Effect of liver failure on the response of ventilation and cerebral criculation to carbon dioxide in man and in the goat.

1. The acid-base state of arterial blood and cerebrospinal fluid, and the ventilatory response to CO2, were measured in twelve patients with liver disease. The CO2 response was also measured in eight goats before and after the experimental production of liver failure. Arterial PCO2 and pH, cerebral blood flow and the cerebral metabolic rate for oxygen were also measured in four of the goats while they breathed air and various CO2-enriched gas mixtures. 2. Liver failure was accompanied by a respiratory alkalosis in both the patients and in the goats. Decreased PCO2 and increased pH occurred in the cerebrospinal fluid and in the arterial blood of the patients. 3. The slope of the ventilatory response to CO2 was reduced when liver failure was severe, in patients and goats alike. In addition there was a reduction in the extrapolated PCO2 at zero ventilation, even when liver failure was mild. 4. Cerebral blood flow and metabolic rate were consistently reduced in the goats during liver failure. There was also less cerebral vasodilatation and a greater reduction in cerebral metabolism during experimental hypercapnia when these animals were in liver failure. 5. The decreases in the ventilatory and cerebral circulatory responsiveness to CO2 indicate that the brain is less well defended against hypercapnia in liver failure, and these changes are especially unfavourable as cerebral function deteriorates when the PCO2 is increased.


Analytical subcellular fractionation of human granulocytes with reference to the localization of vitamin B12-binding proteins.

1. A granulocyte-rich fraction was isolated from blood of a patient with chronic granulocytic leukaemia and from blood of a normal subject and the cells were disrupted in isotonic sucrose. The nuclei were removed by low-speed centrifugation and the post-nuclear supernatant was fractionated by centrifugation on sucrose density gradients. 2. The subcellular organelles in the gradients were detected with marker enzymes by the use of highly sensitive assay techniques. Similar results were obtained with granulocytes from both subjects. 3. Unsaturated vitamin B12-binding proteins were almost exclusively localized to the specific granules. Chromatographic analysis of these proteins showed them to have approximately equal proportions of transcobalamins I and III. Vitamin B12 assayed microbiologically could not be detected in the specific granule fractions.


The influence of cholinoceptor activity on the mitotic rate in the crypts of Lieberkühn in rat jejunum.

1. The influence of cholinoceptor stimulation and cholinoceptor blockade on the mitotic rate in epithelial cells lining the crypts of Lieberkühn in rat jejunum was studied. 2. Cholinoceptor stimulation, either by injection of carbachol or by inhibition of acetylcholinesterase enzymes resulted in an increase in the mitotic rate. This increase in mitotic rate was blocked by tubocurarine, but not by atropine or hexamethonium. Atropine by itself increased the mitotic rate whereas tubocurarine alone or hexamethonium along decreased the mitotic rate. 3. The influence of atropine on crypt cell proliferation appears to involve an adrenergic mechanism since it is blocked by phentolamine.


Calcification of the heart and great vessels.

Calcification of the heart and great vessels is secondary to those diseases which cause a significant alteration of tissue matrix. Metabolic derangements can cause microscopic calcification of heart muscle, but this has not been demonstrated conclusively roentgenographically. The deposition of calcium may follow a single event or be the result of a longstanding process. The etiology and roentgen appearance of calcium deposition in the various tissues which comprise the heart and great vessels are reveiwed. The common as well as the rare cases are considered; if possible, each entity is demonstrated roentgenographically. In addition, an attempt is made to reciew various points of view, which at times are conflicting. The prognostic importance of cardiac, aortic, or pulmonary arterial calcification is underscored whenever possible.


Double-blind trials of clemastine ('Tavegil') in allergic rhinitis.

Two double-blind randomised trials are reported comparing the effectiveness of the antihistamines, clemastine and chlorpheniramine in comparable doses, in relieving the symptoms of allergic rhinitis. In the first trial, treating 58 adults seen in a general practice, both drugs were prescribed in tablet form; l mg. clemastine b.d. and 4 mg. chlorpheniramine b.d. The second trial was carried out in 42 patients attending a children's E.N.T. out-patient department and the drugs were prescribed as clemastine elixir (0.5 mg. b.d.) or as chlorpheniramine syrup (2 mg. b.d.). Both drugs were effective in providing symptomatic relief in a significant number of patients, and the overall efficacy of clemastine was marginally better than that of chlorpheniramine, especially in the second trial in children. Side-effects were minimal and drowsiness was not a problem.


A preliminary evaluation of pipothiazine palmitate in schizophrenia.

A preliminary evaluation of pipothiazine palmitate by injection was carried out in 25 schizophrenic patients to assess the therapeutic effectiveness of the drug, probable dosage schedules and intervals between doses, and to observ any undesirable side-effects. A good overall response was obtained in 64% of the patients. The authors suggest that the optimum maintenance dosage is between 50 and 100 mg. given at 4-weekly intervals. Although extra-pyramidal side-effects were high, only 1 patient had to be withdrawn on this account; the symptoms being well-controlled in mostly by procyclidine. Other adverse side-effects were negligible and there was no evidence of adverse biological effects. It is concluded that pipthiazine palmitate has considerable potential in the treatment of schizophrenia and a longer-term study is being conducted.


The thymoleptic action of two salts of flurazepam ('Dalmane').

A double-blind comparison was made of two dose levels (15 and 30 mg.) of the monohydrochloride and dihydrochloride salts of flurazepam ('Dalmane') in 40 hospitalised patients requiring a night-time hypnotic. Each patient in the four drug/dose groups was his own control and took a placebo for 3 nights followed by the active compound for a further 3 nights. On waking each morning a mood state questionnaire was completed. The results indicate that flurazepam is not only an effective hypnotic, but that it has an apparent anxiolytic effect the following morning. Patients report feeling less anxious and more cheerful following drug administration and this result was most noticeable with the 15 mg. monohydrochloride dose.


The effects of acute severe arterial hypoxemia on the electrocardiogram during exercise.

Sixteen healthy, active men were studied to determine the effects of severe arterial hypoxemia on the electrocardiograms during exercise. The electrocardiograms were all normal at maximum heart rate while the subject breathed ambient air. During maximal exercise breathing ten percent oxygen (mean arterial oxygen pressure [Po2] of 31 mm Hg), only one of the 16 had ST segment changes suggestive of ischemia. These were not present on a repeated study. The widely held view that systemic hypoxemia causes ischemic changes on the electrocardiogram was not confirmed in this study.


Functional characteristics of the lung in chronic uremia treated by renal dialysis therapy.

Ventilatory studies and arterial blood gas analyses were performed in 29 patients with chronic renal failure undergoing renal dialysis therapy (RDT). Twenty-three of the 29 had nearly normal pulmonary function, and the accumulation of body water between dialyses did not influence pulmonary function significantly. A trend was observed towards overinflation and air trapping in the presence of normal airway resistance, indicating narrowing of the small airways. Repeated studies over a 16-month period suggest that these patients develop neither chronic obstructive airway disease nor restrictive ventilatory impairment. Six patients had a restrictive ventilatory impairment with a trend to overdistension and air trapping as in the "normal" patients. This was observed both early and late in the course of RDT and appeared to be irreversible but not progressive. These patients had suffered from uremic pleuropericarditis and either had serous effusions or residual adhesions.


Direct and vagally mediated chronotropic effects of morphine studied by selective perfusion of the sinus node of awake dogs.

Two milliliters of a morphine sulfate solution (1 mg/ml) perfused selectively into the sinus node artery of five trained unanesthetized dogs caused an immediate brief sinus tachycardia followed by a delayed but prolonged sinus bradycardia. Beta-receptor blockade was achieved by selective perfusion of propranolol hydrochloride solution (10 mug/ml) into the sinus node artery and did not prevent the initial sinus tachycardia. Selective perfusion of the sinus node with atropine sulfate solution (1 mug/ml), however, did prevent morphine from causing further sinus rate increase. The immediate positive chronotropic action of morphine was thus attributable to a peripherally located vagolytic action. The exact opposite was true with regard to the delayed bradycardia; it was due to a centrally mediated generalized increase in vagal tone regularly elicited by morphine.


Determination of isonicotinic acid hydrazide in serum.

A method for the determination of isonicotinic acid hydrazide is given. The hydrazide is complexed with cupric ions and condensated with an aromatic aldehyde substituted in ortho-position with a hydroxyl group. The method is adapted to the AutoAnalyzer equipment of first and second generation. Pilot measurements on the pharmacokinetic behaviour of isonicotinic acid hydrazide are presented.


Culture and morphological observations on Trypanosoma melphagium.

The cultural characteristics of Trypanosoma melophagium of sheep were studied. Aspects investigated were size of the inoculum and population growth in Modified Monophasic Medium for Trypanosomes (MMMT), population growth in Medium 199 with 10% inactivated calf serum containing 5, 10, and 15% hemolyzed defibrinated rabbit blood (199-CS-5, 199--CS-10, 199-CS-15) at 27 degrees C, effects on population growth of temperature and hydrogen ion concentration in MMMT, and morphology and morphometrics of the developmental stages found under different experimental conditions. The best growth occurred in medium MMMT at 30 degrees, C, pH 7.25. Temperature seemed to be a critical factor for differentiation of epimastigotes to trypomastigotes. Statistically significant differences were found between the trypomastigotes in MMMT and 199--cs-5 at 37 degrees C on day 4 of incubation for the following measurements: PK (distance from posterior end to kinetoplast), KN (from kinetoplast to middle of nucleus), PN (from posterior end to middle of nucleus), and nuclear and kinetoplastic indices. The trypomastigotes formed in both media were much smaller in size than the blood forms reported by Hoare (1972).


DNA sequence organization in the genomes of five marine invertebrates.

The arrangement of repetitive and non-repetitive sequence was studied in the genomic DNA of the oyster (Crassostrea virginica), the surf clam (Spisula solidissima), the horseshoe crab (Limulus polyphemus), a nemertean worm (Cerebratulus lacteus) and a jelly-fish (Aurelia aurita). Except for the jellyfish these animals belong to the protostomial branch of animal evolution, for which little information regarding DNA sequence organization has previously been available. The reassociation kinetics of short (250-300 nucleotide) and long (2,000-3,000 nucleotide) DNA fragments was studied by the hydroxyapatite method. It was shown that in each case a major fraction of the DNA consists of single copy sequences less than about 3,000 nucleotides in length, interspersed with short repetitive sequences. The lengths of the repetitive sequences were estimated by optical hyperchromicity and S1 nuclease measurements made on renaturation products. All the genomes studied include a prominent fraction of interspersed repetitive sequences about 300 nucleotides in length, as well as longer repetitive sequence regions.


Comparative aspects of DNA organization in Metazoa.

Data on sequence organization in metazoa are reviewed and tabulated. It is shown that the features of sequence organization previously observed in Xenopus DNA are extremely widespread. At least 70% of DNA fragments 2,000-3,000 nucleotides long contain both single copy and repetitive sequence in all the organisms examined except Drosophila.


Energetic aspects of transport of ADP and ATP through the mitochondrial membrane.

Evidence from various sources demonstrates that the release of ATP in exchange for the entry of ADP across the mitochondrial membrane is an active process requiring energy. The necessary energy may be derived from the same source of energy as that used for oxidative phosphorylation. The following results will be discussed:--(1) The exchange is asymmetric with respect to the specificity of ADP and ATP in 'energized' mitochondria. From the outside ADP is much preferred to ATP, but from the inside both exchange with equal specificity. This asymmetry is abolished by de-energization of the membrane. (2) The ADP-ATP exchange is about 50% electrogenic: about half the ATP released against ADP is protonated. The excess of negatively charged ATP might prevent ADP from entering mitochondria against a membrane potential. (3) The ratio of ATP to ADP across the inner mitochondrial membrane is higher outside than inside only in the energized state. Variation of the ATP/ADP ratio maintains this difference. (4) The ADP/ATP ratio apparently varies with changes in the membrane potential as measured by Rb+-distribution. The correlation factor between deltaE changes and this ratio is 0.5, in agreement with predictions from proton-stoichiometry measurements. The deltapH does not significantly change the distribution ratio. (5) By following the P/O ratio, one can show that energy derived from expelling ATP against the ADP/ATP gradient lowers the amount of ATP synthesis. The P/O ratio is lowered as the imbalance of ATP to ADP increases. (6) The energy difference of the phosphorylation potential of ATP is calculated by various methods to be about 8-12 kJ, depending on the conditions. This free energy is the result of the 'active' transport which corresponds to the release of ATP outside the mitochondria. (7) In vivo studies on the distribution of ADP and ATP inside and outside the mitochondria in liver show a corresponding ratio difference of about 15 as predicted from the in vitro studies.


Bioluminescence: from chemical bonds to photons.

The biological transformation of chemical to photic energy involves an enzyme-mediated chemiluminescent reaction, in which one of the products exists in an electronically excited state, emitting a photon as it returns to the ground state. The colour of bioluminescence differs in different organisms, ranging from the deep blue (460 nm) of certain crustacea, through the bluish green (490 nm) of some bacteria, the green (530 nm) of mushrooms to the red (about 600 nm) of the railroad worm. In one case, energy transfer has been demonstrated from the enzyme system to material that emits light with a longer wavelength. The energies involved range from about 165 to 250 kJ/einstein (40 to 60 kcal/einstein). Boyle first showed that air was involved in bioluminescence in 1668 in his experiments with an air pump. Over the past 100 years, it has become clear that most if not all bioluminescent systems require molecular oxygen. The recent isolation and characterization of an oxygen-containing (peroxide) enzyme intermediate from the bacterial system is described and a reaction mechanism is postulated. This scheme is compared with other hypothetical mechanisms, in particular those involving a four-membered ring intermediate, a dioxetane, in which the simultaneous cleavage of two bonds leaves one product in an excited state. I shall discuss the special role of luciferases in bioluminescence, especially in flashing mechanisms involving 'precharged' intermediates.


The purple membrane of Halobacterium halobium: a new system for light energy conversion.

Patches of a distinctly different structure from the rest of the cell membrane occur in Halobacterium halobium. The isolated patches are called the purple membrane. It derives its colour from a retinal-protein complex, bacteriorhodopsin, which is the only protein species in this membrane serving a phototransducing function. Light energy is converted by a photochemical cycle going on continuously under illumination and accompanied by a cyclic release and uptake of protons. In the intact cell, this cycle operates as a vectorial process and therefore builds up an electrochemical gradient across the cell membrane conserving part of the absorbed light energy. The cell apparently uses this electrochemical gradient for the synthesis of ATP. Photophosphorylation is shown to be insensitive to cyanide but sensitive to dicyclohexylcarbodiimide (DCCD) and uncouplers. The concentration of ATP and the pH are tightly coupled but can be uncoupled by DCCD. Bacteriorhodopsin, as a light-driven proton pump, can then be studied in the cell as an isolated process. Quantitation of light energy conversion is possible by the indirect method of inhibition of respiration by light and the comparison of the number of absorbed quanta which prevent consumption of one molecule of oxygen.


Energy transfer in mitochondrial synthesis of ATP; a survey.

The energy transduction in mitochondria, with its principal agent ATP, still represents a major challenge for biological research. In general, the energy transduction process is divided into three sections: (1) the redox processes; (2) a conservation of intermediary energy forms; (3) synthesis of ATP. All three processes are linked to the membrane and are, therefore, as difficult to resolve as are processes linked to other biomembranes. It is probable that the electron transport system is constructed in such a way as to provide energy for synthesis of ATP and related processes. Important for this function is the transversal distribution of these components across the membrane, facilitating generation of membrane potential by electron or proton transfer. The exact composition of the respiratory chain is not yet known, in particular with respect to iron-sulphur proteins. Progress is achieved by defining single species of the respiratory chain, subunit composition, amino acid sequences and genetic derivation from intra- or extra-mitochondrial translation. Energy generated by oxidation can be trapped before ATP is formed by a number of reactions, in particular reversed electron transport, energy-dependent transhydrogenation and uptake of anions or cations into the mitochondria. The latter reaction is of major importance for understanding the intermediate energy form, as it appears to use energy most directly and be driven mainly by membrane potential or proton gradient across the membrane. The formation of ATP is a major problem hindering elucidation of the mechanism of oxidative phosphorylation. The mechanism of this enzymic process is not yet understood although the enzymes have been isolated and the subunits have been defined. Most probably, a concerted reaction between ADP and phosphate, driven by some conformational transition of the complex, leads to the formation of ATP. Release of ATP from a hydrophobic to hydrophilic environment may consume most of the energy.


The active transport of carbohydrates by Escherichia coli.

The active transport of carbohydrates by Escherichia coli is discussed with particular reference to (1) identification of an uptake process as 'active transport', (2) nature and control of transport proteins, and (3) mechanisms of energy transduction. (1) The use of substrate analogues, of mutants blocked in metabolism and of subcellular vesicles in the isolation of the transport process from interference by subsequent metabolic reactions is described. Criteria are outlined for establishing that the solute is taken up against a concentration gradient and that this is energy-dependent. Three types of poisons for energy systems that act primarily on respiration, on ATP formation and as uncoupling ('proton conducting') agents are considered. (2) Methods are described for the selection of mutants impaired in the active uptake of specific carbohydrates. (3) Results show that the uptake of galactose, D-fucose and arabinose by appropriate strains of E. coli is inducible, specific and accompanied by proton uptake. Such and other data support a model based on a chemiosmotic theory of active transport.


Chloroplasts.

Illuminated chloroplast fragments, which can convert light into chemical energy (NADPH2 and ATP), contain a number of soluble and insoluble electron carriers that have been arranged, on the basis of their redox potentials and on kinetic and other evidence, in sequences (analogous to those in mitochondria) to describe the events involved in the light reactions of photosynthesis. Fractionation of chloroplasts allows separation of two light-dependent partial reactions: the evolution of oxygen and the reduction of pyridine nucleotide, accompanied by ATP synthesis. The stoichiometry of the latter reaction is still uncertain. Chloroplasts contain a directional proton-translocating ATPase (CF1) needed for the phosphorylation of ADP in the light. The CF1 also catalyses synthesis of ATP from ADP. When a pH gradient is applied in the dark across the phosphorylating membranes, the amount of ATP synthesized is related to both the change in pH and the electrical potential. During its catalytic activity, the CF1 protein undergoes reversible conformational changes, but this is not the source of the driving force for ATP synthesis.


A double-blind comparison of loxitane--loxapine succinate and trifluoperazine hydrochloride in chronic schizophrenic patients.

Therapeutic efficacy and safety of loxapine succinate (LOX) and trifluoperazine hydrochloride were compared by means of a controlled, double-blind study involving 49 chronic schizophrenic inpatients. The data indicated that LOX in dosages ranging from 40 - 65 mg/day, (usually administered on a b.i.d. schedule) had demonstrable anti-psychotic activity in 14 of 25 chronic schizophrenic patients (56%), while TFP treatment in appropriate dosage showed a similar activity in 9 of 23 patients (39%). Both LOX and TFP displayed essentially the same profile and incidence of side effects. Clinical laboratory abnormalities were minor. On the basis of this study, and in comparison with trifluoperazine, it appears that loxapine is an efficacious and essentially safe medication, suitable for the treatment of chronic schizophrenia.


Pharmacokinetic study of a peripheral analgesic, floctafenin, in man, mouse, rat, and dog.

The pharmacokinetics of floctafenin has been studied in man, mice, rats, and dogs at pharmacological doses. Its absorption, which is exclusively intestinal, is good in man and rodents, but only partial in dogs. Its high plasma clearance rate is primarily due to hepatic hydrolysis to floctafenic acid, which is the main circulating product almost immediately following hydrolysis to floctafenic acid, which is the main circulating product almost immediately following iv administration. The compound binds to two sets of binding sites in animal serum and human plasma with affinity constants of 10(7) M-1 and 10(5) M-1 at 4 degrees C in all species except the dog, where binding is weaker. This binding has been shown to be solely accounted for by albumin. Floctafenin, less protein-bound than floctafenic acid, diffuses more widely into tissues, but very low quantities of the ester and virtually negligible quantities of the acid cross the blood-brain barrier, indicating that their analgesic activity is exclusively peripheral. The elimination of floctafenin and its metabolites is practically complete in 24 hr. The main excretory route is via the bile, biliary excretion being largely predominant in dogs and rats, and somewhat less so in man and mice. There is no enterohepatic cycle of note. The main metabolite in both bile and urine is floctafenic acid. A secondary metabolic pathway, common to all species, leads, by hydroxylation in the postion para to the anthranilic nitrogen, to the corresponding phenols. All products in man and rats are excreted primarily in the form of ether and/or ester O-glucuronides.


Metabolism of propionyl erythromycin lauryl sulfate. I. Fate of the propionyl erythromycin moiety in the rat.

The absorption, excretion, and metabolism of propionyl erythromycin (PE) has been studied in the rat. The major routes of metabolism of PE are ester hydrolysis and N-demethylation. The rates of these two reactions have been examined in vivo using radiolabeled PE. The plasma half-life of the ester is 5.5 hr. The correlation of blood levels of radioactivity with 14CO2 production indicates that the ester is continually hydrolyzed after absorption. The half-life of the dimethyl-amino moiety of the desosamine sugar is estimated at 1.5 hr. This relatively short half-life compared to that of the ester is supported by the fact that at 3.5 hr after dosing there is twice as much desmethyl-PE in plasma as PE. After oral administration of either 14C-PE or 14C-erythromycin, 70% of the radioactivity is absorbed in 6 hr. The major route of excretion is via bile. Approximatley 40% of the absorbed dose is excreted in bile in the first 6 hr after dosing. Tissue levels of radioactivity after administration of 14C-erythromycin or 14C-PE indicate that PE or a metabolite accumulates in the tissue during chronic dosing, whereas erythromycin-related levels are similar after single or multiple doses.


Protection by lead nitrate against carbon tetrachloride hepatotoxicity.

Pretreatment with lead nitrate of rats intoxicated with CCl4 exerted a significant protective effect against several damaging effects of the haloalkane. Liver microsomal lipid peroxidation, one of the earliest phenomena in CCl4 intoxication, was clearly inhibited by pretreating the rats with lead. The heavy metal also ameliorated the polyribosomal disaggregation caused by CCl4. Fatty infiltration in the liver, measured by the triglyceride content in the organ, was less pronounced in CCl4-poisoned rats pretreated with lead than in animals treated with the haloalkane alone. Lipid metabolism was also studied by means of Triton WR 1339-induced hypertriglyceridemia to examine the state of triglyceride secretion from liver into plasma; the higher level of plasma triglyceride in the lead-pretreated rats further strengthened the results obtained on the secretion experiments. Finally, CCl4-induced liver necrosis, as measured by serum transaminases and histological examination, was partially prevented by lead. The mechanism by which lead, an inhibitor of the drug-metabolizing enzyme system, interferes with CCl4 intoxication, is disccused.


Metabolism of propionyl erythromycin lauryl sulfate. II. Fate of the lauryl sulfate moiety in the rat and man.

The absorption, excretion, and metabolism of [1-14C]lauryl sulfate as either the sodium or propionyl erythromycin salt has been studied in the rat and man. In the rat 88% of the radiolabel from propionyl erythromycin [1-14C]lauryl sulfate was excreted in the urine in the 24-hr period following a single oral dose. More than 95% of the radiolabel was excreted as the metabolite butyric acid 4-sulfate. This metabolite was shown to be derived from carbons 1-4 of the lauryl sulfate moiety. There was no evidence of sulfate cleavage in the rat. In man 51-59% of the administered ratioactivity was recovered in the urine. The major excretion product was butyric acid 4-sulfate accounting for approximately 95% of urinary radioactivity. The remainder was unchanged lauryl sulfate. A significant portion of the radiolabeled propionyl erythromycin lauryl sulfate was converted to 14CO2 indicating that the sulfate linkage was cleaved. A minimum value of 9-16% of the dose was metabolized in this manner.


Excretion and stereoselective biotransformations of dl-, d- and l-norgestrel in women.

Excretion data and urinary metabolite patterns of di-, d-, and l-norgestrel were obtained from women who received a single, oral 1.5-mg dose of 14C-labeled racemic norgestrel (Ng) or one of its enantiomers. The average percentage of administered radioactivity +/- SD recovered in the urine after 7 days was 58.1 +/- 7.9% for dl-Ng, 44.8 +/- 8.9% for d-Ng, and 63.6 +/- 15.1% for l-Ng; in feces it was 23.4 +/- 7.7% (dl-Ng), 31.6 +/- 8.2% (d-Ng), and 24.8 +/- 10.7% (l-Ng). Different metabolite patterns were observed for each enantiomer in urine, and the pattern for the racemate appeared to be an approximate composite of the metabolite patterns of the two enantiomers. These differences in the metabolite pattern result from stereoselective transformations; notably 16 beta-hydroxylation of l-Ng and ring A reduction of d-Ng. Other stereoselective pathways noted were: 16 alpha- and 1 beta-hydroxylation as well as D-homoannulation of l-Ng and sulfate conjugation of l-16 beta-hydroxynorgestrel; 2 alpha-hydroxylation of d-Ng, formation of a labile neutral, polar compound which contained the norgestrel moiety in the d-form, and formation of a glucuronide of d-16 beta-hydroxynorgestre. The formation of phenolic derivatives occurred to a very minor degree from transformations of the biologically inactive l-enantiomer. With d-norgestrel, this formation occurred to an even lesser extent, if at all.


Metabolism of p-(cyclopropylcarbonyl)phenylacetic acid (SQ 20,650). Species Differences.

The metabolism of p-(cyclopropylcarbonyl)phenyl[14C]acetic acid (I-14C), a nonsteroidal anti-inflammatory agent, has been studied in rats, dogs, and monkeys. Animals were given single intravenous or oral doses of 5 and 50 mg of I-14C/kg. In all cases, 72-88% of the administered dose was excreted in urine, with most of the radioactivity appearing within 24 hr after dosing; less than 11% was found in feces. The half-life (t1/2) of radioactivity in monkey or dog plasma was 1 and 5 hr. respectively, after the oral or intravenous administration of a 5-mg dose of I-14C per kg. At 50 mg/kg, these half-lives increased to 3.5 and 7.7 hr. respectively. More than 90% of the radioactivity in plasma of both species was associated with unchanged drug. Species differences exist in the biotransformation of I. Rat urine contained 93-97% I; 2-6% (alpha-cyclopropyl-alpha-hydroxy-p-tolyl)acetic acid (II); and approximately 1% as conjugates. Monkey urine contained I-glucuronide (88%) and unconjugated II (7-10%). In the dog, I-taurine accounted for 27% of the radioactivity found in urine; II and its taurine conjugate accounted for 20 and 30%, respectively; a small quantity of II-glycine (3%) was also detected. There are three minor metabolites that have not been identified. Metabolite II isolated from dog urine was shown to be dextrorotatory.


Human metabolism of cyproheptadine.

Gas-liquid chromatographic and mass, nuclear magnetic resonance, and infrared spectrometric techniques were utilized to identify some of the metabolites of cyproheptadine in the urine of human subjects who had ingested radiolabeled drug. Aromatic ring hydroxylation (followed by glucuronide conjugation), N-demethylation, and heterocyclic ring oxidation were shown to occur in man. The principal metabolite, however, was identified tentatively as a quaternary ammonium, glucuronide-like conjugate of cyproheptadine. No evidence was found for metabolic changes at the tricyclic ethylene bridge in this species.


Studies of the disposition and metabolism of mefloquine HCl (WR 142,490), a quinolinemethanol antimalarial, in the rat. Limited studies with an analog, WR 30,090.

The overall fate of the quinolinemethanol antimalarial, mefloquine-HCl [WR 142,490, erythro-DL-alpha-(2-piperidyl)-2,9-bis(trifluoromethyl)-4-quinolinemethanol hydrochloride] was investigated in the rat with 14C-labeled drug. Despite its extensive binding to plasma proteins, high tissue/plasma concentration ratios were found. Fecal excretion accounted for most of the drug and metabolites. This fate was rationalized on the basis of physical properties of mefloquine (and its metabolites) and extensive biliary and gastric secretion, followed by reabsorption. Evidence was obtained for the formation of several metabolites. Limited studies were carried out in rats, dogs, and one human subject with another quinolinemethanol, WR 30,090-14C [DL-6,8-dichloro-2-(3',4'-dichlorophenyl)-alpha-(di-n-butylaminomethyl)-4-quinolinemethanol hydrochloride]. Its general fate and physical properties were similar to mefloquine. The disposition of the two quinoline compounds studied was compared to that of quinine and quinidine reported in the literature. This revealed that modification of the quinine molecule resulted in new drugs which were more highly bound and exhibited longer half-lives than quinine.


[Treatment of healthy salmonella carriers with lactulose beta-galactosido-fructose (author's transl)].

Oral administration of lactulose (beta-galactosido-fructose) of 25 healthy Salmonella carriers cured 21, but failed in four.


The distribution and excretion of 2,4,5,2',5'-pentachlorobiphenyl in the rat.

Rats received single intravenous doses (0.6 or 6.0 mg/kg) of 2,4,5,2',5'-pentachlorobiphenyl-14C (5-CB). The distribution and ecretion of 5-CB-derived material were investigated at periods ranging from 10 min to 42 days after administration and found to be first-order rate processes in the dose range studied. Blood, liver, muscle, skin, and adipose tissues were found to be the most important tissues to the distribution of this compound. More than 90% of the total dose was removed from the blood within 10 min after administration. Most of the radioactivity was initially deposited in the liver and muscle, and then translocated to the skin and adipose tissue. The decay rate of radioactivity from each of the tissues could be described by the sum of two or more exponentials. Metabolism to more polar compounds appears to be a prerequisite to excretion, and most of the radioactivity was excreted in the bile and ultimately in the feces. Excretion in the urine accounted for less than 7% of the total dose and ceased at approximately 8 or 9 days after 5-CB-administration. Studies of the distribution and excretion of 5-CB-derived material over extended periods of time, 1-42 days, demonstrated the importance of the adipose tissue and skin as long-term storage sites, and demonstrated that the decay rate of 5-CB from the tissues paralleled the decreasing rate of excretion in the feces.


Effect of nafenopin (SU-13,437) on liver functions. Hepatic uptake and biliary excretion of ouabain in the rat.

Pretreatment of rats for 2 days with the hypolipidemic drug, nafenopin, 0.5 g/kg, results in an increase in liver weight and bile flow. Despite these changes, hepatic transport of ouabain is reduced. This was demonstrated in the isolated perfused liver as well as in vivo. Although net uptake into liver is diminished in treated rats, the initial rapid phase of the plasma disappearance curve is unaffected. This suggests that the primary uptake process is unaffected by nafenopin or is altered in a manner that is not reflected in this phase of plasma disappearance. Alternatively, nafenopin may increase ouabain efflux from liver to plasma. Biliary excretion of ouabain is markedly suppressed after nafenopin pretreatment and higher liver levels of ouabain were encountered after the first 20 min in treated animals. The inference is drawn that liver to bile transport of ouabain is also suppressed by nafenopin pretreatment.


Effect of chronic cadmium treatment on rat adrenal catecholamines.

Daily intraperitoneal injection of cadmium chloride (1 mg/kg) for 45 days significantly increased adrenal weights and augmented the levels of adrenal norepinephrine and epinephrine as well as the activity of adrenal tyrosine hydroxylase. Discontinuation of the heavy metal treatment for 28 days, in rats previously injected with cadmium for 45 days, restored the activity of tyrosine hydroxylase as well as the amount of norepinephrine and epinephrine. In contrast, adrenal weights were restored only partially following the withdrawal of cadmium treatment. Evidence indicates that the changes in adrenal catecholamine metabolism may be the result of stress induced by chronic exposure to this heavy metal. In addition, some of the untoward effects such as hyperglycemia and arterial hypertension seen during cadmium toxicity might be related to increased synthesis of epinephrine in adrenal glands.


Sleep and hypnotic drugs.

In recent years the effectiveness of hypnotic drugs has had to be assessed in terms of a greatly increased knowledge of the physiology and pathology of sleep. The normal pattern of sleep and wakefulness involves a cyclic alternation between three rather than two basically dissimilar states of the brain and body - alert wakefulness, rapid-eye-movement (REM) sleep and non-rapid-eye-movement (NREM) sleep. The pattern of this alternation in individual people results from the interaction of many influences - biological (including genetic, early developmental and later degenerative influences), psychological, social and environmental factors, various physical and psychiatric disorders, and most drugs which affect the central nervous system. The quality of sleep is not related in any simple or constant manner either to its duration or to the proprotions of time spent in each stage of sleep. Among the disorders of sleep, insomnia is a far more common problem of medical management than are enuresis, narcolepsy, somnambulism or nightmares. With a few exceptions, most hypnotic drugs now in widespread use cease to be effective in treating insomnia after the first few nights. However, the ineffective treatment is often continued because insomnia will be even worse during the initial period of drug withdrawal. These factors and the toxicity of hypnotic drugs when taken in overdose make the long-term treatment of insomnia more difficult than was previously supposed. Barbiturates should no longer be prescribed. Some of the non-barbiturates, such as glutethimide and methaqualone, have no advantage over the barbiturates. The benzodiazepine hypnotics, nitrazepam and flurazepam, are less toxic in overdose and are relatively effective in treating insomnia. Chloral hydrate and its derivates are useful alternative drugs for short-term use. Measures to improve sleep without drugs deserve greater emphasis than they have had in the past.


The effects of adrenalectomy and hydrocortisone replacement on the thyriod of the adult male rat I. Morphometrical data and histochemistry of some oxidative enzymes.

Morphometrical and histochemical studies were performed to determine the effects of adrenalectomy and hydrocortisone replacement on the rat thyroid. It follows from the performed studies that morphometrical and histochemical changes in the rat thyroid induced by adrenalectomy or hydrocortisone replacement depend upon the duration of the experiment. From the 4th day of the experiment adrenalectomy results in an increase in the volume fraction of the epithelium of the thyroid vesicles with a concomitant decrease of the colloid fraction. As compared to sham operated animals on the 10th day of the experiment the increase in the height of the epithelial cells was seen. Hydrocortisone replacement partially prevents the thyroid changes induced by adrenalectomy, however, this hormone markedly increases the volume fraction of the thyroid stroma and the height of the epithelial cells. On the other hand, histochemical reactions for various oxido-reductases do not provide helpful information for evaluation of the secretory activity of the rat thyroid. It follows from our results that adrenalectomy enhances thyroid stimulation by TSH and enhances the secretory activity of this gland, effects partially prevented by hydrocortisone replacement.


Effects of de-icing salt on ground water characteristics.

The effect of "road salt" on the characteristics of Massachusetts drinking water supplies has been significant and cumulative rather than transient or seasonal. De-icing salt is essentially all sodium chloride. Calcium chloride accounted for only three percent of the total salt used. However, hardness content, as well as sodium ion concentration, has increased greatly in ground waters in the past decade. The changing composition of our water supplies has agricultural, economic, and public health implications. This study attempts to quantify the stoichiometry of these changes in concentration, which are in part due to an ion-exchange mechanism in the soil.


The effect of vitamin E on NO2 induced redox changes in the human erythrocyte.

Human erythrocytes admixed with vitamin E supplements (50 mug/ml) were exposed to NO2 in concentrations of 200-600 p.p.m. at 37 degrees C for 2 h.  No protective effect against the increase in the redox ratio [NAD+]/[NADH]normally produced by NO2 was observed.  It is postulated that NO2 increases the redox ratio by a mechanism independent of that producing hemolysis and prevented by vitamin E.  This may involve alteration of NADH dependent enzymes by NO2.


Influences of exercise and endurance training on the oxygen dissociation curve of blood under in vivo and in vitro conditions.

In experiments with graded exercise of 15 men (6 untrained, 3 semitrained, 6 endurance-trained) the trained subjects showed a massive shift to the right of the in vivo O2 dissociation curve (ODC) of femoral venous blood. At a saturation of 20 to 25% (18 mkp/sec) Po2 was about 9 mm Hg higher for the trained than for the untrained group. The following factors play a role: 1. The 2,3-diphosphoglycerate [2,3-DPG] concentration was increased by 15 to 20% in the trained group which explains about 2 mm Hg of the diffenence in Po2-2. Exercise acidosis in the femoral venous blood depends to a large extent on CO2 in the trained, but on lactic acid in the untrained group. At low saturations the CO2-Bohr effect increases sharply thus having a greater importance in the trained subjects. This factor can explain about 2 mm Hg of the difference. However, influence of chloride and 2,3-DPG on the Bohr effect must be taken into consideration. 3. Since the large ODC-shift to the right of the trained group was not reproducible under in vitro conditions, it is suggested that a rapidly decaying unknown substance accounts for the remaining difference in Po2.


Diurnal changes of the 2,3-diphosphoglycerate concentration in human red cells and the influence of posture.

The 2,3-diphosphoglycerate (2,3-DPG) concentration, oxygen half saturation pressure at pH 7.4 (P50), pH in plasma and red cells, and mean corpuscular hemoglobin concentration (MCHC) of venous blood were determined during unrestricted daily activity (series I) throughout 24 hrs as well as during prolonged bed rest until noon (series II). In series I almost synchronous dirunal behavior of P50 2,3-DPG, and plasma pH as well as red cell pH became significantly apparent with highest values in the afternoon. The [2,3-DPG] yielded most pronounced alterations, which made up to 13.5% of the average day value. During prolonged recumbency the [2,3-DPG] showed a nonsignificant tendency to decline; the P50 remained unchanged throughout that period. The possible reason for the missing [2,3-DPG] increase is a reduced change of red cell pH in series II. An influence of a posture dependent aldosterone secretion either directly on the 2,3-DPG metabloism of indirectly via mediating the red cell pH and thus ruling the formation of this organic PHOSPHORIS COMPOUND IS DISCUSSED.


Studies on energy-linked reactions: isolation and properties of mitochondrial venturicidin-resistant mutants of Saccharomyces cerevisiae.

Venturicidin is a specific inhibitor of aerobic growth of yeast and has no effect on fermentative growth, a result which is consistent with its known mode of action on mitochondrial oxidative phosphorylation. Venturicidin-resistant mutants of Saccharomyces cerevisiae have been isolated and form two general classes: class 1, nuclear mutants which are resistant to a variety of mitochondrial inhibitors and uncouplers, and class 2, mitochondrial mutants of phenotype VENR OLYR and VENR TETR in vivo. VENR OLYR mutants show a high degree of resistance to venturicidin and oligomycin at the whole cell and mitochondrial ATPase level but, in contrast, no resistance at the mitochondrial level is observed with VENR TETR mutants. Venturicidin resistance/sensitivity can be correlated with two binding sites on mitochondrial ATPase, one of which is common to the oligomycin binding site and the other is common to the triethyl tin binding site. Biochemical genetic studies indicate that two mitochondrial genes specify venturicidin resistance/sensitivity and that the mitochondrial gene products are components of the mitochondrial ATPase complex.


Catalytically active azoaldolase. Preparation on solid support.

A procedure for the coupling at pH 7.2 of p-carboxy benzene diazonium chloride with rabbit muscle aldolase supported on phosphocellulose is described and some of the spectroscopic, structural and catalytic features of the material obtained are reported. The tetrameric azoenzyme is homogeneous in disc gel electrophoresis even in the presence of 8 M urea. Twelve molecules of the reactant are bound to the protein. Eight azocysteins are identified by both spectroscopic studies and amino acid analysis. The presence of one azohistidine is suggested by the spectroscopic data along with the presence of other, as yet unknown, chromophores. The azoaldolase shows unchanged catalytic properties using both D-fructose 1,6-bisphosphate and D-fructose 1-phosphate as substrates, as compared with the native enzyme. The pH profile of the enzyme activity is broadened towards the alkaline region but no changes occur in the physiological range of pH.


The reversible depolymerization of spinach chloroplast glyceraldehyde-phosphate dehydrogenase. Interaction with nucleotides and dithiothreitol.

The ligand-dependent dissociation of spinach chloroplast glyceraldehyde-phosphate dehydrogenase (Mr 600000) to protomers of Mr about 145000, previously shown by us in 1973, has been further characterized by the technique of velocity sedimentation in sucrose gradients. The process exhibits cooperativity and is accompanied by an increase of the apparent NADP+-dependent activity (reactivation) from a ratio of 0.1-0.2 to a ratio of 1 to 2 with respect to the NAD+-dependent activity. In addition to NADP+ and NADPH, most nucleotide triphosphates and, to some extent, Pi, act as dissociating agents. The enzyme is depolymerized and progressively inactivated in the presence of 2'-AMP. 2. Incubation with 20 mM dithiothreitol or 8-10 mM GTP increases the apparent NADP/H)-dependent activity, although addition of a small amount of a dissociating compound, such as 0.06 mM NADP+, is required for depolymerization. 3. NAD+, NADH and, to a lesser extent, glyceraldehyde 3-phosphate, NMN and cyclic AMP act as inhibitors of the dissociation and reactivation, however induced. They also favour the reassociation of protomers to tetramers. 4. The NADP(H)-linked activity is probably a property of the protomers only. The system described here resembles in many respects the light-dependent regulation of the NADP(H)-linked activity in vivo.


Heme reactivity of hemoglobins. Azide and fluoride binding equilibria of free and mercuriated ferri-gamma chains.

The free gamma chains, isolated from human foetal hemoglobin, are stable when oxidized and thus suitable for ligand binding and subunit equilibrium studies. The metaquo-ferri chains, with cysteine-F9 in the free state II ag gamma SH) possess several properties which are different from those of their p-mercuribenzoate derivative (III aq gammaSHgR); these are: stronger binding of a high-field ligand (N3- minus), altered spin equilibrium and an altered subunit equilibrium. A quantitative assessment of the free energy changes associated with all individual steps involved in changing the metaquo chains to their azide derivatives has been made. The results show that the higher apparent reactivity of III ag gammaSH (compared to IIIaq gammaSHgR) for the azide ion is not solely due to compensatory effects arising from differences of subunit dissociation or of spin equilibrium: other process(es) occurring in the ligand binding site have to be considered.


The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.


The mitochondrial ATPase. Selective modification of a nitrogen residue in the beta subunit.

1. When mitochondrial ATPase, which has been modified on a single tyrosine residue by 4-chloro-7-nitrobenzofurazan, is incubated at pH 9.0, the 7-nitrobenzofurazan group undergoes an intramolecular transfer to a nitrogen residue. The rate of this transfer is sensitive to the binding of adenine nucleotides to the enzyme. The resulting N-nitrobenzofurazan ATPase has little or no activity. 2. The fluorescence of the N-nitrobenzofurazan group in the modified ATPase is quenched on binding of ADP. 3. Electrophoresis of the modified enzyme in sodium dodecyl sulphate on a 10% polyacrylamide gel shows that the fluorescence of the N-nitrobenzofurazan chromophore is exclusively in the beta subunit. 4. The rate of transfer of the nitrobenzofurazan group from tyrosyl oxygen to nitrogen on the enzyme is compared with the rate of transfer between model compounds. 5. The interaction of the N-nitrobenzofurazan ATPase with aurovertin is reported.


Photoinactivation and carbethoxylation of leucine aminopeptidase.

In the present paper the reactivity of histidyl residues of leucine aminopeptidase from bovine eye lens was studied by dye-sensitized photooxidation and by carbethoxylation of the enzyme protein using diethylpyrocarbonate. Of all the different amino acids modified by photooxidation only histidine is connected with the enzymic acticity, whereas tyrosine seems to be involved in structure stabilization. By changing the pH and varying the effectors (Mg2+ and/or dodecylsulfate) of the reaction mixture a different number of histidyl residues of the enzyme protein is caused to react with diethylpyrocarbonate. No secondary reactions with tyrosyl or tryptophyl residues could be observed by spectrophotometric investigations. The enzyme modified by one of the above-mentioned methods shows changes in the capacity of Mn2+ binding measured by autoradiography as well as in the degree of enhancement of enzymic activity by Mn2+ or Mg2+ ions. Of the 48 histidyl residues of the enzyme (Mr = 326000) up to 2 histidyl residues per subunit (Mr = 54000) may be involved in Mn2+ or Mg2+ binding and up to 4 histidyl residues have a strong influence on Zn2+ binding.


The effects of spermine and Mg2+ on the catalytic mechanism of isoleucine: tRNA ligase.

Isoleucyl-tRNA formation catalysed by isoleucine: tRNA ligase is stimulated by both Mg2+ and spermine in the pH-range 7.0 to 8.0 at 310 K. At low [Mg2+] the acceleration caused by both cations together exceeds the sum of their individual effects. 2. The spermine-stimulated reaction has a steeper temperature-dependence than reaction in the presence of Mg2+. Two phases in the kinetics of isoleucyl-tRNA formation are detected in the presence of Mg2+ plus or minus spermine, but only a single step is observed in the presence of spermine alone. Thus the rate-limiting steps under normal assay conditions are different for the two cations. 3. Enzyme-bound isoleucyl-AMP can be formed in the absence of Mg-2+ and plus or minus spermine. 4. It is concluded that there is no evidence for cation-dependent differences in the reaction mechanism of isoleucine: tRNA ligase, though there are certainly differences in the relative rates of some of the individual steps.


Proton-magnetic-resonance studies of the lysine residues of ribonuclease A.

The amino groups of ribonuclease A (RNase-A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase-A. Resonances corresponding to the NH2-terminal alpha-amino and 10 xi-amino N-methyl groups are titrated at 220 MHz to obtain pK values. After correction for the effects of methylation, using values previously derived from model compound studies, a pK of 6.6 is found for the alpha-amino group, a pK of 8.6 for the xi-amino group of lysine-41 and pK values ranging from 10.6 to 11.2 for the other lysine xi-amino groups. Interactions between lysine-7 and lysine-41 or between the alpha-amino and xi-amino groups of lysine-1 have been proposed to account for deviations from simple titration behaviour. The correct continuities for the titration curves of the histidine H-2 proton resonances have been confirmed by selective deuteration of the H-2 protons. Titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A show deviations from the titration curves for the native enzyme, indicating some alteration of the active-site conformation. In the presence of phosphate, titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase-A. The titration curve for the N-methyl resonance of lysine-41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine-41.


Studies on the de novo biosynthesis of NAD in Escherichia coli. The separation of the nadB gene product from the nadA gene product and its purification.

Quinolinic acid (pyridine 2,3-dicarboxylic acid) which is an immediate precursor of the pyridine nucleotides, is synthesised from L-asparate and dihydroxyacetone phosphate in Escherichia coli. Extracts from certain nadB mutants complement the extracts prepared from all nadA mutants for the enzymic synthesis of quinolinate. Using the complementation assay, the quinolinate synthetase B protein has been purified more than 300-fold. The quinolinate synthetase B protein exists in all nadA and nadC mutants examined. The quinolinate synthetase A protein was present in all nadC mutants and most (but not all) nadB mutants. The facile separation of the wild-type quinolinate synthetase A and B proteins out of a nadC mutant suggests that quinolinate synthetase does not exists as a tightly bound complex. The partially purified quinolinate synthetase is inhibited by physiological concetrations of NAD and NADH but not by NADP or NADPH.


Biotin carboxyl carrier protein in barley chloroplast membranes.

Biotin localized in barley chloroplast lamellae is covalently bound to a single protein with an approximate molecular weight of 21 000. It contains one mole of biotin per mole of protein and functions as a carboxyl carrier in the acetyl-CoA carboxylase reaction. The protein was obtained by solubilization of the lamellae in phenol/acetic acid/8 M urea. Feeding barley seedlings with [14C]-biotin revealed that the vitamin is not degraded into respiratory substrates by the plant, but is specifically incorporated into biotin carboxyl carrier protein.


Substrate stereochemistry of the acetyl-CoA acetyltransferase reaction.

1. A specimen of symmetrically tritiated 3S-3-hydroxy[2-3H2]butyryl-CoA was prepared from acetoacetyl-CoA by incubation in tritiated water, followed by enzymic reduction using 3S-specific 3-hydroxyacyl-CoA dehydrogenase. In addition, a specimen of dideuterated, racemic 3RS-3-hydroxy-[2-2H2]butyryl-CoA was prepared chemically from 3RS-3-hydroxy[2-2H2]butyric acid. 2. These acyl-CoA derivatives were incubated respectively with deuterium oxide and tritated water in presence of enoyl-CoA hydratase. Stereospecifically tritiated compounds, respectively 2S,3S-3-hydroxy[2-2H1,3H1]butyryl-CoA and 2R,3S-3-hydroxy[2-1H1,3H1]butyryl-CoA (plus 3R-diastereomer) were formed and isolated. 3. 3S-3-Hydroxy[2-3H2]butyryl-CoA and the 3S-[2-3H2,3-14C]-labelled material were also prepared as described in the preceding paper. The latter substrate was used to establish conditions in which little loss of tritium would occur (found; about 7%) on cleavage to acetyl-CoA in the presence of 3-hydroxyacyl-CoA dehydrogenase and acetyl-CoA acetyltransferase. Little loss of tritium indicates a small degree of racemization of the stereospecifically 2-tritiated 3-hydroxyacyl-CoA derivatives at the level of transiently formed acetoacetyl-CoA and acetyl-CoA. This ensures high optical purity of the chiral acetates generated from the stereospecifically tritiated hydroxybutyryl-CoA specimens listed in paragraph (2). 4. These two acyl-CoA derivatives and the symmetrically tritiated 3S-3-hydroxy[2-3H2]butyryl-CoA listed in paragraph (3) were converted to the acetates enzymically in the sequence hydroxy-butyryl-CoA leads to acetoacetyl-CoA leads to acetyl-CoA leads to acetate. The isolated acetates were assayed for chirality by conversion to the malates and fumarates as usual. 5. The malate formed from 3S-3-hydroxy[2-3H2]butyryl-CoA on incubation with fumarase lost nearly 50%, that derived from 2R,3S-3-hydroxy[2-2H1,3H1]butyryl-CoA (plus 3R-diastereomer) retained about 26% and that derived from 2S,3S-3-hydroxy[2-1H1,3H1]butyryl-CoA retained about 78% of its total tritium content. 6. It was concluded that the detachment of acetyl-CoA from acetoacetyl-CoA on acetyl-CoA acetyltransferase occurs with inversion of configuration at the methylene group which becomes methyl.


The reactive sites of Kunitz bovine-trypsin inhibitor. Role of lysine-15 in the interaction with chymotrypsin.

Kunitz bovine trypsin inhibitor gave with alpha-chymotrypsin a stoichiometric complex stable at neutral pH. The complex has been characteristized by amino acid composition, molecular sieving and zone electrophoresis. Complete dissociation occurred at pH 4.0 as shown by gel filtration, alpha-Chymotrypsin was displaced from the complex by trypsin either in solution or by affinity chromatography on trypsin-Sepharos: alpha-chymotrypsin was recovered in the filtrate (yield about 100%) and the inhibitor was eluted from trypsin-Sepharose with 0.1 M HCl (yield: 83%). Lysine-15 of the inhibitor was shown to be involved in the interaction between alpha-chymotrypsin and the inhibitor. When the complex was maleylated, the maleylated chymotrypsin-bound inhibitor was displaced by affinity chromatography on trypsin-Sepharose. Teh recovered derivative was oxidized, subjected to tryptic hydrolysis and the products separated by peptide mapping and analyzed. The peptides were compared with those obtained with non-maleylated inhibitor and fully maleylated free inhibitor. In the fully maleylated inhibitor, the four lysyl residues of the molecule were blocked but in the maleylated chymotrypsin-bound inhibitor, Lys-15 was unmodified in contrast to Lys-26, Lys-41 and Lys-46; therefore Lys-15 is shielded by chymotrypsin in the complex. On the other hand, when inhibitor with a selectively reduced carboxamidomethylated Cys-14-Cys-38 dislufide bridge was allowed to react with chymotrypsin, cleavage occurred not only at Tyr-21, Tyr-35 and Phe-45 but also at Lys-15, cleavage not observed in the case of the fully oxidized inhibitor. This result shows that under particular conditions the bond Lys-15-Ala-16 can be the substrate for chymotrypsin and the side chain of Lys-15 can be inserted in the chymotrypsin specificity pocket. Apparently the contact area of inhibitor with chymotrypsin seems to be similar to that with trypsin [J. Chauvet and R. Acher (1967) J. Biol. Chem. 242, 4274-4275].


31P nuclear-magnetic-resonance studies of pyridoxal and pyridoxamine phosphates.  Interaction with cytoplasmic aspartate transaminase.

The 31P nuclear magnetic resonance (NMR) spectrum of the phosphate in free pyridoxal or pyridoxamine phosphate reveals a resonance signal that is coupled to the methylene protons of the 5-CH2 with JHP of 6.0 +/- 0.3 Hz. Proton noise decoupling results in a single signal with a pH-dependent chemical shift with deprotonation of the phosphate resulting in a shift of the 31P resonance to lower fields. A single 31P NMR signal at a frequency corresponding to fully ionized phosphate monoesters is observed in aspartate-transaminase-bound pyridoxal or pyridoxamine phosphate. The 31P resonance in the holotransaminase is pH-independent and is unaffected by saturating concentrations of substrates or inhibitors. Only denaturation with 6 M guanidine with HCl results in changes in the 31P of the holoenzyme. It appears that the phosphate group of pyridoxal phosphate is bound to a positive pocket in the holoenzyme and remains fully ionized in the pH range of 5.6 to 9.2. The phosphate-binding properties are present even in the apoenzyme which is able to bind inorganic phosphate which then can be displaced by pyridoxal or pyridoxamine phosphate in the process of holoenzyme formation.


Characterization of a specific transport system for arginine in isolated yeast vacuoles.

The transport of L-arginine was studied in isolated vacuoles of Saccharomyces cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured. Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts. The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined. From the temperature dependence, the apparent energy of activation was calculated to be 9800 cal/mol. Arginine transport shows saturation kinetics with an apparent Km of 30 muM in the isolated vacuoles, and of 1.5 muM in the spheroplasts. Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities. The inhibition of vacuolar L-arginine transport by D-arginine, L-histidine, and L-canavanine was competitive with apparent Ki values of 60 muM, 400 muM and 600 muM respectively.


Excretion of trypsin-like activity, electrolytes and protein in mixed and parotid saliva of patients with cystic fibrosis of the pancreas.

The esterolytic activity of mixed and parotid saliva in cystic fibrosis (CF) patients and normal subjects was determined using BAEE (alpha-Benzoyl-1-arginine-ethylester) as the substrate. Using soybean-trypsin-inhibitor the trypsin-like activity (TLA) was measured and plotted as a function of parotid flow rate. In addition calcium, protein and pH were determined. Trypsin-like activity in mixed and parotid saliva showed large individual variations in CF and normal children. In parotid saliva we could not find any significant difference, whereas a reduction of TLA in mixed saliva of CF patients was observed. The fact that our normal values fell within the range of heterozygotes reported by Rao et al. (19), makes their hypothesis of a close relationship between reduced TLA and the genetic defect very doubtful. Protein, calcium and pH increased with augmented salivation and no difference between CF patients and normal age matched children could be found except for the pH at a flow rate above 0.75 ml/min per m2 body surface where significantly lower pH values resulted. The relevance of reduced TLA to the pathogenesis of cystic fibrosis is discussed.


Effects of aldrin-transdiol on neuromuscular facilitation and depression.

The effects of aldrin-transdiol, one of the active metabolites of the insecticide dieldrin, on evoked transmitter release, neuromuscular facilitation and neuromuscular depression have been studied in frog sartorius nerve-muscle preparations. Conventional techniques of intracellular recordings were used. Aldrin-transdiol caused a marked increase in end-plate potential amplitude under conditions of low quantal content, i.e., in Mg2+-blocked junctions. The increase in end-plate potential amplitude was less pronounced in curarized junctions, in which the transmitter release was not impaired. Concomitant with the increase in end-plate potential size there was a marked enhancement of facilitation during short trains of stimuli applied to the motor nerve. The decay of facilitation was, however, not seriously affected by aldrin-transdiol. These effects may be explained in terms of the known 'calcium hypothesis' by assuring that aldrin-transdiol increases the amount of calcium which enters the nerve terminal during the nerve impulse. The increase in end-plate potential amplitude and in facilitation by aldrin-transdiol was transient. At later stages of poisoning, end-plate potential and facilitation decreased below control level and neuromuscular depression was significantly enhanced. This latter effect may be the result of a direct inhibitory effect of aldrin-transdiol on transmitter mobilization. As far as the fall in end-plate potential amplitude is concerned the known suppressive action of aldrin-transdiol on the chemical sensitivity of the postsynaptic membrane and on the nerve action potential probably plays a part as well. Finally, neuromuscular transmission was completely blocked by aldrin-transdiol.


Effects of psychotropic drugs on methamphetamine-induced behavioral excitation in grouped mice.

When 7 mice injected with methamphetamine (5 mg/kg, s.c.) were placed together into a box with a limited space maintained at 28-30 degrees C, the grouped mice showed extreme behavioral excitation consisting of 2 typical features: fighting behavior with squeak and hyperactive behavior characterized by running and jumping. The effects of various drugs tested, according to the minimal effective doses (mg/kg, p.o.) which inhibited fighting behavior and hyperactive behavior respectively, were: haloperidol, 0.1 (fighting)--0.25 (hyperactivity); clothiapine, 1 - 1; perphenazine, 1 - 1; thiothixene, 1 - 5; chlorpromazine, 5 - 10; diazepam, 5 - 100; reserpine, 100 - 100; phenoxybenzamine, 200 - 200 (no effect). Major tranquilizers except rauwolfia alkaloids completely inhibited both types of excited behavior at low doses, as compared with the taming effects of the drugs on footshock-induced fighting episodes in pairs of mice. On the other hand, minor tranquilizers suppressed only fighting behavior, showing little effect on hyperactive behavior. None of the antidepressants, adrenergic blocking agents and other drugs showed antagonism to both types of excited behavior. The results indicate that this method may be useful for the evaluation of psychotropic drug effects in grouped mice.


Beta-adrenergic blockade and atrio--ventricular conduction impairment.

Atrio--ventricular conduction and its modifications induced by six Beta-adrenergic blocking agents have been investigated in the dog. Premature atrial stimuli (St2) were applied at variable intervals following regular stimuli (St1) ensuring atrial pacing; atrial (AERP), nodoventricular (NERP) and global (GERP) effective refractory periods as well as global functional refractory period (GFRP) were determined before and after administration of each of the six drugs. When Beta-blockade was produced with d,1-propranolol which hwas membrane stabilizing effects (MSE) but no intrinsic sympathomimetic activity (ISA) or with sotalol, which has neither MSE nor ISA, all parameters were significantly increased. When Beta-blockade was achieved with pindolol or practolol, which have only a poor Beta-adrenolytic potency and no ISA. Alprenolol showed intermediate effects. Thus, it appears that Beta-blockade and not MSE, is responsible for the onset of A-V conduction impairment but that ISA, probably through a metabolic mechanism, affords protection against this impairment. On the other hand, measurement of ventricular effective refractory period (VERP) has shown that at the Purkinje-free junction, it is MSE which is mainly involved in conduction impairment.


Control of peripheral vascular resistance in perfused hindquarters of dogs: lack of effect of nicotinic and muscarinic ganglion block.

The effects of nicotinic and muscarinic ganglion blockade were studied on the peripheral resistance of the perfused hindlimbs of the dog. Nicotinic ganglion blockade by chlorisondamine (1 mg/kg) significantly reduced reflex vasomotor responses but did not significantly alter hindlimb peripheral resistance. Combined nicotinic ganglion blockade by chlorisondamine (1 mg/kg) and muscarinic ganglion blockade by atropine (50mug/kg) abolished the reflex vasomotor response but did not alter hindlimb peripheral resistance. It was concluded that autoregulatory mechanisms are capable of maintaining a peripheral resistance after block of both muscarinic and nicotinic ganglion transmission.


Antinociceptive action of oxotremorine and regional turnover of rat brain noradrenaline, dopamine and 5-HT.

In the rat, oxotremorine increases the threshold for vocalisation after-discharge (affective component of pain reactions) dose dependently at subtremor doses (30-67 mug/kg s.c.). Doses of 225-506 mug/kg were needed to elevate the thresholds for vocalisation and motor response. 1-Tryptophan, PCPA, alpha-methyl-p-tyrosine, 1-Dopa, pimozide and LSD-25 did not affect the antinociceptive activity of oxotremorine, while phenocybenzamine slightly increased the threshold for vocalisation. Oxotremorine did not change the endogenous brain concentrations of noradrenaline and dopamine or 5-HT but decreased that of 5-HIAA in all brain regions at the time of maximal analgesia. The decrease of 5-HIAA was still present after pretreatment with probenecid. After inhibition of tyrosine hydroxylase, oxotremorine accelerated the depletion of dopamine in telencephalic cortex during maximal antinociceptive activity and of noradrenaline in all brain regions at a time when this activity had vanished. Atropine significantly antagonized the analgesic activity of oxotremorine. It is concluded that oxotremorine antinociceptive activity in the rat is related to a cholinergic compoent, while a monoaminergic component is not directly involved.


The effect of penfluridol and some psychotropic drugs on monoamine metabolism in central nervous system.

Five neuroleptics: penfluridol, haloperidol, chlorpromazine, pimozide and oxypertine, and two benzodiazepine minor tranquilizers; chlordiazepoxide and diazepam, were studied in mice and rats. When the drugs were injected i.p. in mice, all neuroleptics increased the brain homovanillic acid (HVA) concentration but two benzodiazepines did not change it. Of the drugs tested, only pimozide increased the brain level of 5-hydroxyindoleacetic acid. Penfluridol accelerated the turnover rate of brain dopamine (DA), wheras haloperidol accelerated both DA and noradrenaline turnover rates. In rats, oral administration of penfluridol caused an increase in the striatal HVA and 3,4-dihydroxyphenylacetic acid which persisted longer than that following haloperidol. The concentration of brain 3-methoxy-4-hydroxyphenylethylene glycol, however, was not affected by penfluridol even when the drug was administered at a high dose. Based on these and earlier findings, it is concluded that the effect of penfluridol on DA metabolism resembles that of typical neuroleptic compounds and penfluridol acts only on DA neurons as a long-acting DA receptor blocker.


Effects of some adrenergic and cholinergic drugs on isolated spleen strips from the cod, Gadus morhua.

The effects of drugs on isolated spleen strips from the cod, Gadus morhua, have been investigated. Affinities and intrinsic activities for various agonists were determined from cumulative dose-response curves. Contractions were produced by acetylcholine, methacholine or carbachol. The response to acetylcholine was considerably potentiated by the acetylcholinesterase inhibitor BW 284 C51. Methacholine-induced responses were competitively blocked by atropine (pA2 = 8.2), indicating the presence of muscarinic receptors. Nicotine did not contract the spleen strips. Adrenaline, noradrenaline or phenylephrine contracted the preparations and so did isoprenaline in high concentrations. Competitive blockade of the noradrenaline-induced contraction was obtained with yohimbine (pA2 = 6.1), phentolamine (pA2 = 6.0) and propranolol (pA2 = 3.4), and non-competitive blockade with phenoxybenzamine (pM50 = 7.0), indicating that alpha-adrenoceptors mediate the contractions produced by the adrenergic agonists. Isoprenaline caused a slight relaxation of preparations precontracted with methacholine. This effect was abolished by propranolol suggesting the presence of beta-adrenoceptors. Judged from the pA-values for the competitive antagonist, arterial and capsular/trabecular smooth muscles have slightly different alpha-adrenoceptor properties.


Rates of onset and offset of action of narcotic analgesics in isolated preparations.

In isolated preparations of the myenteric plexus--longitudinal muscle of the guinea-pig ileum and the mouse vas deferens, the rates of onset and offset of action of narcotic analgesics are inversely related to lipid solubility; this effect is probably due to drug binding at secondary lipid-rich binding sites. These findings are in contrast to observations in vivo by A. Herz and his colleagues, who showed that the rates of onset of action and of recovery are directly related to lipid solubility because of the presence of lipid-rich barriers. In view of the opposite effects, an optimum may be expected when a drug has a degree of lipid solubility which ensures rapid penetration of the blood--brain barrier without seriously slowing down of receptor association and dissociation. From the interaction of rapidly acting quaternary antagonists, e.g. N-methylnalorphinium, with slowly acting agonists, e.g. methadone, it is concluded that at least a part of the receptor site is on the surface of the cell membrane, and the possibility of an allosteric agonist--antagonist interaction is considered.


Actions of bile salts and of papaverine and intracellular cyclic AMP in isolated rat uterus.

Both antioxytocin and antiphosphodiesterase activities of desoxycholate were potentiated by lowering the pH of the medium. The results suggest the possibility that it is the bile salt in the non-ionized form which exerts the antioxytocin and antiphosphodiesterase action. The concentration of tissue cyclic AMP was measured at different times during relaxation of the uterus. The results show a significant increase in tissue cyclic AMP concentration at a time when the muscle was just beginning to relax in response to papaverine, but not in response to chenodesoxycholate. The intracellular level of cyclic AMP during relaxation of the uterus produced by papaverine or chenodesoxycholate was, however, significantly increased when relaxation was about 90% complete. The difference between the mode of action of papaverine and that of the bile salts is discussed.


Antagonism by propranolol of the ganglion stimulant action of 5-hydroxytryptamine.

The effects of racemic propranolol and its constituent isomers were studied on ganglionic stimulation produced in situ by close arterial injection of 5-HT and DMPP to the superior cervical ganglion. Ganglion stimulation was recorded in terms of the resultant contraction of the nictitating membrane. d,l-Propranolol caused a biphasic antagonism of the ganglion stimulant effect of 5-HT. At low doses, 0.5-10 mug, the antagonism was surmountable by increasing the amount of 5-HT. The l-isomer (0.2-4 mug) but not d-propranolol also caused antagonism. At higher doses, 0.1-5 mg, both d,l- and d-propranolol caused a second type of blockade of 5-HT which was not surmountable and resembled that seen with procaine. The ganglion stimulant effects of DMPP and acetylcholine were only antagonised by the higher doses of d- and d,l-propranolol. d,l-Propranolol did not reduce the direct stimulation by 5-HT on the muscle of the nictitating membrane.


Effect of benzodiazepine compounds on brain amine metabolism.

In mice, chlordiazepoxide, diazepam and flurazepam reduced 5-HT synthesis and metabolism in the teldiencephalon following an i.v. injection of 3H-tryptophan. Only diazepam produced a significant effect in the brainstem. The steady state level of brain 5-HT or of tryptophan was not altered by any of the drugs. Chlordiazepoxide and diazepam, but not flurazepam, decreased the rate of norepinephrine loss from the brain following inhibition of synthesis with alpha-methyltyrosine.


Attenuation of hydrochlorothiazide-induced hypokalemia in dogs by a beta-adrenergic blocking drug, timolol.

Hydrochlorothiazide, administered at 1, 3 and 9 mg/kg/day p.o. for 4 days, produced a dose-dependent lowering of plasma potassium and an elevation in plasma renin activity in unanesthetized dogs. When plasma renin activity was suppressed in diuretic-treated dogs by the potent beta-adrenergic receptor-blocking drug, timolol (0.5, 2 mg/kg/day p.o. for 4 days), the hypokalemia and hyperreninemia were significantly less pronounced. The data suggest that beta-adrenergic blocking drugs can be used to antagonize these side effects of diuretics.


Stereospecific morphine adsorption to phosphatidyl serine and other membranous components of brain.

In the course of investigating various membranous components for morphine binding, it was found that the major substance responsible was phosphatidyl serine. Other acidic lipids, such as phosphatidic acid and phosphoinositides bind to a considerably lesser extent, while neutral lipids, glycolipids and other phosphatides bind slightly or not at all. Total lipid extracts from a number of regions of rat brain exhibited different degrees of binding to (-)-morphine, such regions as the cerebral cortex and thalamus being the greatest. From an examination of the pH curve for binding and the effect of ionic strength, it was concluded that the binging was largely electrostatic. The method employed was the radioactive measurement of 14C-morphine adsorption to surface films of phosphatidyl serine. When the phosphatide was dispersed in a nonionic detergent near the critical micelle concentration, adsorption was maximal, attaining a value of 1 molecule of morphine adsorbed per molecule of phosphatidyl serine in the surface micelle. The relation between binding affinity and biological potency was not consistent. Morphine adsorption occurred with films prepared from a dispersion of the phosphatide and a hydrophobic protein from synaptic membranes. With the use of levorphanol and dextrorphan it could be shown that binding of morphine was stereospecific.


Reduction of adverse acute cardiovascular actions of 6-hydroxydopamine in dogs by adrenergic receptor blockade.

Injection of dogs with 50 mg/kg of 6-hydroxydopamine (6-OHDA) caused large increases in blood pressure and heart rate, followed after 1 or 2 hr by severe hypotension. Prior administration of the adrenergic receptor blocking agents phentolamine and propranolol attenuated both the hyper- and hypotensive phases of 6-OHDA. Phentolamine and propranolol did not prevent depletionof norepinephrine or development of the characteristic morphological changes induced in adrenergic fibers by 6-OHDA.


Effect of graft-versus-host reaction on the immune response to alloantigens and growth of a syngeneic tumor.

The graft-versus-host reaction (GVHR) was evoked in (C57BL/6J times C3H/6J times DBA/2J) F1 hybrids by grafting parental lymphoid cells. Groups of bothe F1 hybrids were immunized with thymocytes carrying Thy-1.1 (theta AKR) antigen and still other groups of (C57BL/6J times C3H/HeJ) F1 hybrids were immunized with thymocytes carrying the H-2-d antigen. Plaque-forming cells (PFC) producing antibodies to the Thy-1.1 antigen, lytic for AKR thymocytes, or antibodies to the H-2-d antigen, lytic for lymphoblasts carrying the H-2-d antigens, were enumerated in spleens of experimental animals. A pronounced suppression of the immune response to the Thy-1.1 antigen was found in animals suffering from GVHR. The suppression could be demonstrated in some animals as late as 180 days after the onset of GVHR. Significant, although less pronounced suppression was found when response to the H-2-d antigens was assayed. After grafting 10-3 or 10-4 DBA/2J derived lymphoma cells (L5178Y) into (C57BL/6J times DBA/2J) F1 hybrids, clinically detectable tumor growth was found earlier and more frequently in hybrids suffering from GVHR than in control animals. The results obtained lend support to the concept that immunosuppression accompanying GVHR may influence the formation and/or growth of the malignant tumors. In addition, a synergistic effect in the suppression of the responsiveness of F1 recipients was seen in some instances when a mixture of parental bone marrow and thymus cells was grafted. The results are consistent with data indicating that more than one cell type is involved in the initiation of GVHR.


Psychophysical evaluation of toxic effects on sensory systems.

Toxic effects on sensory systems have rarely been evaluated by psychophysical methods. As examples of possible applications four studies are described. Sodium salicylate and kanamycin, both reported to produce hearing deficits in man, have also been demonstrated to affect auditory thresholds in monkeys. With the latter drug the deficits measured were found to be correlated with specific loss of receptor cells in the cochlea. Pheniprazine, known to induce red-green color blindness, was found to disrupt a wavelength discrimination in pigeons. Trans 11-amino-10,11-dihydro-5-(3-dimethylaminopropyl)-5,10-expoxy-5H-dibenzo[a,d]-cycloheptene dihydrochloride, which was found to bleach the tapidum lucidum in dogs when given subacutely, was found to decrease sensitivity to light. The loss in sensitivity measured by behavioral techniques was correlated with the loss of coloration of the tapidum. Monkeys, not having a tapidum, did not show a similar effect.


Evidence for the involvement of alpha-adrenoceptor blockade in the antihypertensive action of diazoxide in the renal hypertensive rat.

The effects of diazoxide on the blood pressure and heart rate of conscious renal hypertensive rats have been investigated. The antihypertensive action of diazoxide has been studied in relation to the effects of diazoxide pretreatment on pressor responses to stimulation of the complete sympathetic outflow and to injections of noradrenaline, phenylephrine, angiotensin and serotonin in pithed rats. In pithed preparations, pressor responses to sympathetic nerve stimulation, and to injected noradrenaline and phenylephrine were significantly reduced by diazoxide pretreatment, at a time corresponding to maximal reduction of blood pressure in conscious animals. At this time there was no significant reduction of pressor responses to injected angiotensin or serotonin. These findings suggest a contribution of alpha-adrenoceptor blockade to the antihypertensive activity of diazoxide.


Determinants of drug effects on punished responding.

The effects of drugs on punished responding depend on interactions among a large number of experimental variables. Among these variables are the drug history of the animal, the dose of the drug administered, the type of stimulus used to punish responding,  the intensity and duration of the punishing stimulus, the schedule of presentation of the punishing stimulus, the control rate and pattern of punished responding, the schedule of positive reinforcement maintaining the punished responding, the species of animal, the deprivation state of the animal, the behavioral history of the animal, and the nature of the required response. Although it is not known how all of these variables interact to determine the effect of drugs on punished responding, there is evidence that many of these variables are important as determinants of drug effects. The task facing behavioral pharmacologists studying drug effects on punished responding is to determine under what conditions drugs produce their characteristic effects on punished responding.


Reinforcement schedules and extrapolations to humans from animals in behavioral pharmacology.

Behavior controlled by various schedules of reinforcement is useful for characterizing drugs as well as for analyzing the mechanisms of action of their effects on behavior. Conditioned avoidance techniques have been useful for studying neuroleptics and for predicting their clinical antipsychotic acitivity; the possible involvement of dopaminergic mechanisms in the effect of neurolpetics on avoidance behavior is discussed. Tricyclic antidepressant agents have been studied in assays involving interactions with other agents, such  as cocaine, amphetamine and tetrabenazine. One type of operant behavior, Sidman avoidance, has been used as particularly sensitive assay for such drug interactions. Another schedule, in which "observing" responses in pigeons are measured. seems to provide a method for studying antidepressants without involving drug interaction phenomena. For tricyclic compounds, facilitation of observing responses and weak potency of conditioned avoidance inhibition constitute a pharmacological profile that seems to have some predictive value for clinical imipramine-like antidepressant activity. "Conflict (punishment) schedules have been useful for predicting antianxiety activity in man. Although the degree of anticonflict effect observed is consistent with Dew's rate dependency hypothesis, this principle does not fully account for the observed drug effects. In the conflict model, the actions of benzodiazepines differ in drug-naive versus drug-experienced animals. Experiments with parachlorophenylalnine have not yet provided clear support for the postulated role of serotonin in related phenomena.


Androgen biosynthesis in experimental cryptorchidism.

This study was conducted to investigate the effects of unilateral cryptorchidism on androgen production and testicular maturation. Experimental cryptorchidism was produced in small and large prepuberal guinea pigs by forcing the testis to remain in the abdomen for a period of 30 days. Small prepuberal animals, which did not reach sexual maturity, showed discrete reductions in the size and weight of the ectopic testes when compared with the scrotal testes. Immaturity of germinal line, tubular size, and intertubular spaces were more pronounced in the ectopic tissue. Both glands produced 4-androstenedione as a primary metabolic product of pregnenolone. Large prepuberal animals, puberal at the time of operation, showed tubular size, spermatic line, and interstitial tissue completely developed in the scrotal gland. Testosterone production was quite similar to that produced in postpuberal testes. The ectopic organ showed only Sertolic cells, some vacuolated, and a few spermatogonia in the tubules. The spaces contained Leydig cells. A significant accumulation of 4-adrenostenedione and a lower testosterone production, compared with that found in the scrotal gland, were observed. The histologic immaturity and the hormonal biosynthesis of the puberal cryptorchid testis parallel findings in scrotal glands from sexually immature guinea pigs.


Activated paper as an immunoabsorbent.

Some workers when using cyanogen bromide activated paper as a solid immunoabsorbent have reported variable success in radio immuno assays. A quantitative investigation has been made of some of the factors which effect the activation of various papers with CNBr, and also of the subsequent coupling reaction with the sensitizing antigen. Different papers have been found to react in different ways, and some papers take up so little antigen that they are unsuitable for use for this purpose.


Standardization of Dermatophagoides pteronyssinus extracts and tyrosine adsorbates.

Human skin test, inhibition of RAST and the radioimmunosorbent technique of Ceska have been shown to be useful assays for the standardization of D. pteronyssinus aqueous extracts. The development of these assay systems for the standardization of D. pteronyssinus tyrosine adsorbed formulations is described. Tyrosine solubilisation procedures are detailed together with the influence of these procedures on the three assays. Good correlation has been shown between the human skin test and the radioimmunosorbent technique. The inhibition of RAST appears to be more sensitive than the other techniques to mild conformational changes in the allergen, induced in this work by acid pH. This may limit its value for the standardization of D. pteronyssinus tyrosine adsorbates but it may prove useful in monitoring manufacturing processes.


The multi-facets of tuberculin standardization.

Some parameters affecting the standardization of a tuberculin preparation have been evaluated and it has been shown that: 1) The loss of tuberculoprotein on the surfaces of containers is in the order of 0.15 mug/cm2 and the 0.18 mug/cm-2 for glass and plastic surfaces respectively, and that this loss can be prevented by the addition of a nonionic anti-adsorption agent such as Tween 80. 2) For non-stabilized tuberculin PPD solutions the losses of PPD due to adsorption increased linearly with the Surface to Volume ration. 3) Exposure to light had a deleterious effect on tuberculin PPD decreasing its biological potency and altering its appearance. This effect increased with the dilution of the tuberculoprotein solution, and can be prevented by storing the tuberculin in amber containers. 4) The potency of tuberculoprotein was dependent on its molecular weight, and the % distribution of tuberculoprotein molecules of different molecular weight varied from batch to batch. This variation can be eliminated by preparing a large batch of tuberculin in a dried form sufficient to fill the demand for many years to come. 5) The route of injection and the species or strains of mycobacteria used for sensitization of guinea pigs affected the relative potency of a tuberculin preparation. 6) The size of a tuberculin skin reaction increased as the volume of tuberculin injected intracutaneously into sensitized guinea pigs was increased although the number of tuberculin units administered was the same. 7) The sizes of skin reactions to certain doses of tuberculin was affected by other doses of tuberculin being injected simultaneously at other sites. 8) Sex and age of guinea pigs affected the size of the tuberculin skin reaction. 9) The genetic make-up of the test animals affected the relative potency of a given tuberculin preparation. 10) Racial differences among various populations might account for different levels of tuberculin reactivity to a given tuberculin preparation. It is hoped that this work has made us aware of the many problems which can be encountered in standardizing tuberculin and it is also to be hoped that the solutions we have offered to some of these problems can be useful to those attempting to standardize allergens other than tuberculin.


[An analysis of the rise of health care costs in institutions in Canada and Quebec].

The author compares increases in institutional care costs in Quebec with the rest of Canada revealing the following: The 14 per cent annual increase reported by federal health minister Marc Lalonde is well above the increase in the Canadian population of 4 per cent. The 12 per cent rise in costs of Quebec health services is far above the increase in the population of 15 per cent. Therefore, population is not a major factor in cost increase. The rate of cost increase in Quebec is below the national rate, 12 per cent against 14 per cent. Cost of institutional care in dollars per capita is appreciably lower in Quebec than in the rest of Canada. The average increase in health care costs in Canada between 1970 and 1973 is actually above the average deseasonized increase rate of the gross national product (GNP). The average cost increase of institutional care remained, however, below the increase of the GNP in Canada. The increase in the number of patients admitted is far above the increase in population, in both Quebec and the other provinces, the difference being more important in Quebec than in the rest of Canada. Utilization has partly been possible through decrease in the average length of stay in both Quebec and the other provinces. The decrease in average length of stay is higher in other Canadian provinces than it is in Quebec. However, it is lower in Quebec for a day. The occupancy rate has consistantly been rising in Quebec while it remained stable in other provinces. The number of patients admitted per 1,000 population has remained much lower in Quebec as compared to the rest of Canda. The difference tends, however, to decrease. The number of institutional care facilities remains higher in other Canadian provinces than in Quebec in proportion to the population The number of visits in emergency units per 1,000 population has doubled in Quebec, as compared to the rest of Canada with half that increase. It would be interesting to establish the trend in institutional care for outpatient departments other than emergency units; complete data about this not as yet availble.


Course and prognosis of 86 episodes of diabetic coma. A five year experience with a uniform schedule of treatment.

The admission data and the course of 58 episodes of severe diabetic ketoacidotic coma and of 28 episodes of non-ketotic coma are compared. The non-ketotic patients were older; initial blood glucose, osmolarity, blood urea and serum sodium concentration were higher than in the ketotic patients. Treatment in the first 24 hrs consisted of similar amounts of insulin in both coma forms, the presence of acidaemia did not increase the insulin needs. Acidaemia was corrected only when pH was below 7.20. The disadvantages of alkali therapy are emphasized. A comparison of the age groups of survivors and those patients who died within 72 hrs showed an increase in mortality with age. However, the mortality rates from ketotic and non-ketotic coma were similar in the age groups above 50 years. On admission, blood glucose, osmolarity and blood urea were higher in the fatal cases. Blood urea was the most important indicator of a fatal outcome. The response of blood glucose to insulin was impaired in the subsequently fatal cases. Insulin was given in "moderate" doses by constant infusion. The use of "small" doses is discussed. Early mortality was 14% in the ketotic and 29% in the non-ketotic cases. The most frequent causes of death were circulatory failure of undetermined origin, infections and thromboembolic complications.


Properties of aryl hydrocarbon (benzo[a]pyrene) hydroxylase in the liver from C3H/He and DBA/2 strains of mice.

Aryl hydrocarbon hydroxylase activity in liver from the C3H/He strain of mice is apparently increased by the administration of 3-methylcholanthrene, but the enzyme activity from the DBA/2 strain of mice is not. The enzyme activity from female mice is higher than that from male mice, even 72 hr after a single application of 3-methylcholanthrene into the mice. The control enzyme in the liver from both strains of mice has a pH optimum at 7.9 and the induced enzyme from C3H/He mice, at 8.2. There are approximately the same levels of the apparent Km for benzo[a]pyrene, NADPH, or NADH in the liver enzyme from both strains of mice even after treatment with 3-methylcholanthrene. The induced enzyme is inhibited by 7,8- or 5,6-benzoflavone non-competitively, and nicotinamide inhibits both the constitutive and induced enzyme uncompetiviely. Cyclohexene oxide and 1,1,1-trichloropropane oxide, known to be inhbitors of epoxide hydrase, inhibit the activity of the constitutive enzyme, but enhance the induced enzyme in the liver. The difference in the properties between the constitutive and induced enzymes from mouse liver is discussed briefly.


Studies on the binding of amylopectin sulfate with gastric mucin.

Amylopectin sulfate, a sulfated polysaccharide that has an antipeptic property, was examined for its ability to bind gastric mucins. After chemically cross-linking the amylopectin sulfate into an insoluble gel, its binding with mucins isolated from antral and fundic mucosa of canine stomachs was studied with chromatography. A component present in both mucin fractions bound to the amylopectin sulfate gel below pH 4.5. This binding was reversible, and the complex dissociated above pH 5. Similar binding properties were found with soluble amylopectin sulfate. The component of the mucine which bound to amylopectin sulfate differed from the one which did not bind in its electrophoretic mobility and in its higher proportion of basic amino acids and a lower hexosamine, serine, and threonine content. This study suggests that amylopectin sulfate may bind to gastric mucins only under conditions of low pH.