Lorazepam kinetics in the elderly.

Lorazepam kinetics in the elderly. Lorazepam is a 3-hydroxy-1,4-benzodiazepine derivative biotransformed by glucuronide conjugation, followed by urinary excretion of the glucuronide metabolite. The kinetic properties of single 1.5- to 3.0-mg doses of intravenous lorazepam were assessed in 15 healthy elderly subjects, 60 to 84 yr of age, and in 15 healthy young subjects, 19 to 38 yr of age. Volumes of distribution for lorazepam in the elderly group (mean, 0.99 1/kg), were slightly but significantly smaller than in the young group (1.11 1/kg), suggesting less extensive drug distribution in the elderly. Values of elimination half-life (t1/2beta) in the elderly (15.9 hr) did not differ significantly from those in the young group (14.1 hr), but total clearance in the elderly (0.77 ml/min/kg) was 22% less (p less than 0.05) than in the young subjects (0.99 ml/min/kg). Age differences in lorazepam clearance were partly explained by more frequent cigarette smoking in the young subjects. Gender had no apparent relationship to kinetics. The rate and completeness of absorption of intramuscular (IM) and oral loraxepam was assessed in 10 of the elderly subjects. Deltoid IM injection and oral administration of tablets in the fasting state led to rapid absorption of lorazepam into the systemic circulation. Peak plasma lorazepam concentrations were always reached within 2.5 hr, and values of absorption half-life (t1/2a) did not exceed 45 min. Absorption of IM and oral lorazepam was 80% to 100% complete. Thus, the aging process is associated with small changes in the kinetics of lorazepam. IM and oral administration of lorazepam in elderly persons, as in the case of young individuals, leads to rapid and nearly complete absorption into the systemic circulation.


Effect of neuroleptic drugs on lithium uptake by the human erythrocyte. We studied the effects of neuroleptic and antidepressant drugs on Li+ uptake in human red cells to examine the reported neurotoxicity reported for lithium-neuroleptic combination treatment and its relationship to altered tissue Li+ levels. All the phenothiazines increased Li+ influx in red cells and steady-state Li+ distribution ratio in vitro. This effect was not observed with other drugs studied. It is suggested that increased levels of Li+ in the tissue caused by phenothiazines may be related to neurotoxic effects observed during Li+ -phenothiazine therapy.


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.


Excretion of kidney brush border antigens as a quantitative indicator of tubular damage. The excretion of antigens and enzymes derived from the brush border region was studied in patients with kidney diseases, after kidney transplantation, during administration of potential nephrotoxic drugs, before and after operations etc. The main portion of membrane constituents was excreted in the urine at an increased rate, compared to healthy persons, and was identical with glycoproteins artificially released from the brush border membrane surface. Antisera against brush border antigens, which had been isolated from urine by affinity chromatography, were used to localise the origin of urinary kidney tissue-proteins applying immunofluorescence microscopy.


Improvement in arterial oxygenation by saralasin infusion in man. Simultaneous hemodynamic and blood gas measurements were performed in 26 hypertensive adults, who were cigarette smokers, before and after a 30-minute infusion of saralasin (5 microgram/kg/min) which is a highly specific competitive antagonist of angiotensin II (AII). The arterial pressure fell in nine, rose in seven and was unchanged in ten patients. The mean cardiac index for the entire group remained unchanged. Pulmonary arterial or wedge pressure, pulmonary vascular resistance, arterial PCO2 and pH did not change. Unrelated to the hemodynamic changes, the mean arterial oxygen pressure (PaO2) increased from 68.6 +/- 2.2 mm Hg to 73.9 +/- 2.1 mm Hg (P less than 0.001). In the absence of a significant increase in alveolar ventilation as indicated by an unchanged mean PCO2 and lacking a hemodynamic explanation, the mechanism for the rise in PaO2 is speculative at this stage. The possibility of an improvement in the distribution of ventilataion by saralasin infusion is under investigation.


Kinetics of theophylline; variability and effect of arterial pH in chronic obstructive lung disease. The pharmacokinetic behavior of theophylline was determined in 12 patients during an acute exacerbation of their chronic obstructive pulmonary disease. A 5.6 mg/kg loading dose of aminophylilne was administered, followed three hours later by a 0.9 mg/kg/hr continuous infusion. The loading dose increased the serum theophylline level an average of only 5.77 microgram/ml. After the loading dose, only five patients had levels greater than 10 microgram/ml. Mean initial drug clearance was 0.77 L/kg/hr, half-life 9.1 hr, and apparent volume of drug distribution .887 L/kg. Wide inter- and intrapatient pharmacokinetic variability was observed. The variability of drug distribution was inversely correlated with the arterial pH. These patients with chronic obstructive pulmonary disease appeared to require more theophylline when acidemic than when alkalemic to achieve similar serum theophylline concentrations.


A microcalorimetric study on the action of penicillins on Staphylococcus aureus and Escherichia coli. The heat generated by cultures of a strain of Escherichia coli and of a beta-lactamase- and a non-beta-lactamase-producing strain of Staphylococcus aureus were registered by a flow microcalorimeter of the heat conduction type. When exposing the non-beta-lactamase-producing strain to benzylpenicillin, an effect that appeared to correspond to the 'paradoxical zone phenomenon' was found, i.e. the inhibitory effect on the bacterial metabolism, as judged from the heat generated, was smaller when using 0.1-0.2 microgram ml-1 of benzyl-penicillin (5 and 10 x minimum inhibitory concentration, MIC) than with 0.04 microgram ml-1 (2 x MIC) and 2.0-200 microgram ml-1 (100-10,000 x MIC). Similar paradoxical response were found when the effects of ampicillin on E. coli and on the non-beta-lactamase-producing strain of S. aureus were studied. In the beta-lactamase-producing strain, no decrease in the heat was obtained when benzylpenicillin was used in concentrations up to 5.0 microgram ml-1 (10 x MIC), whereas a drug concentration of 50 microgram ml-1 (100 x MIC) caused a slight reduction. The addition of dicloxacillin in concentrations of 0.25-12.5 microgram ml-1 (2-100 x MIC) caused a concentration-dependent reduction in the heat developed.


[Changes in brain metabolism in hepatic coma]. After controlling the physiological parameters during 6 hours of experimental hepatic coma in Sprague-Dawley rats the results show 1. No shortage of brain energy supply of the four main cerebral energy reserves, the brain glycogen is significantly elevated, and the phosphocreatine, ATP and glucose unaltered. 2. No alterations in intermediates of the glycolytic pathway and the tricarboxylic acid cycle in brain tissue. 3. A highly significant decrease in the brain content of the "excitatory" transmitter amino acids glutamate and aspartate. These findings do not support the well-known hypothesis of BESSMAN and BESSMANN and later authors on the role of ammonia in the pathogenesis of hepatic coma.


Tyrosine hydroxylase and the conversion of L-thyroxine into 3',3,5-triiodo-L-thyronone in the rat. We have studied the effects of alpha-methyl-p-tyrosine (alpha-MPT), an inhibitor of tyrosine hydroxylase, on the in vivo conversion of L-T4 (T4) to 3',3,5-triiodo-L-thyronine (T3), and on the biological effectiveness of T4. Thyroidectomized rats were used and were injected daily with T4 maintenance doses. Three different types of experiments were carred out. The first involved isotopic equilibration with 125I-labeled T4 and measurement of urinary 125I excretion. The second series involved the injection of a single dose of [125I]T4, with the amounts of [125I]T3 in different tissues being studied 7 or 20 h later. The third series involved daily treatment for 13 days with T4 and alpha-MPT, at the end of ehich the liver alpha-glycerophosphate dehydrogenase activity was measured as a parameter of the biological effects of the hormone. Though the experimental approaches used clearly disclosed the well known effects of 6-propyl-2-thiouracil, no clear-cut effects of alpha-MPT were observed. It is concluded that alpha-MPT neither inhibits the conversion of T4 to T3 in vivo in rats nor affects the biological potency of a given dose of T4, at least to an extent compararble to that observed when 6-propyl-2-thiouracil is used. Thus, present results do not support the hypothesis that tyrosine hydroxylase is involved in the extrathyroidal deiodination of T4 to T3.


Alterations in steroidogenesis and human chorionic gonadotropin binding in the cryptorchid rat testis. One month after the induction of cryptorchidism in adult rats, serum levels of LH and FSH were significantly elevated in comparison with sham-operated controls, whereas serum levels of testosterone remained low to normal. Testis weight in cryptorchid rats was reduced by over 66%, and once the extratubular fluid was removed by decapsulation, the reduction in weight was 78%. The basal production of testosterone, pregnenolone, and estradiol in vitro by testes from cryptorchid rats was similar to controls, whereas significantly less androstenedione was produced. Testicular stimulation in vitro with a high dose of hCG (360 pM) resulted in significantly greater production of testosterone, pregnenolone, and estradiol by cryptorchid than by control rat tissue. The in vitro binding of [125I]hCG per testis was decreased in the cryptorchid state to 40% of control values, probably as a result of down-regulation of LH receptors due to the 4-fold elevation of serum LH levels in the cryptorchid rats.


Increased thermolability of benzodiazepine receptors in cerebral cortex of a baboon with spontaneous seizures: a case report. The benzodiazepine receptor in the cortex of 1 spontaneously epileptic baboon exhibited an increased rate of thermal inactivation at 65 degrees C when compared with those from 3 other baboons. In other respects (receptor concentration, affinities for flunitrazepam and diazepam, and response to changing pH), the benzodiazepine receptor from this animal was very similar to the receptors in the cortex of 3 other baboons. The 3H-QNB (muscarinic) and 3H-naloxone (opiate) binding sites in the brain of all 4 baboons appeared very similar with respect to all parameters studied (thermal stability, concentration, regional distribution, and affinities for respective ligands). An endogenous factor stabilizing the benzodiazepine receptor could be lacking in the spontaneously epileptic baboon.


Cryptorchid human testis after HCG treatment: histological study on Sertoli cells. Histological studies have been carried out on Sertoli cells in cryptorchid patients after long-term HCG treatment. Morphological studies revealed the absence of differentiation of mature Sertoli cells. This behaviour with the persistence of fibroblast cells in the interstitium after long-term HCG treatment suggests that the morphological action of HCG on the Sertoli cells is closely correlated with the maturation of the Leydig cells, particularly with their androgen production.


Secondary hydronephrosis to polyarteritis nodosa. A case of secondary hydronephrosis to lesions from polyarteritis nodosa localized in a short segment of the mid-portion of the ureter together with sclerolipomatosis, periureteritis and functional stenosis is presented. Polyarteritis nodosa is a necrotizing vasculitis affecting small and middle-sized arteries of all organs but more particularly the kidney, heart, gastrointestinal tract, testicles, liver and the striad muscles. Localization in the fatty tissue surrounding the ureter with a periureteritis is not frequent, only 1 case has been published in the literature (1).


[Acid-base changes across the rat jejunum, in vitro (author's transl)]. Everted sacs of rat jejunum are able to adjust to near 6.5 any solution whose pH is situated between 5.5 and 9.5. Beyond these limits the adjustment becomes incomplete. Changes in PCO2 and total CO2 associated with changes in mucosal pH-values suggest that bicarbonate or CO2-movements toward the jejunal lumen involved in this adjustment process are predominant.


Luminal alkalinization by guinea-pig cecum in vitro, an electro-neutral process. Guinea-pig cecum was found to alkalinize its mucosal media in vitro at a chemical equivalents rate greater than the short circuit current (ISC). Alkalinization was inhibited by conditions which did not affect ISC and by low mucosal Na suggesting an electro--neutral process, dependent on Na.


Complexed rheumatoid factor measurements in sera, synovial fluids and in immune complex fractions. Mild acidic treatment increases the rheumatoid factor titre of some sera and synovial fluids (SF) in rheumatoid arthritis (RA), juvenile RA (JRA) and most frequently in rheumatoid vasculitis. This unmasking of 'hidden' RF in serum and SF samples correlated with the RF-immune complexes (RF-IC) and complexed C4 present in the 3% polyethylene glycol (PEG) precipitates, indicating that by means of 'hidden' RF measurements RF-ICs are possibly detected. This method seems to provide a diagnostic tool for detecting RF-ICs in RA and other related diseases.


Diethylpyrocarbonate interferes with lipid-protein interaction and glucose transport in the human red cell membrane. Diethylpyrocarbonate largely diminished both discontinuities in red cell glucose transport and also in red cell membrane ANS fluorescence at about 17--20 degrees C.


Hypotensive mechanism of adrenergic alpha blockers in cats. The hypotensive mechanism of adrenergic alpha blockers is related to their inhibitory effect on the contractile Ca-mechanism in vascular smooth muscles as in nitroglycerin or verapamil.


Effects of adrenergic and cholinergic agonists on adenylate and guanylate cyclase activity of isolated guinea-pig seminal vesicle epithelium. Isolated seminal vesicle epithelium of the guinea-pig contained increased amounts of cAMP and cGMP after treatment with PGE1 and carbachol, respectively. Adrenergic agents had no influence. Possible physiological implications of these results are discussed.


Beta-adrenoceptor blocking drugs, heart rate and genetic hypertension development in rats. In spontaneously hypertensive rats (SHRs) chronically treated during their growth with beta-adrenoceptor blocking drugs, no correlation was found between the reduction in heart rate and the prevention of genetic hypertension development.


Attempts to use carbodiimide (EDCI) to cross-link hemoglobin for transfusions. The polymerization of hemoglobin for use as a blood substitute and an oxygen carrier would be of interest because high-mol. wt macromolecules would have a longer vascular retention time than the monomer. We found that the molecules resulting from the treatment of hemoglobin with ethyldimethylaminopropylcarbodiimide did not have a higher mol. wt than free hemoglobin and also had a dissociation curve resembling that of monomers, but seemed more stable.


[Comparative study of the action of tranquilizers on reinforcement systems]. In experiments on rats the benzodiazepine tranquilizers diazepam (0.5 -- 1 mg/kg), nitrazepam (0.5 -- 5 mg/kg), phenazepam (0.5 -- 5 mg/kg) and grandaxin (5 mg/kg) facilitated the reaction of self-stimulation (RS), with electrodes implanted in the area of the lateral hypothalamus and medial fascicle of the forebrain. Increased doses of the tranquilizers (up to 10 mg/kg) depressed RS, diminished the searching activity and muscle tone. The tranquilizer elroquil belonging to the phenothiazine series depressed RS in doses 15--35 mg/kg. Diazepam (0.2 and 1 mg/kg), nitrazepam (0.2 and 0.5 mg/kg) and phenazepam (0.2 and 1 mg/kg) reduced the latent period of the reaction of active avoidance (RA) during aversive electric stimulation of the medial lemnisk. In doses of 10 mg/kg the drugs inhibited RA. Elroquil and grandaxin raised the latent period of RA within the dosage range studied. The drug effects and efficacious dose ranges varied among the tranquilizers.


[Removal of the optimizing action of neuroleptics on the avoidance behavior of rats after striatectomy]. In an Y-maze rats easier reformed the trajectory of the behavioral response under the effect of haloperidol (0.05--0.1 mg/kg) and aminazin (0.5--1 mg/kg). A restricted (5--10 per cent of the volume) bilateral electrolytic distruction of the striatum fully suppressed the effect of lower doses of the above compounds both early (7--14th day) and late (28--35th day) after the operation. In large doses the neuroleptics continued to facilitate relearning of the rats in the first 1--2 weeks following striatectomy, but later on this effect also disappeared.


Biochemical and physiological studies of long-term synaptic plasticity. High frequency stimulation of fiber systems in the mammalian hippocampus produces a semipermanent increase in synaptic efficacy. This effect, long-term potentiation (LTP), has been of considerable interest as a potential substrate of memory due to its rapid onset and extreme persistence. Experiments are described that indicate that the locus of LTP is confined to the synaptic complex of the fibers stimulated; further, Ca2+ is shown to be essential for the initiation of LTP and may play a role in triggering this increase in synaptic efficiency. Data from biochemical analyses of LTP indicate that a 40,000 dalton synaptic membrane protein shows a highly reliable change in its endogenous phosphorylation following high frequency hippocampal stimulation. Phosphorylase kinase, a Ca2+ sensitive enzyme, is shown to specifically catalyse the phosphorylation of this 40,000 dalton protein. The data are discussed in terms of a working model in which the Ca2+ dependent phosphorylation of the 40,000 dalton protein produced by high frequency stimulation is a biochemical intermediate in the production of LTP.


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.


Cyclic nucleotides, phosphorylated proteins, and the nervous system. Some postsynaptic effects of several classes of neurotransmitters appear to be mediated or modulated through the cyclic nucleotides, cyclic AMP and cyclic GMP. Available evidence suggests that the molecular mechanism by which the cyclic nucleotides carry out this second messenger role in nerve cells involves regulation of the state of phosphorylation of specific neuronal proteins. Phosphorylated proteins also appear to be involved in mediating certain of the actions of several other classes of regulatory agents, including calcium and the steroid hormones.


Reversible toxic effect of salicylazosulfapyridine on semen quality. The semen qualities of six patients with salicylazosulfapyridine-treated chronic ulcerative colitis are discussed. Over-all poor parameters were observed during the treatment period. Reduced motility and a shift in morphology were the most significant abnormalities in the specimens. A characteristic megalo cell type occurred in the morphology with a frequency of 3% to 8%. In three patients when no medical contraindication existed, discontinuation of the drug resulted in a dramatic improvement in the semen quality, and all three reported pregnancies within the ensuing 3 months. A possible reversible toxic effect of salicylazosulfapyridine on semen quality is discussed.


Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. When unfertilized hamster eggs are placed in highly concentrated solutions of neutral salts (e.g., 2 M ammonium sulfate), the egg proper, or vitellus, shrinks, creating a large perivitelline space; the zona pellucida remains unchanged in appearance under the light microscope. When these eggs are inseminated, many spermatozoa attach to and penetrate the zona. The specificity as well as several physical and chemical characteristics of the zona seem to remain unchanged during storage of the eggs in salt solutions. The properties of the human zona pellucida which allow the attachment and penetration of human spermatozoa are also retained in concentrated salt solutions. Shipment of salt-stored human eggs at ambient temperature to any part of the world is feasible and inexpensive. The present study suggests that salt-stored eggs can be used as substitutes for fresh living eggs in the preliminary assessment of fertilizing capacity of spermatozoa when collection of a large number of fresh unfertilized eggs, particularly in humans, is not practical.


Angiotensin-induced steroidogenesis in rabbit adrenal: effects of pH and calcium. The effect of variations in pH and Ca2+ on angiotensin II (A-II)-induced steroidogenesis was tested on isolated adrenal glomerulosa cell suspensions. The results show that a reduction in pH from 7.4 to 6.5 produces both a shift to the left of the A-II dose-response curve as well as an increase in maximum steroid production. In contrast, removal of Ca2+ from the incubation medium virtually abolished steroidogenesis to A-II (5 X 10(-9)M(, KCl(10mM) and ACTH (250 microU/ml). The Ca2+ antagonist D-600, however, was less effective than simple removal of Ca2+ as 10(-4) M was required to block the steroidogenic response to these same agonists. The results indicate that the response characteristics of this system to A-II resemble most closely those seen with isolated arterial smooth muscle - especially rabbit aortic strips.


The regulation of adenylate cyclase of the adrenal cortex. This short review summarizes some of the data concerning the regulation of adrenocortical adenylate cyclase by ACTH and other putative effectors, such as guanosine and nucleotides, divalent cations and adenosine. The available information on ACTH-sensitive adenylate cyclase of the adrenal cortex is discussed in comparison to other cyclase systems and the possible biochemical mechanisms of action of ACTH on the adrenal cortex.


Stimulation effect of neurotransmitters on the aromatization of testosterone by Sertoli cell-enriched cultures. The aromatization of testosterone has been studied in Sertoli cell-enriched cultures derived from 19-day-old rats. It was found that, besides dbcAMP and FSH, several neurotransmitters have a stimulatory effect. At a concentration of 10(-5) M, the strongest effects were noted with L-isoproterenol, L-norepinephrine, L-epinephrine and D,L-synephrine. Some stimulation was observed with dopamine, whereas histamine, melatonin and serotonin as well as acetylcholine and pilocarpine were ineffective. Experiments with alpha- and beta-adrenergic antagonists suggest that beta 2- and maybe also alpha-receptors are involved. The same maximal level of stimulation was generally observed for dbcAMP, FSH and isoproterenol. No further increase in activity was obtained with combinations of these compounds. It is concluded that adrenergic agonists may play a role in testicular development and function.


Some biochemical characteristics of hormone-secreting pituitary tumors and of the host's anterior pituitary gland. The activity of some glycolytic, oxidative, and degradative enzymes was studied in transplanted rat hormone-secreting pituitary tumors MtTW15 and 7315a and in the host pituitary gland. The elevated serum-hormone concentrations produced by 7315a tumor decreased the size of the host's pituitary gland, its hormone content, and G6P-DH, LDH, PK, and ICDH, but produced no changes in MDH, acid phosphatase, cathepsin-D, and LYSAR enzyme activities (mU/mg tissue). LDH and PK activities were greater in unit weight of pituitary tumors than in pituitary glands. Although more G6P-DH was found in MtTW15 tumor than in normal pituitary tissue, less of the enzyme was detected in 7315a pituitary tumor. It is concluded that elevated serum pituitary hormones selectively decrease hormone production and the activity of some enzymes in the pituitary gland, presumably through a feedback mechanism.


Effect of leucine on the pyridine nucleotide contents of islets and on the insulin released--interactions in vitro with methylene blue, thiol oxidants, and p-chloromercuribenzoate. In the presence of glucose (2 mg/ml), leucine (10 mM) noticeably increased islets' NADPH contents as well as the NADPH:NADP ratio; the changes occurred as soon as 1 min after its addition. NADH concentrations were also increased by leucine. The NADPH:NADP ratio as well as insulin release stimulated by glucose plus leucine were markedly decreased by methylene blue. The thiol oxidants diamide and tert-butyl hydroperoxide also inhibited insulin secretion in response to glucose plus leucine. Employing the perfused pancreas technique, the insulin-releasing action of p-chloromercuribenzoate was further enhanced by leucine. The combined effects were inhibited by tert-butyl hydroperoxide, however. Our data suggest that the insulin-releasing action of leucine depends on the islets' NADPH and reduced glutathione (GSH); in addition, leucine may contribute to insulin secretion by increasing the islet NADPH:NADP ratio and the NADH:NAD ratio. From the data, we assume that the observed increase of NADPH may lead via GSH to an increase in the number of such thiol groups in the beta-cell membrane, which are believed to be related to stimulation of insulin release and, thus, to increase the sensitivity of the beta-cell to stimulation by glucose and/or leucine.


Acid-base disturbance in patients with fulminant hepatic failure. Forty-two arterial blood pH and gas determinations were carried out on 11 patients with fulminant hepatic failure. The most common type of acid-base disturbance was that of respiratory alkalosis in 22 cases (52.4%). This was partially compensated in 13 subjects (31.0%) while an accompanying metabolic alkalosis was present in 9 (21.5%). Partially compensated metabolic acidosis was observed on 15 occasions (35.7%), all of which were in patients with laboratory evidence of impaired renal failure. The mental status of the patients was evaluated in each of the categories of acid-base disturbances. Some degree of correlation was evident between the PCO2 and the magnitude of base excess and that of the severity of the encephalopathy. The lower PCO2 and greater negative base excess values tended to be nearly always present in totally comatose subjects. By contrast, there was no clear cut relationship between pH and mental state.


Are epinephrine and gastrin accelerative factors of acute cinchophen ulcer?: Studies on gastric mucosal microcirculation and gastric secretion. Measurements of serum epinephrine and gastrin, simultaneously, gastric mucosal blood flow and gastric secretion were carried out in cinchophen treated dogs. No significant changes in either serum concentrations of epinephrine and gastrin or fundic mucosal microcirculation after a single 100 mg/kg cinchophen administration were found while gastric secretion increased markedly after the medication. On the other hand, a significant increase in serum epinephrine and gastrin levels was observed while gastric secretion decreased significantly after large doses of cinchophen (300 mg/kg) were injected intravenously. Here gastric mucosal microcirculation is decreased. Repeated administration of 100 mg/kg cinchophen for 3 to 7 days brought about an increase in epinephrine and gastrin levels and caused an occurrence of fundic mucosal hemorrhage. Sympathetic discharge and gastrin release were not seen after a 3-week period of cinchophen administration. Cinchophen ulcers were produced, even when contact between the bile and the stomach mucosa was avoided. Vagotomy had no connection with ulceration and gastric secretion.


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.


Small bowel absorption of sulfasalazine and its hepatic metabolism in human beings, cats, and rats. To elucidate the role of the small bowel and liver in sulfasalazine (SASP) metabolism, we performed studies in patients, cats, and rats. The role of the small bowel in absorption and metabolism of SASP was determined by the amount of administered SASP excreted in ileostomy effluents, and the concentration of serum and urinary SASP and its metabolites, sulfapyridine and 5-amino salicylic acid. Seventy-five to ninety percent of the drug was excreted in ileostomy effluents of 6 patients as SASP, and only 5% of the dose was sulfapyridine. In cats, ileostomy and portal venous cannulations revealed that 20--30% of administered SASP is absorbed from the small bowel without being metabolized. The role of the liver in SASP metabolism was established in vivo and in vitro. SASP metabolites were measured in bile, serum, and urine of 2 patients with a choledochal T-tube and in serum and bile in six cats and four rats. Twenty to fifty percent of the absorbed drug was excreted in bile as SASP and no detectable sulfapyridine appeared in the bile. SASP concentration in peripheral blood and urine ranged between 3 and 12 microgram/ml, and no significant amount of sulfapyridine metabolites were detected in the bile, serum, or urine of animals with an ileostomy. In vitro experiments with liver from cats and rats showed nearly complete reduction of SASP into metabolites. These studies reveal that SASP is not metabolized by the liver in vivo, and after absorption from the small bowel, 25--50% of the SASP is excreted unchanged in the bile, and the rest enters the circulation. Implications of these results in the treatment of patients with Crohn's disease of the small intestine are discussed.


Protection by histamine receptor antagonists and prostaglandin against gastric mucosal barrier disruption in the rat. This study was undertaken to determine if the cytoprotective effect of prostaglandin and the H2 histamine receptor antagonist cimetidine involves protection against disruption of the gastric mucosal barrier. Groups of anesthetized, vagotomized rats received one of the following parenterally: saline (control), mepyramine--an H1 histamine receptor antagonist, cimetidine, cimetidine and mepyramine, or 16,16 dimethyl prostaglandin E2. Parameters of barrier disruption were then determined before and after exposure of the gastric mucosa to 40mM acetylsalicylic acid. At the end of the study, gastric lesions were scored according to size and number. Lesion score and fall in potential difference were significantly lower in rats receiving cimetidine, cimetidine and mepyramine, and prostaglandin. Other parameters of barrier disruption--H+ back diffusion, Na+ and K+ influx, and protein outpouring--exhibited the same pattern and correlated with change in potential difference. We conclude that both prostaglandin and cimetidine, but not mepyramine, protect against barrier disruption by topical aspirin, and this may be a factor in the mechanism of their cytoprotective action.


How to minimize side effects of psychotropic drugs. Aging has a direct effect on drug absorption, distribution, and elimination. With aging, there is an alteration in the neurotransmitters whereby dopaminergic and catecholaminergic systems are relatively depleted of monoamines while acetylcholine is largely unaffected. Pharmacotherapy in the elderly requires an understanding of the physiologic effects of medications. Dosage generally should be lower for the elderly patient on the basis of a milligram per kilogram of body weight, but determining the appropriate dosage required more than allowing for "smaller body mass" or "less body water." Because of lengthened half-life and slowed distribution of medications, it is necessary to start with very low dosages and observe the patient for relatively longer periods of time before giving another dose, increasing the medication, or adding another drug.


Neuroleptic drugs: how to reduce the risk of tardive dyskinesia. Prescribing neuroleptic drugs to control disruptive behavior in geriatric patients exposes them to the risk of tardive dyskinesia. Neuroleptics must be administered in the lowest effective dose for the shortest possible period, and should be given only for specific indications, usually psychotic disorders.


Influence of intestinal transit time on azo-reduction of salicylazosulphapyridine (Salazopyrin). During a normal and an accelerated intestinal transit, in seven healthy volunteers, the recoveries of salicylazosulphapyridine (SASP) and its split products sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) were determined in urine and faeces. The azo-reduction of SASP and consequently the recovery of 5-ASA in the faeces was found to be substantially decreased during an accelerated intestinal transit. In addition, in 18 patients with inflammatory disease of the colon during maintenance therapy of SASP it could be demonstrated that the serum SP levels were related to the diarrhoeal state and did not correlate with disease activity. As recent studies have reported that 5-ASA is possibly the active therapeutic moiety of SASP, the ineffectiveness of SASP therapy in patients with active colitis may be ascribed to the reduced azo reduction of SASP as the result of profuse diarrhoea.


Experimental transmission of Tahyna virus (California group) to wild rabbits (Oryctolagus cuniculus) by mosquitoes. Two wild rabbits (Oryctolagus cuniculus) were subcutaneously infected with a dose of 2.6 dex. i.c. LD50/0.02 ml of extraneurally passaged Tahyna virus strain "236". During the 24-72 hours interval p.i. viremia was demonstrated in both animals ranging from 1.0 to 3.42 dex. i.e. LD50/0.02. Mosquitoes Aedes vexans, which had fed on them, transmitted the virus to one of three other rabbits. During 24-96 hours interval after terminated feeding of infectious mosquitoes viremia was detected in this animal ranging from 0.49 to 4.08 dex i.c. LD50/0.02 ml. Seroconversion was revealed by means of plaque reduction neutralization test 11 days after infection.


Effects of drugs on brain neurotransmitter and pituitary-testicular function in male rats. The effects of different drugs influencing brain neurotransmitter contents have been tested on the pituitary-testicular function in male rats. L-dopa (200 mg/kg body weight, i.p.) increased the dopamine and noradrenaline contents of the hypothalamus, amygdala, striatum and mesencephalon, but it was ineffective as regards the 5-hydroxytryptamine contents of the same brain areas, and increased the plasma testosterone level. alpha-Methyl-p-tyrosine (250 mg/kg b.w., i.p.) decreased the dopamine and noradrenaline contents of these brain areas, but it was ineffective to 5-hydroxytryptamine, and decreased the plasma testosterone level. Diethyldithiocarbamate (400 mg/kg b.w., i.p. twice a day) increased the dopamine levels in the hypothalamus, amygdala, striatum and mesencephalon, decreased the noradrenaline contents in the same brain regions but had no effect on the 5-hydroxytryptamine contents of these brain areas or on the testosterone level in the peripheral blood. p-Chlorophenylalanine (300 mg/kg b.w., i.p.) decreased the 5-hydroxytryptamine contents of the different brain areas, while it had no effect on the dopamine and noradrenaline levels or on the plasma testosterone level. 5-Hydroxytryptophan (200 mg/kg b.w., i.p.) increased the 5-hydroxytryptamine contents of all brain areas studied, but was without effect on the dopamine and noradrenaline contents or the plasma testosterone level. The data suggest that both dopamine and noradrenaline may be involved in the regulation of the pituitary-testicular function, and the ratio of the two transmitters might be more important that their actual levels in definite brain areas.


The board-and-care home: does it deserve a bad press? A survey of 46 randomly selected schizophrenic residents of six board-and-care homes in the Los Angeles area found that the schizophrenic who adjusts to the setting experiences a schizoid-compliant pattern of outcome on antipsychotic drugs that is characterized by blunted affect, passivity, and lack of initiative, interest, and spontaneity. The authors conclude that it is those negative symptoms of schizophrenia, mistakenly attributed to the presumed inadequacies of the board-and-care environment, that have given the board-and-care home a bad press both in the newspapers and in the psychiatric literature.


Tardive dyskinesia and the long-term patient. A psychiatric patient's long-term use of antipsychotic medication often results in the irreversible movement disorder, tardive dyskinesia. The author uses a composite case history as a basis for discussing the symptoms, diagnosis, epidemiology, and treatment of tardive dyskinesia. A number of drugs have been used to treat the disorder, but so far none have been effective. While tardive dyskinesia cannot be cured at this time, the author believes that it can be prevented by treating psychoses with the lowest possible dose of the least toxic drug for the shortest length of time.


Innovators gather at conference.

Innovators gather at conference. The AHA's Center for Health Promotion invited innovators in the field of community health education programming to exchange ideas and learn from expert consultants.


Meeting examines productivity.

Meeting examines productivity. The 12th annual meeting of ASHFSA focused on means of increasing productivity, staffing analysis, and use of management by objectives/Ethical considerations also were discussed.


Enhancement of IgE antibody formation in the rabbit by adrenergic antagonists. The effects of adrenergic antagonists on the humoral immune response of rabbits to egg albumin was studied. Propranolol, a beta-adrenergic antagonist, when administered at a dosage of 20 mg/kg of body weight, enhanced both passive hemagglutinating and IgE (passive cutaneous anaphylaxis) antibody formation. Phenoxybenzamine, an alpha-antagonist, at 0.4--40 mg/kg had a similar adjuvant effect. A marked delay in the immune response was noted with high dosages of phenoxybenzamine. Possible mechanisms of the adjuvant effects of these drugs are discussed.


Studies on competitive inflammatory sites. I. Influence of the size of sensitizing skin implants on second-set corneal graft reactions. A dependence of second-set corneal graft reactions on the size of the sensitizing skin allografts was established in different rat donor-recipient pairs, each of which involved a response to the B2 major histocompatibility antigen. Sensitizing skin sizes larger or smaller than the optimal size were found to result in less intense reaction in subsequently placed second-set corneal allografts. The data obtained support the view that a persisting skin allograft not only sensitizes, but constitutes a competitive inflammatory site. The latter role of a first-set skin allograft may be facilitated by being both more accessible to inflammatory cells and larger than corneal grafts. The relative strength of the sensitizing and competitive effects of a first-set skin allograft apparently influences the fate of a second-set corneal allograft.


Effect of complement inactivated rabbit antichick brain serum on thymic and splenic lymphocytes. The effects on in vitro exposure of chicken thymic-dependent lymphocytes to complement inactivated rabbit antichick brain serum (RACB) previously absorbed with normal serum (ns) and kidney (k) [RACBns,k] was investigated. Spleen lymphocytes which normally initiate a 'graft vs. host' reaction give rise to much more intense reactions when treated with (RACB)ns,k. This may be due to increased antigenicity of the donor cell and/or stimulation of the donor T cell. Migration studies showed that chicken thymocytes are capable of in vitro migration which can be inhibited by exposure to (RACB)ns,k. Thymocytes pretreated with (RACB)ns,k were found to express factor(s) capable of inhibiting thymocyte migration. The correlation between lymphokines and increased splenomegaly was discussed.


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.


Role of alkaline endonucleases in the release of soluble chromatin from thymus, spleen and liver nuclei of normal and irradiated mice. Thymus, spleen and liver nuclei released a large fraction of soluble chromatin in vitro when incubation was carried out in sucrose media containing low concentrations of CaCl2 and/or MgCl2. A significant fraction of deoxyribopolynucleotides (DPN) was also extracted from nuclei. After 30 min of incubation at 37 degrees C, the maximum release of soluble chromatin was observed near a pH of 8, which corresponds to the optimum pH of the alkaline endonuclease activity from thymus, spleen and liver. The soluble chromatin and DPN were precipitated by increasing the bivalent ion concentration of the medium. The protein/DNA ratio and the molecular weight of DNA suggest that the soluble chromatin and DPN represent nucleosome-like particles. The release of soluble chromatin in the first 4 hours of incubation was significantly increased if the nuclear fraction was isolated from the thymus and spleen of whole-body irradiated mice (1000 rad). This effect was absent in the liver nuclei.


Dibenzepin as an antifibrillatory agent for spontaneously terminating electrically induced ventricular fibrillation. The effect of dibenzepin, a tricyclic antidepressant drug, on electrically induced ventricular fibrillation was studied in 20 cats and nine dogs. The ventricular fibrillation threshold and the ability of the ventricle to defibrillate spontaneously were determined before and after the administration of dibenzepin, with each animal serving as its own control. The drug raised the ventricular fibrillation threshold in all the animals tested. Before treatment, spontaneous ventricular defibrillation occurred in only 8 of the 20 cats and in none of the dogs. After treatment, all of the cats and eight of the dogs exhibited spontaneous ventricular defibrillation. The study of drugs that have a self-defibrillatory effect may serve to further understanding of the mechanism of ventricular fibrillation and its spontaneous termination.


Assessment of clinical status of schizophrenic patients by the WAIS comprehension subtest. A previous study indicated that the Comprehension subtest of the Wechsler Adult Intelligence Scale (WAIS) might be a sensitive instrument for evaluating mental status in schizophrenic patients. To test this hypothesis, 3 groups of subjects were given the WAIS Comprehension subtest repeatedly at brief intervals. The results indicate that for a given individual (normal control or stable chronic schizophrenic), scores do not increase even with 4 consecutive weeks of testing. In contrast, a group of schizophrenic patients tested shortly after readmission and again after being established on neuroleptic medication showed a marked improvement in scores which, somewhat surprisingly, were not correlated with changes in global ratings of disordered thinking by the treating psychiatrist. This standardized test may offer a convenient objective technique for quantifying some aspects of thinking in schizophrenic patients.


Urinary incontinence in patients receiving neuroleptics. This paper presents a series of case reports making the observation of a possible association between neuroleptic drug therapy and urinary incontinence. The incontinence was limited and not of the overflow or stress variety. Anticholinergic compounds or drug action did not seem to influence or account for the findings. A central basis is postulated for consideration and warrants further study.


Purification and characterization of rat pepsinogens whose contents increase with developmental progress. Two pepsinogens, the contents of which increase with developmental progress, were purified from the gastric mucosa of the adult rat by ammonium sulfate fractionation and chromatography on DEAE-cellulose and DEAE-Sepharose CL-6B columns. The purified zymogens, designated as pepsinogens I and II, were each shown to be homogeneous by polyacrylamide gel disc electrophoresis. Pepsinogen II had a greater electrophoretic mobility toward the anode at pH 8.0 than pepsinogen I. The molecular weights of both zymogens were estimated to be 38,000 by SDS-polyacrylamide gel electrophoresis. The activated enzymes, pepsins I and II, each had the same molecular weight of 32,000. The pH optima for both enzymes were found to be 2.0. The enzymes showed high stabilities at pH 8.0, while they lost their activities within 60 min at pH 10.0. The enzymes were inhibited by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN). The activities of the enzymes in hydrolyzing N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine (APDT) were about 1/8 of that of porcine pepsin. These results suggest that pepsins I and II are very similar.


Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin. Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.


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.


Latent proteinase activity of gamma-glutamyl transpeptidase light subunit. gamma-Glutamyl transpeptidase, which is composed of two unequal subunits, exhibits proteinase activity when treated with agents such as urea and sodium dodecyl sulfate. The heavy subunit is preferentially and rapidly degraded. The enzyme also degraded bovine serum albumin in the presence of urea; however, several other proteins and model proteinase substrates were not cleaved. Treatment of the enzyme with 6-diazo-5-oxo-L-norleucine, a gamma-glutamyl analog, results in parallel loss of transpeptidase and proteinase activities indicating that the site at which gamma-glutamylation of the enzyme occurs (presumably a hydroxyl group on the light subunit) is also involved in proteinase activity. The purified light subunit, but not the heavy subunit, exhibits proteinase activity even in the absence of urea. Results suggest that dissociation of the enzyme unmasks the proteinase activity of the light subunit involving the site at which gamma-glutamylation of the enzyme occurs, and that the heavy subunit may impose transpeptidase reaction specificity by contributing the binding domains for gamma-glutamyl substrates.


The metabolic fate of 13N-labeled ammonia in rat brain. 13N-labeled ammonia was used to study the cerebral uptake and metabolism of ammonia in conscious rats. After infusion of physiological concentrations of [13N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (alpha-amino), and glutamine (amide) in brain were approximately 1:5:400, respectively. The data are consistent with the concept that ammonia, entering the brain from the blood, is metabolized in a small pool of glutamate that is both rapidly turning over and distinct from a larger tissue glutamate pool (Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H. (1962) J. Biol. Chem. 237, 2562-2569). Analysis of 13N-metabolites, after infusion of [13N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment of rats with methionine sulfoximine led to a decrease in the label present in brain glutamine (amide) following carotid artery infusion of [13N]ammonia. On the other hand, 13N activity in brain glutamate was greater than that in the alpha-amino group of glutamine, i.e. following methionine sulfoximine treatment the expected precursor-product relationship was observed, indicating that the two pools of glutamate in the brain were no longer metabolically distinct. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [13N]ammonia via the right common carotid artery, was found to be independent of ammonia concentration within the bolus over a 1000-fold range. This finding indicates that ammonia enters the brain from the blood largely by diffusion. In normal rats that were killed by a freeze-blowing technique 5 s after injection of an [13N]ammonia bolus, approximately 60% of the label recovered in brain had already been incorporated into glutamine, indicating that the t1/2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered.


Stereospecificity of sodium borohydride reduction of tyrosine decarboxylase from Streptococcus faecalis. Sodium boro[3H]hydride reduction of tyrosine decarboxylase from Streptococcus faecalis followed by complete hydrolysis of the enzyme produces epsilon-[3H]pyridoxyllysine. Degradation of this material to [4'-3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase shows that the re side at C-4' of the cofactor is exposed to solvent at pH 5.5 and 7.0. After binding of L-tyrosine at pH 5.5 or tyramine at pH 7.0 to the holoenzyme, sodium boro[3H]hydride reduction proceeds from the si face at C-4' of the substrate . cofactor complex. This indicates one of two conformational changes occurs upon binding of substrate; either rotation about the C-4 to C-4' bond in the cofactor or rotation about the axis through the C-5 and C-5' bond.


Metal-free southern bean mosaic virus crystals. Native southern bean mosaic virus contains a significant number of Mg2+ and Ca2+ ions. These can be removed by treatment with EDTA causing the virus to swell by 7% in radius at alkaline pH values. The swollen virions are susceptible to protease and nuclease digestion. They are likely to be an intermediate during assembly and disassembly. Crystals of the metal-free virus have been grown and were found to be approximately isomorphous with the orthorhombic type III southern bean mosaic virus crystals (Akimoto, T., Wagner, M.A., Johnson, J.E., and Rossmann, M.G. (1975) J. Ultrastruct. Res. 53, 306-318), although the cell dimensions are longer by 2%. Native rhombohedral type II crystals disintegrate on changing the pH or increasing the ionic strength of the mother liquor. Damage can be prevented by addition of ethylene glycol. At alkaline pH values, these crystals also show a 2% increase in their cell dimensions as well as a significant alteration in their diffraction patterns. In the type II and III crystals, the viruses pack with only their 5-fold axes in contact. Thus, the difference of the apparent swelling in solution and in the crystals may be one of differential swelling over the virus surface.


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).


Specific perturbation by Ca2+ of tyrosyl residue 138 of calmodulin. Calmodulin that was selectively nitrated at tyrosyl residue 99 or 138 was used as a regional spectral probe of the structural changes associated with Ca2+ or Mg2+ binding to the protein. Perturbations of the spectral properties of the nitrotyrosyl residues correlated well with those of the tyrosyl residues in unmodified calmodulin. The spectral properties of tyrosyl residue 99 were sensitive to ionic strength, but not to Ca2+ binding to the protein. The spectrum of tyrosyl residue 138, in contrast, was markedly affected both by ionic strength and Ca2+ binding. Although Mg2+ caused spectral changes, they were clearly different from those produced by Ca2+ and resembled instead the less specific changes elicited by Na+. Conformational changes associated with Ca2+ but not Mg2+ binding to calmodulin seem to affect selectively the microenvironment around tyrosyl residue 138.


Conversion of glutathione to glutathione disulfide, a catalytic function of gamma-glutamyl transpeptidase. A purification procedure, based on that previously used for rat kidney gamma-glutamyl transpeptidase, was used for the purification of glutathione oxidase (which converts glutathione to gluthathione disulfide). The two activities co-purified, the ratio of the activities remaining constant through all steps of the isolation procedure. The purified enzyme was separable into 12 isozymic species by isoelectric focusing. All 12 isozymes exhibited a constant ratio of transpeptidase to glutathione oxidase activities, strongly supporting the conclusion that conversion of glutathione to glutathione disulfide is a catalytic function of gamma-glutamyl transpeptidase. Modulation of oxidase activity by inhibitors and acceptor substrates of transpeptidase is discussed in relation to the possible glutathione binding sites involved in gamma-glutamyl transfer and oxidase activities of the enzyme.


The mechanism of NADPH-dependent lipid peroxidation. The propagation of lipid peroxidation. NADPH-dependent lipid peroxidation occurs in two distinct sequential radical steps. The first step, initiation, is the ADP-perferryl ion-catalyzed formation of low levels of lipid hydroperoxides. The second step, propagation, is the iron-catalyzed breakdown of lipid hydroperoxides formed during initiation generating reactive intermediates and products characteristic of lipid peroxidation. Propagation results in the rapid formation of thiobarbituric acid-reactive material and lipid hydroperoxides. Propagation can be catalyzed by ethylenediamine tetraacetate-chelated ferrous ion, diethylenetriamine pentaacetic acid-chelated ferrous ion, or by ferric cytochrome P-450. However, cytochrome P-450 is destroyed during propagation.


Acid ribonuclease from HeLa cell lysosomes. An acid ribonuclease has been purified from HeLa cell lysosomes. The specific activity of the RNase in lysosomes is 8-fold higher than that in nuclei and 15-fold higher than that in the postlysosomal fraction. The purified enzyme showed no detectable DNase, phosphodiesterase, phosphatase, or alkaline RNase activity. The acid RNase binds to Con A-agarose and is inferred to be a glycoprotein. It has a low isoelectric point at pH 3.0 to 3.5, and the optimal pH for activity is between 5.0 and 5.5. The enzyme requires no divalent cation for optimal activity and is totally inhibited by 1 mM Cu2+ or Hg2+. Monovalent cations including Na+, K+, and NH4+ stimulate the activity in low ionic strength buffer. The enzyme degrades rRNA faster than tRNA, and tRNA faster than poly(U); poly(A) and poly(C) are highly resistant. The products from rRNA are mostly oligonucleotides with 3'-phosphate ends. An acid RNase is also present in the lysosomes of L-cells grown in a medium free of serum; it is probably identical to the one described here.


Degradation of bis(monoacylglycero)phosphate by an acid phosphodiesterase in rat liver lysosomes. Bis(monoacylglycero)phosphate was purified from the livers of chloroquine-treated rats and labeled with tritium by a nonreductive catalytic exchange procedure. The mechanism of its degradation by rat liver lysosomes has been examined. A substantial amount of bis(monoacylglycero)P is degraded to monoglyceride and lysophosphatidic acid by a lysosomal phosphodiesterase having an acid pH optimum. Some bis(monoacylglycero)P is degraded to lysophosphatidylglycerol by lysosomal phospholipase A. In contrast, other phosphoglycerides have been reported to be degraded by sequential deacylation in lysosomes. The initial rate of breakdown of bis(monoacylglycero)P is only 10% of the rate observed for dioleoylphosphatidylcholine. [3H]Lysophosphatidylglycerol conversion to [3H]bis(monoacylglycero)P is stimulated by unlabeled bis(monoacylglycero)P, resulting in a futile cycle which allows the resynthesis of bis(monoacylglycero)P from its breakdown product, lysophosphatidylglycerol. This futile cycle and the unusual sn-1-glycerophospho-sn-1'-glycerol stereoconfiguration of the water-soluble backbone (Joutti, A., Brotherus, J., Renkonen, O., Laine, R., and Fischer, W. (1976) Biochim. Biophys. Acta 450, 206-209) may be important factors in the marked resistance of bis(monoacylglycero)P to degradation by lysosomal acid hydrolases.


Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport. Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.


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.


Ultraviolet and laser Raman investigation of the buried tyrosines in fd phage. The tyrosines of the filamentous phage fd have been found to be inaccessible to solvent by pH titration while monitoring the ultraviolet spectrum or the laser Raman spectrum. The uv spectra suggest that the tyrosines do not become deprotonated unless the phage becomes disrupted. One possible explanation of the Raman spectra is that the tyrosine OH groups are the recipients of hydrogen-bonded protons arising from fairly acidic donors, yet these acidic donors do not become titrated over the pH 7 to 12 range.


The liver microsomal FAD-containing monooxygenase. Spectral characterization and kinetic studies. A FAD-containing monooxygenase isolated from pig liver microsomes migrates as a single band upon electrophoresis in polyacrylamide gels in the presence of dodecyl sulfate. The minimum molecular weight based on mass of amino acids per mole of flavin is 64,000. However, the catalytically active enzyme exists as aggregating units of the monomer. Neither oxygen nor organic substrates perturbed the spectrum of the oxidized flavoprotein and their binding to this form of the enzyme could not be detected. Anaerobically NADPH bleaches the flavoprotein, and in the presence of both NADPH and oxygen a remarkably stable intermediate form of the enzyme, with an absorption band at 375 nm, is observed. The spectrum of the intermediate resembles that of a peroxyflavin. The monooxygenase catalyzes NADPH- and oxygen-dependent oxygenations of nucleophilic nitrogen- or sulfur-containing compounds. Kinetic studies carried out with a model organic nitrogen substrate (trimethylamine) and a sulfur substrate (methimazole) gave similar patterns. The kinetic data are consistent with an ordered Ter-Bi mechanism with an irreversible step between the second and third substrate where NADPH is added first, followed by oxygen, and the oxidizable organic substrate is added last. If NADPH is the first substrate added, then NADP+ must be the last product released since NADP+ is competitive with NADPH.


Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions. The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.


Cytosolic phosphorylation potential.

Cytosolic phosphorylation potential. The tissue contents of the reactants of the myokinase (EC 2.7.4.3) and the combined glyceraldehyde-3-phophate dehydrogenase (EC 1.1.1.29)-3-phosphoglycerate kinase (EC 2.7.2.3) reactions were measured in rapidly inactivated samples of human blood and rat brain, muscle, and liver. The tissue contents of the reactants of the creatine kinase (EC 2.7.3.2) reaction were measured in rat brain and muscle. In vitro the value of the expression: KG+G = [sigma3PG] . [sigmaATP] . [sigmalactate] KLDH = [sigmaHAP]/22] . [sigmaADP][sigmaPi] . [sigmaRUVATE] (1) was found to be 0.725 x 10(7) M-1 at I = 0.25, T = 38 degrees C, and free [Mg2+] = 0.15 mM and the value measured in vivo in red cell was 0.699 x 10(7) M-1. The value of the expression KMYK = ([sigma ATP] [sigma AMP]/[ADP2]) measured under the above conditions and at pH 7.2 was found to be 0.744 while the value found in red cell was 0.784 +/- 0.037. These reactions, therefore, appear to be in a state of near-equilibrium in the red cell and the measured tissue contents of ATP and ADP, which are common reactants in both reactions, approximate closely the activity of these reactants in vivo. In brain and muscle, the value of KG + G/KLDH calculated from the measured tissue contents of the reactants was a factor of 20 or more lower than that expected at equilibrium as was the measured value of the expression: KCK = [sigma ATP] [sigma creatine] divided by [sigma ADP] [sigma creatine-P] [H+] (2) Substitution of calculated free [sigma ADP] values in the expression of KG + G/KLDH gave values of 0.83  +/- 0.19 x 10(7) M-1 for brain and muscle, respectively, which agreed well with the value of 1.65 x 10(7) M-1 measured in vitro at I = 0.25, free [Mg2+] = 1 mM, T = 38 degrees C. This agreement between two highly active enzyme systems in the same compartment is taken as evidence of the existence of near-equilibrium in both these systems and suggests that free cytosolic [sigma ADP] is probably 20-fold lower than measured cell ADP content in mitochondrial-containing tissues.


Chick embryo liver beta-glucuronidase. Comparison of activity on natural and artificial substrates. The beta-glucuronidase in homogenates of 12-day chick embryo livers catalyzed the release of glucuronic acid from 4-methylumbelliferyl-beta-D-glucuronide and from the nonreducing terminals of the hexasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4 at rates of 143, 114, and 108 nmol of glucuronic acid/h/mg of protein, respectively, when assayed at pH 3.5 in 0.05 M sodium acetate buffer. During a 60-fold purification of the enzyme, the ratios of the activities on these substrates did not change. When 4-methylumbelliferyl-beta-D-glucuronide was used as substrate the enzyme was active at pH values from 3.0 to 5.5, with maximal activity between pH values 4.0 and 4.5. Concentrations of NaCl from 0.15 to 0.3 M inhibited the activity at low pH values but activated the enzyme between pH 4.0 and 5.5. The enzyme was active on the chondroitin-6-SO4 hexasaccharide from pH 3.0 to 5.5, with a broad optimum between 3.0 and 4.5. NaCl inhibited the activity on the oligosaccharide substrate at all pH values. Eadie-Scatchard plots of rates of 4-methylumbelliferyl-beta-D-glucuronide hydrolysis at substrate concentrations ranging from 2 to 1000 microM showed multiple kinetic forms of the enzyme, a form with a Km of approximately 11 microM, and a second form with a Km of approximately 225 microM. The pH optimum of the low Km form was 3.5 to 4.0; that of the high Km form was pH 4.5. NaCl inhibited the activity of the low Km form, but activated the high Km form of the enzyme. Chondroitin SO4 oligosaccharides competed with 4-methylumbelliferyl-beta-D-glucuronide for the low Km form of the enzyme but had little effect on the hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide by the high Km form of the enzyme. The activities of the beta-glucuronidase on tetra-, hexa-, octa-, and decasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4, measured using a new assay procedure which can detect the formation of 1 nmol of product, were similar, although rates were somewhat lower for the higher oligosaccharides. With the exception of the chondroitin-4-SO4 tetrasaccharide, all of the oligosaccharide substrates saturated the enzyme at concentrations of 20 to 30 microM, indicating Km values of less than 10 to 15 microM for the oligosaccharides. Highly purified beta-glcuronidases from human placenta and from rat preputial gland also showed multiple kinetic forms when assayed using 4-methylumbelliferyl-beta-D-glucuronide as substrate.


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.


In vitro stimulation of adenosine 3',5'-monophosphate in unilateral undescended testes of humans by follicle-stimulating hormone and luteinizing hormone. The in vitro effects of FSH and LH on cAMP generation by unilateral undescended testes and contralateral descended testes were investigated. Biopsy tissue was preincubated at 37 C for 5 min in 1 ml Krebs-Ringer bicarbonate buffer (KRB) containing 10 mM aminophylline. Half of each specimen was frozen in liquid nitrogen to assess basal levels of cAMP. The other half was incubated for 20 min at 37 C in 1 ml fresh KRB containing aminophylline and FSH (10 microgram) or LH (10 microgram). The mean concentration of cAMP in the descended testes was 1.03 +/- 0.04 pmol/mg protein, whereas in the undescended testes the level was 1.98 +/- 0.07 pmol/mg protein (P less than or equal to 0.01). After the 20-min incubation period with FSH the concentrations of cAMP in the descended and undescended testes were 10.85 +/- 0.86 pmol/mg protein and 11.21 +/- 1.1 mol/mg protein, respectively. Incubation with LH resulted in the generation of equivalent levels of cAMP in both the descended (2.76 +/- 0.30 pmol/mg protein) and undescended (3.0 +/- 0.17 pmol/mg protein) testes. Although marked histological differences were observed in the germinal epithelium, the appearance and numbers of Leydig and Sertoli cells were comparable. The data suggest that the Sertoli cell is the primary receptor for FSH. Leydig and Sertoli cell functions of the undescended testes, at least in terms of cAMP generation, are similar to normal descended testes.


Regulation of the interaction of purified human erythrocyte AMP deaminase and the human erythrocyte membrane. The binding of purified human erythrocyte AMP deaminase to human erythrocyte membranes and the effect of binding on enzyme catalytic activity was investigated. AMP deaminase binds preferentially and specifically to the cytoplasmic surface of the erythrocyte membrane. The binding is saturable, reversible, and responsive to alterations of pH, of ionic strength, and of ATP and AMP concentrations. A limited number (approximately equal to 2.2 X 10(4) per erythrocyte) of apparently homogeneous high affinity (Ka approximately equal to 2.6 X 10(7) M-1) binding sites is present. The stability of purified and endogenously bound AMP deaminase is markedly improved by the interaction with the membrane, whereas the catalytic activity of AMP deaminase is sharply reduced. AMP deaminase displaces membrane bound glyceraldehyde 3-phosphate dehydrogenase in roughly a dose-response manner. No evidence for binding of AMP deaminase to spectrin or band 3 (the G3PD binding protein) was found in sucrose gradients, however. The interaction of AMP deaminase with the erythrocyte membrane may play an important role in the regulation of cellular adenine nucleotide metabolism.


An extrarenal role for parathyroid hormone in the disposal of acute acid loads in rats and dogs. Acid infusion studies were performed in nephrectomized rats and dogs with either intact parathyroid glands (intact) or after thyroparathyroidectomy (thyroparathyroidectomized [TPTX]) to determine the role of parathyroid hormone (PTH) in extrarenal disposal and buffering of acutely administered acid. 29 intact rats given 5 mM/kg HCl and 6 intact dogs given 7 mM/kg HCl developed severe metabolic acidosis but all survived. However, each of 12 TPTX rats and 4 TPTX dogs given the same acid loads died. Intact rats and dogs buffered 39 and 50% of administered acid extracellularly, respectively, whereas extracellular buffering of administered acid was 97 and 78% in TPTX rats and dogs, respectively. 17 TPTX rats and 6 TPTX dogs given synthetic PTH 2 h before acid infusion survived. The blood bicarbonate and extracellular buffering in these animals, measured 2 h after acid infusion, was similar to intact animals. Changes in liver, heart, and skeletal muscle pH determined from [(14)C]5,5-dimethyl-2,4 oxazolidinedione distribution seemed insufficient to account for the increased cell buffering of PTH-replaced animals. Indeed, muscle pH in TPTX dogs given PTH and acid was only 0.06 pH units lower than in control dogs given no acid, suggesting that another tissue, presumably bone, was the target for PTH-mediated increased cell buffering. This conclusion was supported by the observation that PTH did not alter the pH of intact rat diaphragms in vitro. These results indicate that PTH is necessary for the optimal buffering of large, acute acid loads presumably by increasing bone buffering.


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.


Observations on the factors that control the generation of triiodothyronine from thyroxine in rat liver and the nature of the defect induced by fasting. Studies were performed to explore the mechanism underlying the impaired generation of 125-I-3,5,3'-triiodothyronine (T3) from 125I-thyroxine (T4) (T3-neogenesis)) in preparations of liver from rats fasted for 48 h and the prevention of this effect by the feeding of glucose. T3-neogenesis in livers from fasted animals and those fed chow or glucose was assessed in various mixtures of crude microsomal fractions with either buffer or cytosols. T3-neogenesis was mediated by an enzyme present in the microsomal fraction whose activity was enhanced by cytosolic cofactor(s). In livers from animals fasted for 48 h, the supporting activity of cytosol was decreased, whereas the activity of the enzyme was unaffected. Administration of glucose as the sole nutritional source prevented the decrease in the supporting activity of hepatic cytosol that was regularly observed in the case of animals totally deprived of food. The diminished supporting activity for T3-neogenesis provided by liver cytosol from fasted animals was restored to normal by enrichment with either NADPH or GSH, but the two cofactors appeared to act at different loci. GSH stimulated T3-neogenesis in microsomes incubated in the absence of cytosol, i.e., in buffer, whereas NADPH did not. The stimulatory effect of both agents was blocked by the sulfhydryl oxidant, diamide, which also inhibited T3-neogenesis in mixtures of microsomes with cytosols. Taken together, these observations suggest that GSH acts directly on the enzyme in the crude microsomal fraction, whereas NADPH acts within the cytosol, possibly by increasing the concentration of GSH through the action of the enzyme glutathione reductase, for which NADPH is a cofactor. In this light, the decreased supporting activity of hepatic cytosol from starved animals appears to reflect, at least partly, a decreased concentration of one or both cofactors. The direct stimulation of enzyme activity by GSH, and the apparent lack of inhibition of unstimulated activity by diamide, suggests that the 5'-monodeiodinase for thyroxine that mediates T3-neogenesis may be a GSH transhydrogenase.


Beta-adrenergic potentiation of the increased in vitro accumulation of cycloleucine by rat thymocytes induced by triiodothyronine. We have previously demonstrated that 3,5,3'-triiodothyronine (T(3)), whether administered in vivo or added to suspending media in vitro, promptly stimulates the in vitro accumulation of the nonmetabolized amino acids, alpha-aminoisobutyric acid, and cycloleucine (CLE) by thymocytes isolated from weanling rats. In these studies, we have examined the in vitro interaction between catecholamines and T(3) with respect to this effect. The previously reported enhancement of CLE accumulation in thymocytes by T(3) in vitro (1 muM) was confirmed. When added alone in concentrations ranging between 10 nM and 0.1 mM, the adrenergic agonists, epinephrine and norepinephrine, had no effect on CLE accumulation. At a concentration of 1 muM, isoproterenol, terbutaline, and phenylephrine were also without effect. However, the effect of T(3) was clearly potentiated by the concomitant addition of epinephrine, norepinephrine, and possibly isoproterenol, whereas terbutaline and phenylephrine were without effect. Neither basal nor T(3)-enhanced CLE accumulation was affected by the addition alone of the adrenergic blocking agents, propranolol (0.1 mM), phentolamine (10 muM), or practolol (0.1 mM). Nevertheless, the beta(1)- and beta(2)-antagonist, propranol, and the beta(1)-antagonist, practolol, blocked the increment in CLE accumulation produced by epinephrine; the alpha-antagonist, phentolamine, was without effect. The enhancement of CLE accumulation that occurred in the presence of T(3), with or without epinephrine, was seen to be a result of an inhibition of CLE efflux, because T(3) alone inhibited CLE efflux, and this effect was increased when epinephrine was also present. On the other hand, neither T(3) alone nor T(3) plus epinephrine appreciably altered the rate of inward transport of CLE. As judged from studies of the ability of thymocytes to exclude trypan blue, neither T(3) alone nor T(3) plus epinephrine either enhanced or impaired viability of cells during 3-h periods of incubation. Cell water content, measured with [(3)H]urea, was unaffected by T(3), either alone or in the presence of epinephrine. In confirmation of previous results, the stimulatory effect of T(3) on CLE accumulation was unaffected by concentrations of puromycin sufficient to inhibit protein synthesis by at least 95%, and the potentiating action of epinephrine on the response to T(3) was similarly unaffected. From these findings, it is concluded that the effect of T(3) to increase CLE accumulation by thymocytes in vitro, though itself independent of adrenergic mediation, is potentiated by beta(1)-adrenergic stimulation. This interaction appears distinctly different from other thyroid hormone-catecholamine interactions, in which thyroid hormones enhance physiological responses to catecholamines. Its mechanism remains unclear, but the properties of the T(3) effect, and possibly the interaction itself, suggest that T(3) enhances CLE accumulation by an action at the level of the cell membrane.


Circulating DNA:anti-DNA complexes in systemic lupus erythematosus. Detection and characterization by ultracentrifugation. Although it is generally accepted that DNA:anti-DNA immune complexes play a significant role in the pathogenesis of tissue injury in systemic lupus erythematosus, their presence in the circulation is still a matter of controversy. In this study, we detected DNA:anti-DNA compexes by identification of both the antigen and(or) the antibody, the necessary requisites for immune complex definition, in 14 of 24 plasmas (7 of 11 patients). These antibodies were specific for native DNA and could be adsorbed by anti-immunoglobulin (Ig)G antisera. The DNA recovered was, at least in part, of low molecular weight. The presence of DNA:anti-DNA compexes was not related to high molecular weight IgG, cryoprecipitins, positive polyethylene glycol precipitation, or low plasma C3 levels. It was related significantly to low plasma C4 levels and to the presence of diffuse proliferative nephritis. The lack of correlation with other methods of detection of immune complexes and with the presence of heavy IgG (above 13 S) is in favor of the existence of other antigen-antibody systems (or aggregated immunoglobulins) in systemic lupus erythematosus plasmas. From the results, it appears that methods directed towards the demonstration of specific immune complexes are more informative than those detecting heavy or altered immunoglobulins.


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.


Synthesis of hemoglobin Aic and related minor hemoglobin by erythrocytes. In vitro study of regulation. Factors that influence hemoglobin (Hb)A(Ic) synthesis by intact erythrocytes were studied in vitro. After incubation cells were lysed, and hemoglobins were separated by isoelectric focusing on polyacrylamide slab gels and quantitated by microdensitometry. HbA(Ic) increased with time, glucose concentrations (5-500 mM), and incubation temperature (4 degrees -37 degrees C). Low temperatures allowed prolonged incubations with minimal hemolysis. At 4 degrees C HbA(Ic) increased linearly with time for 6 wk; after incubation at the highest glucose concentration, HbA(Ic) comprised 50% of total hemoglobin. Insulin (1 and 0.1 mU/ml) did not affect HbA(Ic) synthesis in vitro. In addition to glucose, galactose and mannose, but not fructose, served as precursors to HbA(Ic). A good substrate for hexokinase (2-deoxyglucose) and a poor hexokinase substrate (3-O-methylglucose), were better precursors for HbA(Ic) synthesis than glucose, suggesting that enzymatic phosphorylation of glucose is not required for HbA(Ic) synthesis. Autoradiography after erythrocyte incubation with (32)P-phosphate showed incorporation of radioactivity into HbA(Ia1) and A(Ia2), but not HbA(Ib), A(Ic), or A. Acetylated HbA, generated during incubation with acetylsalicylate, migrated anodal to HbA(Ic) and clearly separated from it. Erythrocytes from patients with insulinopenic diabetes mellitus synthesized HbA(Ic) at the same rate as controls when incubated with identical glucose concentrations. Likewise, the rate of HbA(Ic) synthesis by erythrocytes from patients with cystic fibrosis and congenital spherocytosis paralleled controls. When erythrocytes from cord blood and from HbC and sickle cell anemia patients were incubated with elevated concentrations of glucose, fetal Hb, HbC, and sickle Hb decreased, whereas hemoglobins focusing at isoelectric points near those expected for the corresponding glycosylated derivatives appeared in proportionately increased amounts.