy. It was shown that the small cells have morphologic characteristics of APUD cells. Also presented are the histologic features of a carcinoma of the lung with large cell undifferentiated carcinoma, adenocarcinoma, squamous cell carcinoma, and giant cell carcinoma areas in the primary site and in several metastatic foci. Two of the renal metastases showed small cell carcinoma. The combined tumors and the numerous other similar neoplasms described in the literature and reviewed here suggest an endodermal origin for digestive and respiratory tract APUD cells based on the hypothesis that cancer is a clonal proliferation, and mucous and squamous cell differentiation is an endodermal rather than neural crest characteristic. The ultrastructural features of tumors of cells of known neural crest origin, including a medullary carcinoma of the thyroid, three carotid body tumors, a pheochromocytoma, and two cutaneous melanomas were compared with those of other APUD cell tumors including small cell carcinomas of the lung, two bronchial carcinoids, a carcinoid of the appendix, and a carcinoid of the kidney. Cells of the latter group sometimes possessed cytoplasmic tonofibrils, round compact masses of cytoplasmic microfilaments, and ductal lumina. These features were lacking in the former group and may signify a different embryologic origin. The histologic, histopathologic, and embryologic evidence regarding the origin of digestive and respiratory tract APUD cells is reviewed, showing that the former are, and the latter probably are, of endodermal and not neuroectodermal origin.

[Doppler ultrasonic measurement for diagnosing carotid occlusions and stenoses (author's transl)].

Doppler ultrasonic measurements were performed in 74 patients suffering from cerebrovascular disorders. Flow direction was determined in the supratrochlear artery by using a directional Doppler sonographic instrument according to the method of Keller et al. 15 patients presented, unilateral or bilateral, with a flow reversal (i.e. direction of flow into the orbita via supratrochlear artery). In 8 of these patients carotis or aoitic arch angiographies were performed and in 4 cases necropsy reports were available. In all instances where flow reversal occurred severe carotid stenoses or occlusions were demonstrable. Thus 9 internal carotid occlusions, 1 common carotid occlusion, and 3 severe internal carotid stenoses were proved. One internal carotid occlusion and 3 internal carotid stenoses were operated on. Check-up sonograms after endarterectomy of carotid occlusions or stenoses showed a normal antegrade flow thus demonstrating patency of the carotid vessels. Obviously, supratrochlear flow reversal as measured by ultrasonic technique signals ipsilateral carotid vessel occlusion or stenoses. Furthermore, the method seems to be a reliable means for controlling carotid endarterectomy.