Appearance of tyrosine hydroxylase, aromatic amino-acid decarboxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase during the ontogenesis of the adrenal medulla: an immunohistochemical study in the rat. The cellular localization of the enzymes tyrosine hydroxylase (TH), aromatic amino-acid decarboxylase (or dopa decarboxylase, DDC), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of adult rats and rat fetuses (14th, 17th, 18th, 19th and 21st day) was examined. In the prenatal stages the medullary blastema and an adjacent part of the primitive sympathetic trunk were also investigated. Tissues were fixed in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Cryostat sections (10 micron in thickness) were stained by the indirect immunofluorescence technique. Rabbit antibodies to TH (isolated from human pheochromocytoma), DDC, DBH and PNMT (the latter three isolated from bovine adrenal medulla) were used. Sections incubated with serum of non-immunized rabbits were used as controls. In the adult adrenal medulla, two cell types can be distinguished. One cell type contains only TH, DDC and DBH. The other cell type contains PNMT in addition. It is concluded that these cells correspond to the noradrenaline-(NA-) and adrenaline- (A-)storing cells respectively. In all prenatal stages TH, DDC and DBH are found in the primitive sympathetic trunk, in the medullary blastema, and in the medullary cells which have migrated into the cortical "anlage". PNMT is observed for the first time on the 18th day. Moreover, PNMT could only be demonstrated inside the adrenal gland. From these observations it is concluded that the capacity to synthesize NA is developed even before the "medullary" cells have reached the cortical "anlage". On the contrary, the capacity to synthesize A seems to be acquired only after this contact is established. The hypothesis is put forward that this phenomenon might indicate the induction of PNMT by glucocorticoids secreted by the fetal cortex.


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


The interaction of aliphatic analogs of methylene-dioxyphenyl compounds with cytochromes P-450 and P-420. The spectra resulting from the interaction of a series of substituted dioxolanes with microsomal cytochromes P-450 or P-420, as well as purified cytochrome P-450, were measured. With the exception of dioxolane, 4-methyldioxolane and 4-ethyldioxolane, these compounds interacted with ferric cytochrome P-450 to give complexes exhibiting type I optical difference spectra, and, after incubation with NADPH, spectra with peaks at about 430 nm. These complexes, as well as those formed from dioxolanes in the presence of cumene hydroperoxide, inhibit the binding of CO to the cytochrome. Consideration of the known chemistry of dioxolanes, together with recent advances in the understanding of double Soret spectra, lead to a possible explanation for the differences between the spectra of dioxolanes and their aromatic analogs, the methylenedioxyphenyl compounds.


[Can blockade of presynaptic adrenergic beta receptors explain the antihypertensive effects of propranolol?]. Pretreatment with 6-hydroxydopamine abolished the antihypertensive effects of intravenously and intracisternally administered dl-propranolol in dogs with acute neurogenic hypertension. These results show that the anti-hypertensive action of this drug is dependant on the integrity of central catecholaminergic neurones and suggest an action on presynaptic bêta-adrenoceptors.


The effect of methylprednisolone on hepatic oxygen supply and plasma lactate and glucose in endotoxemia. This study was designed to determine the effect of methylprednisolone on the profile of hepatic oxygen supply and selected blood parameters in fasted, male rats administered an LD85 dosage of E coli endotoxin intraperitoneally. Mortality rates within 24 hours were 85% in rats receiving endotoxin only, 9% in rats receiving a 30 mg/kg dosage of methylprednisolone intraarterially one hour subsequent to endotoxin insult, and 0% in methylprednisolone controls. Beginning with the fourth hour, untreated endotoxin rats had significantly higher heart rates and lower plasma glucose; by the sixth or eighth hour there was significantly greater hypocapnia, lower blood pH, and higher plasma lactate levels in comparison to endotoxic rats receiving methylprednisolone. In addition, mean hepatic pO2 between the sixth and seventh hours was 2.6 mm Hg in endotoxic rats, 10.6 mm Hg in endotoxic methylprednisolone rats, and 17.7 mm Hg in methylprednisolone controls. Methylprednisolone controls showed a steady increase of plasma glucose levels through eight hours but were otherwise stable. Maintenance of hepatic circulation is cited as the probable basis for differences of morbidity and mortality between treated and glucocorticoid-treated endotoxic rats.


A controlled study on the preventive effect of ketotifen, an antiallergic agent, on methacholine-induced bronchoconstriction in asthmatics. We studied the preventive effect of ketotifen, an oral drug with antianaphylactic and antihistaminic properties on methacholine-induced bronchoconstriction in controlled cross-over experiments in twenty-six adult patients with extrinsic asthma. Both a single dose of 1 mg ketotifen and 4 weeks treatment of ketotifen, 1 mg twice daily, failed to reduce the methacholine-induced drop in peak expiratory flow. The spirometric findings remained unchanged during ketotifen treatment. There was no difference between treatments with ketotifen and placebo with regard to the patients assessment of the severity of asthma or airway sensitivity to tobacco smoke, fumes or dusts, or exercise. The results suggest that treatment during 4 weeks with ketotifen does not reduce unspecific broncial hyperreactivity in patients with extrinsic asthma.


Improvements in and clinical utility of a continuous-flow method for routine measurement of dialyzable (ultrafiltrable) calcium. We describe modifications to the original continuous-flow procedure for dialyzable calcium (Clin. Chem. 23: 1258, 1977) needed to make the method more suitable for routine clinical laboratory use. The modifications simplify the continuous-flow (AutoAnalyzer) manifold, decrease baseline noise, increase the sensitivity, and permit use of a less-expensive fluorometer. Bias due to variation in serum processing is minimized by use of serum samples minimally exposed to air and a pH 7.40 buffer in place of the routinely processed sera and pH 7.30 buffer used formerly. Day-to-day precision (CV) during the past year for samples that included three different lots of quality-control sera was 2 to 3%. The analysis requires 200 micro L of serum, collected with minor additional precautions. We find that dialyzable calcium can be dependably measured in the routine service laboratory and show how this information is clinically more useful than is information on total calcium in serum.


Determination of lysosomal enzymes in saliva. Confirmation of the diagnosis of metachromatic leukodystrophy and fucosidosis by enzyme analysis. This paper deals with the measurement of acid hydrolases in uncentrifuged and centrifuged saliva of normal controls and the determination of some of their physical characteristics such as pH optimum, thermal stability, and residual activity after freezing and storage. From this study it appears that beta-D-glucosidase sediments totally, whereas the other enzymes show varying residual activity in the supernatant after centrifugation. In the saliva from 2 patients with metachromatic leukodystrophy and 2 patients with fucosidosis a deficiency of arylsulphatase A and alpha-L-fucosidase, respectively, was found.


Carboxypeptidase B activity of cultured skin fibroblasts and relationship to cystic fibrosis. Enzyme activity against hippuryl-L-arginine was studied in cultured skin fibroblasts from controls and cystic fibrosis patients. The enzyme had a lysosomal distribution and an acid optimum of pH 4.5 with little or no activity present above pH 7.0. Dithiothreitol was required for full activity and the kinetics of thiol activation were different for the control and cystic fibrosis enzyme. The properties and lysosomal distribution of the enzyme indicated that it was a carboxypeptidase B. Substrate affinity, thermolability, pH stability, the fall and rise in activity with subculture, the cyclical pattern of activity through serial passage and the level of activity were similar for the control and cystic fibrosis enzyme.


Human erythrocyte thiol methyltransferase: radiochemical microassay and biochemical properties. A radiochemical microassay for the measurement of thiol methyltransferase (TMT) activity in human red blood cell (RBC) membranes has been developed. Both 2-mercaptoethanol and dithiothreitol were used as substrates for the enzyme. The pH optimum of the reaction was approximately 9.0 when glycine NaOH was used as a buffer. The apparent Michaelis-Menten (KM) value for the methyl donor for the reaction, S-adenosyl-L-methionine, was 43 mumol/l. Human RBC TMT activity was neither activated nor inhibited by Ca2+, Mg2+, or tropolone, but the enzyme was inhibited by SKF 525A and by reagents that react with sulfhydryl groups. The mean TMT activity in blood from 289 randomly selected adult white subjects was 10.93 +/- 3.22 units per mg protein (mean +/- S.D.). The activity was the same in samples from men and women. The results of experiments in which TMT activity was measured in mextures of RBC membranes with relatively "low" and relatively "high" activities provided no evidence that individual variations in the enzyme activity were due to variations in endogenous TMT activators or inhibitors.


Studies in metachromatic leukodystrophy. XIV. Purification and subunit structure of human liver arylsulfatase A. Arylsulfatase A was purified to apparent homogeneity from normal human livers obtained at autopsy. According to gel electrophoresis in sodium dodecyl sulfate, purified arylsulfatase A consistently contained two subunits of slightly different sizes: approximately 69 000 and 57 000 daltons, but were not present in stoichiometrically equal amounts. Peptide maps of the entire enzyme and of the two individual subunits showed that the two polypeptides share similar if not identical sequences. These observations raise the possibility that the smaller polypeptide might be derived from the larger one. The sensitive peptide mapping procedures employed will make feasible future studies with the abnormal enzyme found in metachromatic leukodystrophy.


Erythrocyte glucose metabolism in Duchenne muscular dystrophy. We compared glucose metabolism by erythrocytes from patients with Duchenne muscular dystrophy and by erythrocytes from control individuals. There was a significant decrease in the rate of lactate production at pH 7.2 in the dystrophic group. When the pH of the incubation medium was changed to 8.0, we found that the increase in the rate of lactate production for the dystrophic group was significantly larger. We measured the concentrations of key glycolytic metabolites and adenine nucleotides and determined the values of the energy charge during these incubations. We also determined the concentrations of polyol pathway intermediates, the activities of the oxidative portion of the pentose phosphate pathway and the activities and kinetics of phosphofructokinase from both cell groups. There were no significant differences between groups for any of these variables.


An automated continuous flow procedure for the determination of enterokinase. A continuous flow method has been developed for the automatic determination of enterokinase in rat small intesstine mucosa and/or luminal content. Trypsinogen was first hydrolysed by enterokinase under conditions which minimize autocatalytic activation. L-benzoyl-arginine paranitroanilide was then added and split to paranitroaniline by the trypsin so formed. Liberated paranitroalinine was diazotized and converted by the Bratton-Marshall reagent (N-naphthyl ethylene diamine) to an azodye, with maximum absorption at 550 nm. This method of determination was found to be six times more sensitive than the direct p-nitroaniline determination method. 36 determinations can be made hourly.


A fluorimetric assay of the phosphoribosylation of 6-mercaptopurine in human blood cells. A new fluorimetric method to assay HGPRT (hypoxanthine-guanine phosphoribosyl transferase, EC 2.4.2.8) from human red cell lysates using 6MP (6-mercaptopurine) as substrate is described. This assay is compared with an existing spectrophotometric method using hypoxanthine as substrate. Precision, sensitivity limits and kinetic differences of these assays are reported.


Monoclonal IgG1 immunoglobulinaemia with strictly pH dependent cryoprecipitability. A monoclonal IgG1 lambda protein with peculiar cryoprecipitable characteristics was found in the serum of a patient with advanced multiple myeloma. The cryoprecipitate was observed only when the serum was exposed to the air. Further studies defined this phenomenon as being strictly pH-dependent. No antiglobulin activity (rheumatoid factor) was detected in the cryoglobulin. The isoelctric focussing of isolated cryoglobulin showed four distinct fractions around the pH 7.8 zone, which was comparable to the pH at which the cryoprecipitate began to form. Electrostatic bonds probably contribute to the mechanism involved in the formation of cryoprecipitate in this case.


Kinetics of pamatolol, a cardioselective beta adrenoreceptor blocker. The systemic bioavailability, elimination half-life (t1/2), and plasma concentration--response relationships of pamatolol, a relatively cardioselective beta adrenoceptor blocker, have been measured in healthy subjects. Pamatolol is rapidly and completely absorbed after oral dosing. Elimination t1/2 ranged from 2.9 to 4.6 hr after oral doses and from 2.2 to 5.6 hr after intravenous doses. There was a clear relationship between log plasma pamatolol concentration and sympathetic blockade assessed by reduction of exercise heart rate. Concentration-response curves were essentially identical after oral and intravenous doses. There is no evidence of a first-pass effect, nor is there any evidence of metabolite activity.


Effects of extracellular potassium on the pressure response to noradrenaline in the perfused hindquarters of the rat. 1. The hindquarters of the rat were perfused with oxygenated Tyrode-Ficoll solution at constant flow and the blood pressure in the caudal artery was measured. All experiments were started with the resistance vessels maximally dilated (arterial pressure 20 mmHg). 2. Variations in the extracellular potassium concentration (from 0.5 mmol/l to 22 mmol/l) had no direct effect on the arterial pressure. 3. Noradrenaline in the range 1.6-17 mumol/l increased the arterial pressure in a dose-dependent manner at all potassium concentrations. 4. Variations in the potassium concentration within the physiological range did not affect the dose-dependent responses to noradrenaline.


Alpha- and beta-receptor blocking drugs in the treatment of hypertension. The wide variety of antihypertensive agents now available allows considerable flexibility in the pharmacologic management of hypertension. The newly available alpha- and beta-adrenergic blocking agents have added considerably to this flexibility. Their safety and efficacy insure that they will find increasing use in the treatment of hypertension. The new postsynaptic alpha-receptor blocker prazosin, is generally free of the side effects that precluded the use of alpha-blockers which affected both pre- and postsynaptic receptors. Is is moderately effective and, excepting the problem of "first-dose" hypotension, it is usually well tolerated. Labetalol, a compound possessing both alpha- and beta-receptor blocking properties, has been found to be effective both as an oral therapy for chronic hypertension and as an intravenous agent in treating hypertensive crisis. Further experience with labetolol will determine its safety and efficacy for the long-term management of hypertension. Although the usefulness of beta-adrenergic receptor blocking agents in hypertension is unquestionable, the mechanism by which they lower the blood pressure remains in question. Most of our experience has been with propranolol, but other beta-blockers, now used in England and Europe, are likely to become available in the United States. As of January, 1979 only metoprolol has been added to propranolol as approved beta-blockers for the treatment of hypertension. General guidelines for the use of propranolol are given in Table 6. Metoprolol will likely find increasing use since it seems to work as well as propranolol and probably causes fewer side effects. Nevertheless, the experiences with one beta-blocker, both good and bad, cannot be interpolated to another beta-blocker since there are obvious and subtle differences in their actions. Patients doing well on propranolol therapy should continue to receive that drug; patients being started on a beta-blocker therapy can be given the choice of metoprolol and, in the near future, other beta-blockers as well. Beyond their efficacy and freedom from side effects in about 80% of patients, beta-blockers may offer a special advantage in protecting from coronary heart disease. Such protection has been suggested in limited trials but needs further documentation. In the meantime, the known advantages of beta-blockers ensure their increasing use in the treatment of hypertension. Regardless of which alpha- or beta-blocker is chosen, a diuretic should be used concomitantly to enhance and preserve the effectiveness of the adrenergic blocker.


A rare cause of ischemic colitis: polyarteritis nodosa. This paper details the first case report of a patient with fulminant, gangrenous, ischemic colitis caused by polyarteritis nodosa which was successfully treated surgically. Ischemic colitis is a rare complication of polyarteritis nodosa. It should be suspected in patients with a past history of polyarteritis nodosa who develop abdominal pain and rectal bleeding. The identification of cutaneous lesions preoperatively and or mesenteric or serosal vascular beading at operation are helpful in establishing this diagnosis. Prognosis is generally determined by the extent of systemic involvement by polyarteritis nodosa, and death is generally associated with renal failure.


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


Effects of isoniazid treatment on selected hepatic mixed-function oxidases. The hepatic microsomal content of cytochromes P-450 and b5, the defluorination rates of four volatile fluorinated ether anesthetics, and the activities of selected mixed-function oxidases were compared following administration of either isoniazid, phenobarbital, beta-naphthoflavone, or saline to male Fischer 344 rats. Isoniazid treatment significantly increased the rate of metabolism of p-nitroanisole, ethoxyresorufin, aniline, methoxyflurane, enflurane, isoflurane, and sevoflurane, significantly decreased the rate of metabolism of aminopyrine, and did not alter the activity of NADPH-cytochrome c reductase or the microsomal contents of cytochromes b5 and P-450 per mg of microsomal protein. The pattern of catalytic activities associated with isoniazid induction did not resemble that of either phenobarbital or beta-naphthoflavone induction. Furthermore, isoniazid treatment resulted in a shift in the (reduced cytochrome P-450 plus CO) absorption maximum from 450 to 451 nm. This shift in absorption, coupled with the observation that the total microsomal cytochrome P-450 content is not elevated, suggests that there is an increased production of one species of cytochrome P-450. The great enhancement of enflurane defluorination following isoniazid treatment was of particular interest because other enzyme-inducing agents, including phenobarbital, 3-methylcholanthrene, phenytoin, and beta-naphthoflavone, have not been found to increase enflurane defluorination to a clinically significant level.


Strain differences in adrenal xenobiotic metabolism in guinea pigs. Studies were carried out to compare adrenal and hepatic xenobiotic metabolism in various strains of guinea pigs. In all strains studied (Hartley, English Short Hair, NIH, Strain 2, Strain 13), microsomal protein and cytochrome P-450 levels and NADPH-cytochrome c reductase activities were greater in adrenals than livers. Neither adrenal values nor hepatic values for these parameters differed across strains. Ethylmorphine (EM) demethylase and benzo[a]pyrene (BP) hydroxylase activities were also greater in adrenals than livers in all strains. However, the rates of adrenal xenobiotic metabolism were far greater in the highly inbred Strain 2 and Strain 13 guinea pigs than in other strains. In contrast, hepatic metabolism of EM and BP was not strain-dependent. Adrenal steroid 21-hydroxylase activity was also similar in all strains. The results indicate that strain is an important determinant of adrenal but not hepatic xenobiotic metabolism in the guinea pig. In addition, genetic control of adrenal microsomal drug and steroid metabolism appear to be independent of one another.


Rodent models of the human isoniazid-acetylator polymorphism. Inbred strains and subpopulations of rats, laboratory mice, and deer mice were examined for individual variation in the ability to metabolize several arylamines (p-aminobenzoic acid, sulfamethazine, aniline, alpha-naphthylamine, and aminofluorene) by N-acetylation. Individual differences within species were found to be dependent upon the tissue source of N-acetyltransferase activity and the acetyl acceptor employed. Long-Evans rats possessed about 2-fold more p-aminobenzoic acid N-acetyltransferase activity in blood and liver than Sprague-Dawley rats; no strain differences could be found with sulfamethazine. Nine strains of laboratory mice (Mus musculus) were found to have considerable liver p-aminobenzoic acid N-acetyltransferase activity but only slight activity towards sulfamethazine. No strain differences were apparent in regard to liver N-acetyltransferase activity. Blood p-aminobenzoic acid N-acetyltransferase activity was distinctly polymorphic in laboratory mice; of the nine strains tested, only A/J mice did not have this activity. Partially inbred deer mice (Peromyscus maniculatus) showed a narrower phenotypic range than random-bred stock from which they were obtained, which suggests the existence of distinct subpopulations with respect to N-acetylation capacity. Presumptive evidence for multiple forms of N-acetyltransferase in liver and blood was obtained through a study of substrate specificity.


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


Production of a dimer of 2-acetylaminofluorene during the sulfation of N-hydroxy-2-acetylaminofluorene in vitro. During the sulfation of N-hydroxy-2-acetylaminofluorene (NOH-2AAF) by rat liver 100,000 g supernatant fraction in vitro, an unidentified metabolite is produced which accounts for 22% of the N-OH-2AAF metabolized. This product has been characterized as the 2AAF dimer, 1-(N-2'-fluorenylacetamido-2-acetylaminofluorene) by comparing its TLC, HPLC, UV, and mass spectral properties with a synthetic standard which was prepared from the reaction of N-acetoxy-2-acetylaminofluorene (N-AcO-2AAF) with 2AAF. Increasing amounts of 2AAF added to the incubation mixture of N-OH-[acetyl-14C]2AAF and rat liver 100,000 g supernatant fraction decreased the irreversible binding of 14C to protein, and increased the formation of 2AAF dimer proportionately. This suggests that the 2AAF dimer is formed from the reaction of 2AAF and the electrophilic species produced from the sulfated N-OH-2AAF. In the presence of the 9,000 g fraction of rat liver, the dimer of 2AAF was aroximately 1/25 as active as 2AAF in producing mutations in the Salmonella mutagenesis test system.


Aldrin epoxidation, a highly sensitive indicator specific for cytochrome P-450-dependent mono-oxygenase activities. Aldrin epoxidation was studied in rat liver microsomes. The assay is very sensitive; amounts of less than 1 microgram of microsomal protein were sufficient for activity determination. The very low background, stability of the metabolite, and the complete separation of substrate and metabolite permit estimation of mono-oxygenase activities of less than 1 pmol per mg of protein per min by a simple procedure. Pretreatment of animals with the mono-oxygenase inducer phenobarbital (PB) increased the epoxidation rate 3-fold, whereas 3-methylcholanthrene (MC) treatment markedly depressed enzyme activity. Induction with MC did not change the apparent Km of the reaction, which was 18 muM. The Km in microsomes from PB-treated animals was 28 muM. These data suggest that the same or (a) similar form(s) of mono-oxygenase catalyze(s) the epoxidation in the three different microsomal preparations. SKF 525-A, metyrapone, and 7,8-benzoflavone were almost similarly active as inhibitors in microsomes from control and inducer-treated rats. Sensitivity to these inhibitors was low; 0.7 mM SKF 525-A and 0.4 mM 7,8-benzoflavone were necessary to reduce enzyme activity by 50%, whereas 0.5 mM metyrapone caused an inhibition of 10-45%. The activity of aldrin epoxidation in untreated rats increased almost parallel to the activity of ethylmorphine demethylation between 3 and 10 weeks of age. The rate of benzo[a]pyrene hydroxylation remained unchanged during this period. The results demonstrate that aldrin epoxidation offers a selective and sensitive assay for the activity of mono-oxygenases dependent on cytochrome P-450 forms.


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


The comparative metabolism and disposition of penfluridol-3H in the rat, rabbit, dog, and man. Penfluridol, 1-[4,4-bis(4-fluorophenyl)butyl]-4-[4-chloro-3-(trifluoromethyl)phenyl]-4-piperidinol, was well absorbed by the rat, rabbit, dog, and man after single oral doses of drug in solution. Penfluridol and its metabolites were widely distributed in tissues of male rats and cleared slowly. The terminal plasma t 1/2 for penfluridol was greater than 40 hr for the rabbit, 227 hr for the dog, and 199 hr for man. Fecal excretion of total radioactivity predominated in the rat, rabbit, and dog whereas in man excretion was evenly divided between urine and feces. The major biotransformation pathways of penfluridol in the rat, rabbit, dog, and man were oxidative N-dealkylation followed by beta-oxidation, conjugation of penfluridol, and conjugation of the acidic metabolites.


Physiological disposition and metabolism of 2-aminomethyl-4-(1,1-dimethylethyl)-6-iodophenol hydrochloride. MK-447-(14)C [2-aminomethyl-4-(1,1-dimethylethyl)-6-iodophenol hydrochloride] was well absorbed and metabolized in man, rats, and dogs. Peak plasma levels of radioactivity were observed in these species 1-2 hr after oral administration of 2 mg/kg to rats and dogs and 25 mg to man. At the peak, parent drug represented about 15% of the radioactivity in human plasma and only approximately 5% in rat and dog plasma. The half-life of the parent drug in human plasma was approximately 4 h. Human subjects excreted 96% of the dose, with 76% in the urine and 20% in the feces, in 3 days. Rats excreted 80% of an oral and 82% of an intravenous 2-mg/kg dose in 72 hr, with 66% in the urine and 12-16% in the feces. In dogs given a 2-mg/kg dose intravenously, the recovery of radioactivity in 72 hr was approximately 99%, with 85% in the urine and 14% in the feces. The major metabolite in rat and dog urine, constituting approximately 90% of the urine radioactivity, was the O-sulfate conjugate of MK-447. In man, this metabolite accounted for 17% of the radioactivity in the urine. The major metabolite in human urine, constituting approximately 73% of the urine radioactivity, was tentatively identified as the N-glucuronide of MK-447. Less than 1% of the radioactivity in the urine of the three species was in intact MK-447.


Metabolic fate of phenobarbital in man. N-Glucoside formation. 1-(beta-D-Glucopyranosyl)phenobarbital was identified as the major metabolite of phenobarbital in man. Proof of structure was based on the comparison of the UV, NMR, and mass spectrometry and TLC data for the acetylated metabolite with an authentic compound. The previous erroneous structure assignment of this metabolite as N-hydroxyphenobarbital was based on insufficient data. After oral administration of 14C-labeled phenobarbital to two healthy male subjects, most of the radioactivity (87 and 78% of the dose) was recovered in urine over a period of 16 days. The N-glucopyranoside, p-hydroxyphenobarbital, and unchanged phenobarbital accounted for 30 and 24%, 18 and 19%, and 33 and 25% of the dose, respectively.


Metabolism of deflazacort in the rat, dog and man. The metabolism of [2'-14C]deflazacort, (11 beta, 16 beta)-21-(acetoxyl)-11-hydroxy-2'-methyl-5'H-pregna-1, 4-dieno[17,16-d]oxazole-3,20-dione, orally given to rats, dogs, and humans, has been studied. From the urine of the three species and from rat bile and liver preparations, five main metabolites I-V have been isolated and their structures investigated by physicochemical analysis: 1,(5 beta,11 beta,16 beta)-11,21-dihydroxy-2'-methyl-5'H-pregn-1-eno[17,16-d]oxazole-3,20-dione; II, (11 beta,16 beta)-11,21-dihydroxy-2'-methyl-5'H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione; III, (6 beta,11 beta,16 beta)-6,11,21-trihydroxy-2'-methyl-5'H-pregna-1,4-dieno[17,16-d]oxazole-3,20-dione; IV, (3 epsilon,11 beta,16 beta)-3,11,21-trihydroxy-2'-methyl-5'H-pregn-5-eno[17,16-d]oxazol-20-one. Metabolites II and III are quantitatively the most important in the urine of the rat, dog, and man; metabolite V, whose structure is uncertain, has been found in human and rat urine. In the formation of metabolites I-V the fused 2-methyloxazoline ring is unmetabolized, whereas the steroid moiety follows the general metabolic pathways reported for other related corticosteroids.


Metabolites of feprazone in man.

Metabolites of feprazone in man. Two male volunteers received 400 mg of nonlabeled feprazone, 1,2-diphenyl-3,5-dioxo-4-(3'-methyl-but-2'-enyl)pyrazolidine. Metabolites were extracted and isolated by chromatography on silica gel. Isolated metabolites were unchanged drug, E-1,2-diphenyl-3,5-dioxo-4-(3'-hydroxymethyl-but-2'-enyl)pyrazolidine, and feprazone (C4)-beta-glucuronide. Feprazone (C4)-beta-glucuronide was the main metabolite and it belongs to a novel type of conjugate, (C)-beta-glucuronide.


[Kawasaki syndrome (author's transl)].

[Kawasaki syndrome (author's transl)]. In a 2-year 4-month-old boy endarteritic changes and spindle-shaped aneurysmas of the coronary arteries were found as a complication of Kawasaki syndrome. The vascular alteration, probably immunopathologically based, must be differentiated as a primary endarteritic process from primary necrotising periateritis nodosa which also occurs in childhood. The fate of the affected children is determined by the complications of coronary artery changes which however occur only in 1--2% of affected persons.


A neurophysiological analysis of the effects of hypercapnia on the embryonic spinal cord. Progressive hypercapnia in the normal chick embryo late in incubation (14-19 days) is temporally associated with a gradual decline in motor activity and the corresponding frequency of polyneuronal (burst) activity in the spinal cord. We have studied the possible correlation between the increasing hypercapnia and the declining frequency of burst activity seen during these later stages of incubation by systematic manipulation of CO2 levels. Burst frequency was seen to decrease as a result of a 5-min exposure to different carbon dioxide environments at all ages studied. The magnitude of this inhibition and the ability to recover from consecutive bouts of hypercapnia (pulses) is pulse and age dependent. These short-term (less than 5.0 min) changes differ qualitatively from the long-term (greater than 2.5 h) effects of subsequent hypercapnic episodes. This evidence suggests a role for metabolic factors in the normal developmental changes in motility and electrophysiological activity in the chicken embryo spinal cord.


[Effect of beta-adrenergic drugs on humoral immune response factors]. The authors examined the influence of beta-adrenergic stimulators Isoprenaline and Salbutamol as well as beta-adrenergic blockers Propranolol, Practolol and Pindolol on the proliferation of cells, forming plaques, 7S-immunoglobulins, hemagglutination titres and hemolytic activity of the complement. Primary stimulation with subsequent inhibition was induced by beta-adrenergic agonists and inhibition--by beta blocker Propronanol in a base of 10 mg/kg on the proliferation of cells, forming antibodies. The results were in correlation with hemagglutination titres. Salbutamol and beta-blockers inhibited immune hemolysis. The mechanisms of the observed effect are discussed in respect to the specific and nonspecific effect of the drugs used.


[Antihistamine and antiserotonin action of 2 aminotetralin derivatives]. The authors performs studies under the conditions of whole organism in vivo and on isolatid organs in vitro and examined antihistamine and antiserotonine activity of two derivatives of 2-aminotetraline, synthesized by D. Dancev and Iv. Ivanov, namely: N-(trans-e-hydroxy-1, 2, 3, 4-tetrahydro-2-naphtyl (-N-) 3-oxo-3-phenylpropyl) piperasine dichloride and N-(trans-3-hydroxy-1, 2, 3, 4-tetrahydro-2-naphtyl (-N-) 3-oxo-3-3phenyl-2-methylpropyl (piperasine dichloride) indicated respectively as P10 and P11. The experiments on isolated ileum of a guinea pig showed that the compound P11 (in concentration of 1.10(-7), 1.10(-6), and 1.10(-5) gm/ml) and P11 (in concentration of 1.10(-6) and 1.10(-5) gm/ml) inhibited the spasomogenic effect of histamine (1.10(-8) and 1.10(-7) gm/ml) as better antihistaminev effect revealed P10. Both compounds inconcentrations of 1.10(-6) and 1.10(-5) gm/ml inhibited the spasmogenic effect of serotonine (1.10(-7) gm/ml), as more significant changes occurred under the influence of the compound P11. The compound P10, administered subcutaneously in a guinea pig in a dose of 0.5 mg/kg of body weight 30 minutes before the occurrence of histamine shock, prevented its development. The same compound inhibited also the development of enterocolitis in mice, induced by serotonine (in a dose of 1 mg/kg of body weight). Both examined compounds inhibited the development of serotonine oedema of the hind paw of a rat as better effect showed the compound P11. The established facts are discussed.


Studies on isoamyalse formation in biological fluids. Storage of human serum, saliva and duodenal secretion transformed amylase fractions on cellulose acetate membranes into more anionic forms. Incubation with lectins, proteases, glucosidases, neuraminidase and some effectors did not modify this conversion, which was promoted by rising temperature and pH values. Increasing concentrations of ammonium ions delayed the transformation of amylolytic fractions, thus indicating nonenzymatic deamidation as the reason for isoamylase development. A change of molecular weight could be excluded.


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


The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect. The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.


Determination by radioimmunoassay of the sum of oxidized and reduced forms of NAD and NADP in picomole quantities from the same acid extract. The sum of the amounts of NAD + NADH was determined from the same acid tissue extract with the aid of a highly specific radioimmunoassay for 5'-AMP. NAD was converted to 5'-AMP via ADP-ribose by alkaline treatment while NADH was converted first to ADP-ribose by incubation of the acid extract at 25 degrees C followed by alkaline conversion to 5'-AMP. Removal of phosphate groups in NADP and NADPH by treatment of the extracts with alkaline phosphatase extended the procedure to the quantification of NADP(H). When combined with enzymic analyses of the oxidized coenzyme forms, NAD/NADH and NADP/NADPH ratios could also be obtained from the same extracts. The sensitivity of the test allows quantification of pyridine nucleotides in the range of 0.1--10 pmol.


Simple, fast preparation of gallium-68-labelled human serum albumin microspheres. Following a study of the main factors involved in the 68-Ga labelling of human serum albumin microspheres (H.S.A.M.), especially methods of production and preparation of active solution and conditions of radioelement fixation on the protein support, the practical details of a fast technique (60 min) based on the process described by Hnatowich are presented. This method gives high labelling yields (93 +/- 3%), and after washing of the microspheres leads to a radiopharmaceutical product almost without free 68Ga (less than 2%). The spheres ready for use carry a total radioactivity corresponding to about 35%, including decay, of the activity originally recovered in the generator eluate and to more than 98% of that, found in the final suspension. The labelled product is sterile, non-pyrogenic and non-toxic. When it is injected in animals by left ventrical catheterization the uptake rates in the heart, lungs, spleen, left kidney and right kidney are similar to those observed with reference 85Sr-labelled carbonized microspheres. This radiopharmaceutical, easy to prepare and having excellent biological and nuclear properties, seems ideally suited for the scanning of organs by position emission tomoscintigraphy.


Graft-versus-host reaction after blood transfusion in a patient with cellular immunodeficiency: the role of histocompatibility testing. A patient with an inborn cellular immunodeficiency syndrome developed a graft-versus-host reaction after a transfusion with packed red cells. This diagnosis was confirmed by skin biopsy and finally proved by tissue typing.


Blood pressure and plasma noradrenaline during single high-dose beta adrenoceptor blockade. The acute effects upon blood pressure and sympathetic outflow of two beta adrenoceptor blocking drugs, propranolol and atenolol, are described in five healthy normotensive subjects. Supine blood pressure, heart rate, plasma noradrenaline, and urinary catecholamine excretion were measured before and at intervals for 24 h after a single oral dose of either propranolol 200 mg, atenolol 100 mg, or placebo. Propranolol caused a fall in blood pressure and heart rate of 17.2/14.1 mm Hg and 20.4 beats/min respectively two hours after dose. Atenolol caused a fall in blood pressure of 11.4/18.6 mm Hg within 7 h of the dose, and a fall in heart rate of 13.8 beats/min after 2 h. The reduction in blood pressure after single high dose beta adrenoceptor blockade is established. The synchronous reduction in blood pressure and heart rate after propranolol was not associated with an increase in peripheral sympathietic activity as assessed by the biochemical indices. It is conceivable that the reduction in blood pressure during beta adrenoceptor blockade may be due in part to inappropriately low sympathetic activity but this cannot be the main mechanism of pressure reduction.


Effect of beta-adrenergic blockade on haemodynamic responses to dynamic and isometric exercise in angina pectoris. The effect of treatment for 1--4 weeks with metoprolol, a beta 1-selective blocking agent, or alprenolol, on the heart rate and blood pressure response to isometric exercise was studied in two groups of 12 patients with angina. Measurements were made during the peak effect of metoprolol 10, 40 or 50 mg, and alprenolol 200 mg as Aptin Durules. After 1 min of sustained handgrip at 50% of maximal voluntary contraction, systolic (6--15%) and diastolic (8--12%) blood pressure after both drugs was significantly lower than without any beta-blockade; Heart rate was decreased by 19--22% by metroprolol but not by alprenolol. The blood pressure rise during handgrip was not attenuated by either drug. The rise in heart rate was significantly reduced (by 36--50%) by metoprolol 40 and 50 mg and alprenolol 200 mg. No patient experienced angina during handgrip. In contrast, all but one were restricted by angina during bycycle exercise without treatment, at a level that produced the same increase in heart rate as the handgrip test, vis. 3 min at a load of 33 W). The cardiovascular response to sustained handgrip is too small to provide a useful challenge for determinatin of the anti-anginal efficacy of drugs. However, slight ECG changes of ischaemia did occur during handgrip, which were reversed by beta-blockade.


A preliminary study of the effects of prepeated doses of clobazam on aspects of performance, arousal and behaviour in a group of anxiety rated volunteers. Repeated doses of a 1,5 benzodiazepine, clobazam, were administered to a group of consenting volunteers. Performance on a psychomotor performance task was not impaired by repeated doses of the drug. Subjective assessments of anxiety were reduced commensurate with an increase in the level of cortical integration, measured via critical flicker fusion thresholds, suggesting that the anxiolytic activity of the drug is related to its effect on arousal. Important differences were found between the responses of high and low anxiety subjects on objective measures of performance and arousal and on self-completion analogue rating scales.


Pharmacodynamic studies in man of the beta-adrenoceptor antagonist DL 071 IT. DL 071 IT, a new potent non-selective beta-adrenergic blocking drug with intrinsic sympathomimetic activity and weak membrane stabilizing activity, was evaluated alone and in comparison with oxprenolol, in six volunteers, at rest and during an exercise test. Heart rate and systolic pressure were monitored for up to 7 h after oral administration of the drugs. Exercise heart rate and systolic pressure were significantly reduced by both drugs, but only DL 071 IT caused a significant reduction in resting heart rate. As compared to oxprenolol, DL 071 IT has a longer duration of action and is from 5.0 to 13.5 times more potent.


Do neuroleptics prevent the penetration of dopamine agonists into the brain? A new, specific and highly sensitive method for the determination of apomorphine based on high performance liquid chromatography on a C18 reverse-phase column, coupled with electrochemical detection has been developed. The limit of detection of the assay is approximately 0.5 ng/sample (2 pmol). Haloperidol, cis-flupenthixol, metoclopramide and reserpine prevented the accumulation of apomorphine equally in "dopaminergic" as well as in "non-dopaminergic" brain areas. The non-neuroleptic trans-isomer of flupenthixol was without effect. Suppression of the accumulation of the dopamine agonist 6,7-ADTN (administered as the prodrug dibenzoyl-6,7-ADTN) was seen after combined treatment with haloperidol or reserpine, whereas cis- and trans-flupenthixol were without effect. The results imply that certain behavioural, biochemical and neuropharmacological studies, based on apomorphine in combination with other drugs, may need reinterpretation.


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


Ibotenic acid-induced neuronal degeneration: a morphological and neurochemical study. Possible neurotoxic actions of intracerebral injections of ibotenic acid, a conformationally restricted analogue of glutamic acid, have been evaluated in rat brain and compared with those of kainic acid. Light microscopical analysis revealed that ibotenic acid produced a marked disappearance of nerve cells in all areas studied, namely striatum, the hippocampal formation, substantia nigra and piriform cortex. Lesions in areas distant to the injection site were not seen. Axons of passage and nerve terminals of extrinsic origin did not seem to be damaged, since, e.g., no apparent degeneration of the dopaminergic terminals in the neostriatum was observed except for a small area surrounding the cannula. In the neostriatum, enkephalin immunoreactive neuronal cell bodies as well as nerve terminals disappeared after injection of ibotenic acid into this nucleus. After injection into the substantia nigra tyrosine hydroxylase immunoreactive cell bodies in the zona compacta disappeared, whereas no certain effect could be seen on the enkephalin immunoreactive nerve fibers. In vitro experiments, conducted with striatal synaptosomal and membrane preparations, showed that ibotenic acid differed from kainic acid by being devoid of a significant inhibitory effect on high affinity glutamate uptake and by having a low affinity for 3H-kainic acid binding sites. Furthermore, ibotenic acid did not interfere with the binding of a number of radioligands for other transmitter receptors. As compared to kainic acid, ibotenic acid has the advantage of being less toxic to the animals and of producing more discrete lesions, possibly due to faster metabolism and/or other fundamental biochemical differences. Because of these special features, ibotenic acid seems to represent a valuable new tool in the morphological and functional analysis of central neuronal systems.


Conformation of ribonuclease S-protein.

Conformation of ribonuclease S-protein. Ribonuclease S-protein exhibits a pH-dependent conformational transition between folded and unfolded states, and some unfolded S-protein persists up to pH 8. The histidine C2 proton resonance of the unfolded species was erroneously assigned by Bradbury et al. to histidine residue 119 of the folded species.


The effect of proctolin on the adenylate and guanylate cyclases in the Locusta brain at various developmental stages. Proctolin at concentrations 10(-8)-10(-7) M elevated by 40% brain adenylate cyclase activity of adult Locusta migratoria migratoriodes R.F. In moulting individuals, proctolin caused a decrease in brain adenylate cyclase activity, and it proved to be ineffective in the larvae. Proctolin caused only a slight decrease on guanylate cyclase activity of the brain at every developmental stage.


Stereospecificity of hydrogen transfer of aldehyde reductase. Aldehyde reductase from human liver catalyzes the hydrogen transfer from the pro-4R position on the dihydronicotinamide ring of the coenzyme to the re face of the carbonyl carbon atom of the substrate.


Development of gamma-glutamylcysteine synthetase and oxoprolinase in rat kidney. gamma-Glutamylcysteine synthetase is present in barely detectable amounts in foetal kidney. Its activity starts to increase in postnatal life. In contrast, oxoproline is already found in significant quantities in the foetal tissue. Both enzymes show marked elevation in activities during the weaning period.


Photosynthesis by isolated chloroplasts of Sorghum vulgare. The ambiguous location of photosynthetic carboxylases of mesophyll and bundle sheath chloroplasts of sorghum was investigated after successful homogeneous preparation. The phosphoenol pyruvate carboxylase was found as a particulate enzyme in the mesophyll cell chloroplasts and ribulose 1,5-biphosphate carboxylase in the stroma of the bundle sheath cell chloroplasts. Extensive characterization was carried out on these 2 enzymes for better understanding of the enzyme action.


Acid-base equilibrium in the blood of sheep. The acid-base equilibrium in the blood of sheep is different from that of human blood mainly because of a lower concentration of 2,3-DPG. A nomogram relating pH, pCO2, total CO2 content and base excess has been developed.


[Comparative study of the sedative and tranquilizing effect of phenazepama and seduxen and their influence on human evoked brain potentials]. Phenazepam was compared to diazepam in a double-blind study made in 32 patients. The drugs were administered by month in doses of 0.0025 mg/kg and 0.005 mg/kg. respectively 1--2 days before surgery with the use of general anesthesia. Anxiety assessed by means of the brain evoked potential test and by a clinical test according to Gologorsky's scale reduced 2 hours following both phenazepam and diazepam administration. Phenazepam elicited a more pronounced and more lasting tranquilizing effect as compared to that of diazepam. Phenazepam was concluded to be a powerful anxiolytic and sedative agent recommended for premedication before surgical interventions.


[Effect of tranquilizers on animal resistance to adequate stimulus exposure of the vestibular apparatus]. The effect of tranquilizers on the intensity of vestibulospinal reflexes and motor activity was studied in 900 centrifuged albino mice. Chemically heterogenous tranquilizers (meprobamate, elenium, nicolit) were applied in therapeutic doses. Actometric studies have shown that the tranquilizers possess group capacity for increasing animal resistance to the action of adequate stimuli to the vestibular apparatus. Meprobamate has exhibited the most pronounced protective effect.


[2-14C-phenazepam metabolism in vitro].

[2-14C-phenazepam metabolism in vitro]. Phenazepam undergoes aromatic and C3-hydroxylation in the microsomes of albino rats. Saturation of phenazepam oxidising enzymes proceeds at different substrate concentrations for both processes. Interaction of cytochrome P-450 with phenazepam is accompanied by spectral changes in the type II hemoprotein.


Regulation of fatty acid synthesis.

Regulation of fatty acid synthesis. Acetyl-CoA carboxylase and fatty acid synthetase are the two major enzymes involved in the synthesis of fatty acids in animals. The activities of both enzymes are affected by nutritional manipulations. Although acetyl-CoA carboxylase is considered generally to be the rate-limiting step in lipogenesis, there is evidence that suggests that fatty acid synthetase may become rate limiting under certain conditions. The principal support for the view that acetyl-CoA carboxylase is the rate-limiting enzyme for lipogenesis is that the activity of the enzyme is controlled by allosteric effectors that change the catalytic efficiency of the enzyme. Until recently, the only known control of fatty acid synthetase was through changes in rate of enzyme synthesis. Data are reviewed that show that fatty acid synthetase can exist in forms possessing different catalytic activities. Thus fatty acid synthetase appears to be subject to the type of control necessary for an enzyme to serve as a regulator of the rate of a biological process over a short term.


The origin of orchiopexy-induced testicular lesions in the pig. Cooling experiments of abdominal testes in adult, naturally cryptorchid pigs indicate that spermatogenic arrest in abdominal testes is not due to an inborn defect, but is caused solely by maintenance of the testis at abdominal temperature. It is postulated that failure of spermatogenic cells to differentiate after orchiopexy results from surgical trauma. Evaluation of the orchiopexy procedure revealed that simple manipulation of a normally descended testis may give rise to damage to the spermatogenic epithelium. Furthermore, it appeared that, in normally descended testes of naturally unilaterally cryptorchid pigs subjected to orchiopexy, the spermatogenic epithelium was poorly developed as compared with that of scrotal testes of unilaterally cryptorchid pigs that had not undergone a surgical procedure.


Functional differentiation of mouse T lymphocytes. GVHR-precursors: tissue origin and specificity of activity inducer. For theoretical and practical reasons, it is important to find out whether the differentiation of T cell precursors to the functional lymphocytes can be induced under in vitro conditions. Using the local GVHR assay (based on the enlargement of the popliteal lymph node), the inducibility of the precursors of reactive cells was studied with bone marrow, thymus, spleen, and lymph node cell suspensions submitted to short-term incubation with cell-free extracts from calf thymus, spleen or brain. GVHR-precursors from bone marrow were inducible not only specifically (i.e., with thymus extract) but also--and even to a higher degree--with spleen or brain extract. Thymus and spleen cell suspensions (the latter also depleted of the reactive subpopulation by treatment with anti-Thy 1.2 serum and complement) were, on the other hand, inducible mainly specifically, whereas lymph node cells were refractory to induction. The inductive action of tissue extracts obviously depends on the tissue origin of T cell precursors; their effects on pre- and postthymic differentiation of T lymphocytes are discussed.


Euhypnos Forte (temazepam) for resistant insomnia: post-marketing surveillance, an interim report. An interim report at six months of a post-marketing surveillance study of Euhypnos Forte, a new high-dose temazepam preparation for the treatment of insomniac patients resistant to conventional hypnotic dosage. The analysis includes 2,043 First Reports (FRs) of two weeks treatment and 669 Second Reports (SRs) of three months treatment. More than 95% of the patients took a nightly dose of two capsules, temazepam 40 mg. Adverse reactions were generally acceptable, consisting mainly of headache, vivid dreams, gastro-intestinal disturbances and hangover effects. The preparation was effective in 88.6% of patients at two weeks and 95.8% at three months. All patients had previously found other hypnotics ineffective. Euhypnos Forte was rated effective by 85.5% of the 874 patients who had previously found nitrazepam unsatisfactory, and by 90.0% of the 201 who found barbiturates unsatisfactory.


A three-month double-blind study of clobazam versus diazepam in out-patients suffering from neurotic disturbances. Efficacy and safety of the 1,5 benzodiazepine, clobazam, in comparison to the 1,4 benzodiazepine, diazepam, were controlled in sixty psychiatric out-patients over a period of three months. In the course of this long treatment period data were obtained confirming findings of shorter-lasting studies. Global assessment of the therapeutic efficacy and the total scores of the Hamilton Anxiety Scale revealed no significant the compounds. Both groups showed a significant (p less than 0.01) improvement in the total scores of the Hamilton Anxiety Scale after two weeks of treatment. Scores of the individual items indicated distinct spectra of action: Clobazam was more effective in diminishing anxious mood, whereas diazepam was better able to influence muscular symptoms of anxiety. The relevance of the findings for a more individualized therapy is pointed out.


Consultation evaluation: I. Psychotropic drug recommendations. A follow-up chart review was undertaken of 100 consecutive patients seen in psychiatric consultation for whom psychotropic medication was recommended. Significant differences in implementation (P less than 0.01) by class of drugs (neuroleptics, tricyclics, benzodiazepines) were found. Most often no discernible reason for consultee disagreement could be found or inferred. The importance of such follow-up studies for consultation work is stressed.


Catalytic and regulatory properties of the Triton and trypsin forms of the brush border hydrolases. Amphipathic enzymes, invertase (EC 3.2.1.26), 8-amylase (EC 3.2.1.3), and alkaline phosphatase (EC 3.1.3.1), were purified from the rat small intestinal mucosa as trypsin and Triton forms, the catalytic and regulatory characteristics of which were compared in rats and in drosophila. Differences in the catalytic propertiis of the two enzyme forms were demonstrated, which suggested that the hydrophobic part of the enzyme was involved in maintaining optimal conformation of the catalytic part. Many modifiers have beenfound to influence the Triton rather than the trypsin form of the enzyme. It is therefore suggested that the hydrophobic sub-units of the enzymes might be involved in transmitting information from the cytoplasm into the external surface of the membrane, the cell in this way regulating the activity of surface enzymes. If this is indeed the case, it is suggested that the hydrophobic part performs functions not only of external but also of internal regulation.


Prostaglandin synthetase activity in acute ulcerative colitis: effects of treatment with sulphasalazine, codeine phosphate and prednisolone. Prostaglandin synthetase activity in rectal biopsy specimens from patients with ulcerative colitis has been shown to fall on treatment with sulphasalazine, local steroids, and codeine phosphate. In vitro studies have shown that sulphasalazine is an inhibitor of prostaglandin synthetase, although less potent than indomethacin, whereas prednisolone and codeine phosphate were inactive. It is suggested that the therapeutic action of sulphasalazine may be related in part to its action in inhibiting prostaglandin biosynthesis.


Endothelial permeability increase produced by angiographic contrast media. Permeability changes of the thoracic aortic wall of the rat were examined under light microscopy using the silver staining method. Methylglucamine salts of common angiographic contrast media and metrizamide were tested using a constant iodine concentration. Statistically significant differences between these media were found. The contrast agents were divided into three groups according to the extent of increasing endothelial damage: 1. Metrizamide and methylglucamine diatrizoate, 2. Methylglucamine metrizoate in the presence of calcium and methylglucamine iodamide, 3. Methylglucamine iothalamate. The osmolality of the contrast media was determined. The ionic contrast agents were equal in hypertonicity and metrizamide was nearly isotonic. The endothelial damage caused by the radiographic contrast media seems to depend both on chemical structure and on hypertonicity.


[Maldescensus testis, therapy and prognosis]. Since histologic changes are found in undescended testes after the second year of life, treatment is necessary. Often therapy with human choriongonadotrophin is successful, otherwise operative treamtnet is indicated. Infertility at a high rate is found in boys treated for maldescence of the testicles in later childhood. Therapy before the age of two years improves the prognosis.


[Long-term effect of calcium carbonate containing antacids on basally and peptone-stimulated hydrochloric acid secretion]. Basal and meal-stimulated gastric acid secretion was measured before and after a 3-week-period of regular consumption of a calcium carbonate-containing antacid with 6 therapeutic doses per day in normal healthy volunteers. Furthermore the effect of a single large dose of 2000 mg calcium ions was studied on meal-stimulated gastric acid secretion before and after this period. The results suggest that (1) even a large single dose of calcium ions seems to have no major effect on meal-stimulated gastric acid secretion and (2) chronic consumption of calcium carbonate containing antacids over a 3-week-period does not change basal- and meal-stimulated gastric acid secretion in healthy volunteers nor does it increase the response to 2 g calcium ions.


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


Plasma triiodothyronines in fetal sheep: effects of illness and thyroidectomy. Plasma 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine concentrations were measured in fetal sheep prior to death in utero and after thyroidectomy. In six fetal sheep who subsequently died in utero, plasma rT3 concentrations were elevated in all for 2 to 13 days prior to death. There were no consistent changes in plasma T4 concentrations. In two thyroidectomized fetal sheep, plasma T4 and rT3 concentrations fell to low levels. Plasma T3 concentrations remained low and there was no increase in plasma T3 in the last week prior to parturition like that which occurs in normal fetal sheep. Parturition was preceded by the normal increase in fetal plasma cortisol concentrations and occurred at the normal time. These data indicate that plasma rT3 concentrations are increased as a result of illness in fetal sheep and that such measurements may be useful as an indicator of fetal distress. The normal increase in plasma T3 late in gestation is not necessary for the late gestational cortisol surge or for normal parturition.


Studies on the interaction of tiamulin with the phospholipids in model membranes and with microsomes. The drug tiamulin interacts with phospholipid membranes mainly in a nonelectrostatic way. At pH-values where the drug possesses a net positive charge only small binding is observed. In the presence of cholesterol tiamulin is excluded from the membranes. The interaction of tiamulin with membranes cannot be explained by a simple partitioning but is governed by structural rearrangements of the lipid phase. At low drug concentrations we observe sigmoidal binding characteristics in the rigid as well as in the fluid state up to a level of about 2-3 mol drug bound per 1000 mol phospholipid. The range in which this cooperative interaction occurs can be compared with the drug concentration in the erythrocyte membrane which protects from hypotonic lysis. Further addition of tiamulin to the rigid membrane leads to fluidization. Saturation of the membranes with tiamulin is completely in parallel to their fluidization. The relevance of the cooperative interaction at low drug concentration and of the subsequent fluidization at elevated concentration for the microsomal membrane is discussed.


Isolation of the catalytic subunits of cyclic AMP-dependent protein kinases from different mammalian tissues on the basis of charge differences of their subunits. Both, the experimental data and a literature survey presented reveal common charge differences in the subunit composition of cAMP-dependent protein kinases from a variety of mammalian tissues. In general, the holoenzymes (type I and II) focus at pH 4.5-5.5, the cAMP-binding regulatory subunit below pH 4, whereas catalytic subunits are found at pH 6.7-9.1. For the purification of the catalytic subunits, the anionic holoenzymes of a variety of rat and rabbit organs as well as mouse and human-derived tissue culture cells were adsorbed to DEAE-cellulose. The catalytic subunit from the particular isoenzyme were selectively eluted at an appropriate ionic strength depending on the isoenzyme elution pattern by small amounts of cAMP. Extraction of tissues with Triton X-100 increased enzyme yield. Improved elution of the enzyme was accomplished by 8-(4-aminobutyl)amino-cAMP instead of cyclic AMP. Carboxymethyl-cellulose chromatography leads to both, concentration and, if necessary, to further purification of the enzyme.


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


Human platelet arylsulphatases: identification and capacity to destroy SRS-A. Arylsulphatases IIA and IIB have been separately identified in human platelets by use of anion exchange chromatography and gel filtration. Arylsulphatase IIA had a molecular weight of 160,000 and a pH optimum of 4.5. Arylsulphatase IIB had a molecular weight of 60,000 and a pH optimum of 5.5. Both arylsulphatases IIA and IIB were inhibited by phosphate and sulphate ions characteristic of this enzyme class. Platelets, upon exposure to ionophore A-23187 or thrombin, discharged arylsulphatase coincident with beta-glcuronidase release. Partially purified platelet arylsulphatase IIB inactivated rat SRS-A.


Assessment of attachment of Neisseria gonorrhoeae to HeLa cells by double radiolabeling. Attachment of Neisseria gonorrhoeae to HeLa cells was assessed by a technique using double radioisotopic labeling. Piliated, virulent bacteria from colony type 2 attached to HeLa cells to a greater extent than nonpiliated, avirulent bacteria from colony type 4. Maximal attachment rates for bacteria from both colony types occurred during the early incubation periods at 37 degrees C, and the HeLa cells appeared saturated at 4 h. Attachment was maximum at pH 6.5 and dependent upon the multiplicity of infection. Treatment of the HeLa cells with trypsin diminished the degree of attachment, but this effect substantially disappeared by 24 h after trypsin treatment. Scanning electron microscopy revealed bacteria of colony types 2 and 4 adhered to the HeLa cell surface. Thin-section transmission electron microscopy showed that bacteria were associated with the surface of the HeLa cell but not ingested.


Ultraviolet irradiation disrupts somatic pili structure and function. Three piliated bacterial species were exposed to ultraviolet light (7 X 10(3) microW/cm2), and the effect of increasing duration of irradiation on the integrity of the somatic pili was quantitated by negative-stain electron microscopy. Heavily piliated Proteus mirabilis became devoid of pili after 20 min of irradiation, but Escherichia coli and Neisseria gonorrhoeae required 40 min for complete depiliation. Partially purified proteus pili underwent progressive loss of structural integrity with increasing doses of irradiation as determined by negative staining and nephelometry, suggesting that ultraviolet light exerted an effect directly on the pili themselves. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that new, small molecular weight fragments appeared after irradiation of purified E. coli pili, suggesting that cleavage of the peptide chain rather than disassociation of pilin monomers accounted for the loss of pili structure. Ultraviolet irradiation also inhibited the ability of piliated bacteria to bind to human buccal epithelial cells. These observations indicate that the ultrastructural integrity and function of pili can be disrupted by ultraviolet light.


Immunochemical cross-reactions between type III group B Streptococcus and type 14 Streptococcus pneumoniae. Serological cross-reactions between certain streptococci and some serotypes of Streptococcus pneumoniae have been reported. These studies detail the serological cross-reactivity observed between hot HCl-extracted group b streptococcus type III (GBS III) antigens and S. pneumoniae type 14 (Pn 14) polysaccharide. Similar electrophoretic migration patterns of GBS III and Pn 14 were observed when either type-specific BGS III antisera or pneumococcal omniserum was utilized to precipitate these antigens. Both the GBS III antigen and the Pn 14 polysaccharide migrated toward the cathode, whereas all other pneumococcal polysaccharides migrated toward the anode. No cross-reactions were observed between GBS III antisera and the 11 other types of pneumococcal polysaccharides. Lines of identity were observed between type-specific GBS III antisera and monospecific Pn 14 antiserum with either GBS III antigens or purified Pn 14 polysaccharide. The cross-reacting antigens of GBS III and Pn 14 appear to be identical by immunodiffusion and immunoelectrophoresis.


Influence of lipopolysaccharide on graft versus host reactivity of lipopolysaccharide-unresponsive C3H/HeJ mice. It was initially reported that lipopolysaccharide (LPS)-unresponsive C3H/HeJ mice are refractory to LPS at the B-lymphocyte level, but more recently it has been shown that other cells are similarly unaffected. The current study was undertaken to study an in vivo LPS-modulated disease process involving macrophage-T cell interactions. Adult CBA/J and C3H/HeJ mice were used as spleen donors, and graft versus host reactions were induced in BALB/c neonates. Prior LPS treatment of CBA/J adults decreased the ability of their spleen cells to cause fatal graft versus host disease in BALB/c neonates, whereas no difference was found between injection of spleen cells from normal or LPS-treated C3H/HeJ mice. Similar results were obtained with these cell types when the mouse spleen mixed leukocyte culture system was used. In a carbon clearance assay for stimulation of the reticuloendothelial system with LPS, it was found that the rate of phagocytosis was significantly increased in BALB/c and CBA/J mice 72 h after inoculation of LPS. No stimulation was seen in rate of carbon uptake in the C3H/HeJ animals after treatment with phenol-extracted LPS or with butanol-extracted LPS. An LPS-induced protective serum factor was produced only in the LPS-responsive CBA/J mice and was specific for the syngeneic cells.


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


Bacterial adherence to pharyngeal cells in smokers, nonsmokers, and chronic bronchitics. Selective adherence to host mucosal surfaces is probably a requirement for colonization and infection by bacteria. Since pharyngeal colonization may be an important determinant in the pathogenesis of pneumonia, we studied the adherence of 10 different bacteria to pharyngeal cells obtained from nonsmokers, smokers, and chronic bronchitics. Various patterns of adherence among the different groups of subjects were found. Young healthy smokers had increased adherence of Streptococcus pneumoniae type I and, to a lesser extent, S. pneumoniae type III and Staphylococcus aureus when compared with nonsmokers. Middle-aged smokers with a long history of chronic bronchitis had significantly increased adherence only of untypable Haemophilus influenzae when compared with age-matched nonsmokers. The acquisition of pneumococcal pneumonia by smokers and the role of nontypable Haemophilus species in chronic bronchitis may be determined, in part, by bacterial adherence to pharyngeal cells.


Immunoglobulin A1 protease production by Haemophilus influenzae and Streptococcus pneumoniae. Bacterial strains of Haemophilus species and Streptococcus pneumoniae were examined for synthesis of the enzyme immunoglobulin A1 (IgA1) protease. Of 36 H. influenzae strains examined, 35 produced IgA1 protease; strains included all six capsular types, unencapsulated variants of types b and d, and untypable H. influenzae. Eight Haemophilus strains (non-H. influenzae) were studied, and two produced IgA1 protease. All 10 strains of S. pneumoniae produced IgA1 protease; these strains included 9 different capsular polysaccharide types and 1 untypable strain. Both IgA1 proteases cleaved myeloma IgA1 and secretory IgA but not myeloma IgA2, IgM, or IgG as determined by immunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes cleaved IgA1 myeloma sera, but not IgA2, into two fragments. The apparent molecular weight of the cleaved fragments was dependent both on the apparent molecular weight of the cleaved fragments was dependent both on the specific IgA1 protease assayed and the specific IgA1 substrate utilized. It is postulated that both carbohydrate variation between the IgA1 substrates studied and the ability of S. pneumoniae glycosidases to cleave carbohydrates from glycoprotein offer an explanation for the different fragment sizes observed.


Large-scale production and physicochemical characterization of human immune interferon. Large-scale production of crude high-titered (10(2.3) to 10(4) U/ml) human immune interferon (type II) was carried out in roller bottle cultures of human peripheral lymphocytes by using the T-cell mitogen staphylococcal enterotoxin A. Over 99% of human immune interferon was destroyed by pH 2 or heat at 56 degrees C for 1 h. The interferon was not neutralized by antibody to human leukocyte interferon. The kinetics of development of the antiviral state were slow for immune interferon relative to those for leukocyte interferon. Ultrogel AcA 54 chromatography of crude or the concentrated interferon resulted in two peaks of activity, a major one (87% of recovered activity) with a molecular weight of 40,000 to 46,000 and a minor peak of molecular weight 65,000 to 70,000. The column elution buffer consisting of 18% ethylene glycol and 1 M NaCl in phosphate-buffered saline resulted in at least 100% recovery of added interferon. The data suggest, then, that the interferon produced under large-scale conditions was immune (type II). The efficiency of the production was comparable to that described for large-scale production of human leukocyte interferon. Our large-scale production system for human immune interferon offers a feasible approach to preparation of large quantities of purified immune interferon for structure studies, antibody production, and clinical application.


Attachment pili from enterotoxigenic Escherichia coli pathogenic for humans. Pili from enterotoxigenic Escherichia coli pathogenic for humans have been isolated by adsorption to the surface of erythrocytes followed by thermal elution. The pili are composed of two protein subunits with molecular weights of 13,100 and 12,500 as determined by sodium dodecyl sulfate-gel electrophoresis. These pili also bind to human buccal cells under temperature conditions (37 degrees C) which prevent the binding of these pili to the erythrocytes. Analogous temperature effects on binding have previously been observed with whole bacterial cells. This binding can be inhibited by antiserum prepared against the isolated pili.


Adherence of Mycoplasma pneumoniae to glass surfaces. Attachment of M. pneumoniae to glass was quantitated in an experimental system enabling the settling down of [3H]palmitic acid-labeled cells onto glass cover slips. Attachment of mycoplasmas suspended in buffer increased with temperature, decreased with higher ionic strength, and showed a maximum at about pH 5.5. The findings suggest a participation of ionic bonds in the attachment process. Trypsin did not detach glass-bound mycoplasmas, and treatment of the cells with glutaraldehyde did not reduce their attachment to glass, suggesting that membrane components other than proteins may be involved in the attachment. Low concentrations (up to 20 mg/ml) of bovine serum albumin buffer. However, during the next few hours, attachment increased far above the bovine serum albumin control. This marked increase was reduced by more than half in the presence of chloramphenicol. Increased attachment was also observed when glucose (0.1 to 2 mg/ml) was added to the bovine serum albumin-containing buffer. The findings suggest different mechanisms for the attachment in protein-free buffer and in growth medium or glucose-containing bovine serum albumin buffer, respectively. The latter apparently requires metabolic activity of the mycoplasmas.


Progestational potency of oral contraceptives: a polemic. Dickey and Stone have attempted an evaluation of progestational potencies of oral contraceptives based upon such uterine criteria as subnuclear vacuolization and delay of menses. Their review, unfortunately, is marred by numerous errors which vitiate the potency estimates. The actions of progestagens in target organs depend upon the specific binding of the compounds to a protein receptor that is produced by estrogen treatment. Potency of hormones depends ultimately upon this binding. Since it seems unlikely that specific binding occurs in such nontarget sites as the blood vessels, direct potency relationships are highly improbable between diverse phenomena. Possible relationships between oral contraceptives and specific side effects must be studied in relationship to individual side effects and particular contraceptive products. Possible coincidental activities, no matter how analyzed, are unlikely to contribute meaningfully to our understanding of these drugs.


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


Clinical evaluation of two biphasic and one triphasic norgestrel/ethinyl estradiol regimens. Four hundred and fifty-eight women were followed for 3,586 cycles in a controlled, randomized, open trial designed to compare two biphasic and one triphasic norgestrel/ethinyl estradiol (ee) regimens. The three regimens are effective and no pregnancies were reported. Cycle control was good with all three regimens but the incidence of intermenstrual bleeding was higher with the triphasic. With this regimen, total hormonal ingestion is well below the lowest fixed dose norgestrel/ethinyl estradiol preparation (300 mcg norgestrel and 30 mcg ee) in the original 10:1 ratio. It is concluded that the staggering of hormonal doses opens the possibility of contraception with less hormone intake than is possible when a fixed dose regimen is used.


Antibodies to spermatozoa. VIII. Correlation of sperm antibody activity with postcoital tests in infertile couples. A study of the correlation between results of postcoital tests (PCTs) and presence of sperm antibodies in sera of either male or female partners or in the cervical mucus of seminal plasma, was made in 127 infertile couples. The percentage of positive antibody tests increased progressively as PCT results deteriorated from excellent to poor. Thus, the percentage of positive serum tests, by either method used, was 7% in the excellent group as compared to 52% in the poor group. This progressive increase was evident only in the females; the antibody tests in the males did not correlate directly with the PCT results. Nonetheless, even with excellent PCT results, 7% of women and 21% of men showed presence of antibodies. On the other hand, in the group showing poor PCTs, 48% of women had an absence of antibodies.


Chromosomal anomalies of preimplantation rabbit blastocysts and human artificial abortuses in relation to maternal age. It is well established that in man there is a higher frequency of chromosomally and phenotypically abnormal offspring and a decline in fertility as maternal age increases. This paper describes preliminary work on some chromosomal aspects of 5- and 6-day old preimplantation rabbit embryos, with attention to maternal age dependence of chromosome anomalies. Chromosome preparations were made by a modification of the method of Shaver and Carr (1967). Two trisomic blastocysts were observed among 50 embryos examined from 10 does of 40 to 52 months of age, that is, 45XY with an extra submetacentric chromosome and 45XX with an extra acrocentric chromosome. In man, three trisomies (47XX, G+, 47XX, D+, and 47XY, G+), one monosomy (45X or 45XX, C-) and one double trisomy (48XY, C+, E+ or 48XXY, E+) were karyotyped out of 32 early induced abortuses from women older than 40. Among the 22, induced abortuses analyzed from women between 35 and 39, one trisomic embryo of 47XY, G+ was observed.


Plasma gonadotropin response to D-Ala6-LH-RH propylamide. A long-acting analog of LH-RH, D-Ala6-desGly10-LH-RH propylamide (D-Ala6-LH-RH PA) was administered intramuscularly in a dose of 250 micrograms to 11 women with amenorrhea in whom the determinations of urinary steroid secretion, progesterone challenge, clomiphene, HMG, and LH-RH tests had been performed previously. Blood samples were taken twice before the injection and at 0.5, 1, 2, 4, 8, 12, 24, 26, 28, 30, and 32 h. No visible side effects were observed. Plasma levels of LH and FSH were determined by radioimmunoassays and expressed in mIU/2nd-IRP HMG/ml. The analog caused a great elevation in plasma LH and FSH levels. The maximal absolute increment for LH was between 3.93 and 115.89 mIU/ml, and the maximal increment in percentage was between 220 and 4,300. The time of the LH peak varied between 0.5 and 12 h. For FSH, the maximal absolute increment was between 6.45 and 50.92 mIU/ml and the maximal increment in percentage was between 63 and 1,442. The peak of FSH occurred in most cases at 4 to 8 h.


Evaluation of the hypothalamic-pituitary function in a female with hypogonadotropic hypogonadism and anosmia. A case of Kallmann's syndrome in a woman is presented to evaluate the hypothalamic-pituitary function and status of the genital organs. The diagnosis of Kallmann's syndrome was performed under the determination of low circulating level of estradiol-17 beta and gonadotropins and low sensitivity to an olfactometer. Pituitary response to LRH was observed. Plasma LH response to LRH was observed in a lesser degree compared with the less impaired response of FSH. Daily injection of LRH for 13 days increased pituitary response of gonadotropin secretion. Diminished prolactin secretion and the dissociation of response to TRH and chlorpromazine were shown. Laparoscopic findings revealed a hypoplastic uterus and nearly normal sized ovaries. Ovarian response to exogenous gonadotropins was affirmed by increased estradiol-17 beta level. These results indicated disturbed hypothalamic control of gonadotropins and prolactin, and a reserved pituitary function in this case. Low circulating level of estradiol-17 beta might affect the heterogenous response of the hypothalamic-pituitary axis.


Attempts to induce ovulation in amenorrheic patients using D-Ala-6-LH-RH propylamide. Thirteen amenorrheic patients, two with primary amenorrhea, and eleven with secondary amenorrhea, including five with anorexia nervosa, were treated with an analog of LH-RH, D-Ala-6-desGly-10-LH-RH propylamide (D-Ala-6-LH-RH PA). The patients were follwed with checks of daily basal body temperature, cervical mucus characteristics, urinary total estrogens and pregnanediol, plasma LH and FSH, and twice-weekly clinical checkups. D-Ala-6-LH-RH PA was given continuously for 11 days at a dose of 50 microgram im from the 4th day of a steroid-induced cycle. Ethynyl estradiol (50 microgram orally) was given twice on day 14 followed by 250 microgram of D-Ala-6-LH-RH PA for the following 3 days. All the treated cycles were monophasic. Withdrawal bleeding occured in nine patients from the 4th to 6th days posttreatment. A significant total maximal increment in estrogens was found in seven patients, which was higher than the values obtained previously in the same patients with human menopausal gonadotropin. One patient conceived in the cycle immediately following discontinuation of treatment, indicating a possible effect of analog on the follicular development during the treatment; a normal baby was delivered. A second patient conceived three cycles afterwards, and two other patients began normal menstrual cycles.


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


Distribution of ATPase in isolated human spermatozoa nuclei: a high resolution cytochemical study. The presence of ATPase activity was demonstrated in isolated nuclei of human spermatozoa by high resolution cytochemical methods. The Wachstein and Meisell technique as modified by Marchesi and Palade was used. ATPase activity was identified as dense and irregularly distributed granules confined to the exposed surface of spermatozoa nuclei. Within the nucleus the reaction product appeared as electron dense precipitates randomly distributed. Control experiments were negative. Deposits of lead phosphate specifically restricted to the exposed surface of nuclei were interpreted as an indication of a glucose-6-phosphatase and/or phosphohydrolase activity. Whether this activity is located in remnants of the inner leaflet of the nuclear envelope is not known. The presence of the enzyme activity within the nucleus is thought to be related to aerobic ATP synthesis previously suggested. If so, this function may be involved in establishing and/or maintaining the highly complex structural organization of spermatozoa nuclei.


Prostaglandins E and F in the uterine tissue of cyclic rats. Prostaglandins E and F (PGE and PGF) levels and the biosynthetic ability of rat uterine tissue have been estimated in the various phases of oestrous cycle by radioimmunoassay technique. Significantly high PGF concentration was found during metaoestrous phase. In comparison, PGE levels did not show as high elevation. The metaoestrous phase also showed significantly higher biosynthesis of prostaglandins E and F. Our experimental findings support the role of prstaglandins in ovulation and luteolysis of the corpus luteum.


Detection of beta subunit HCG in plasma of IUD users as an indication of frequency of conception. One hundred and two women using an intrauterine copper device (Cu IUD) were studied for determination of HCG in blood plasma specimens obtained during the last 5 days of the menstrual cycle. HCG, LH, and progesterone were measured by radioimmunoassay (RIA). Two positive HCG values were found, indicating conception. The methods by which the frequency of fertilization during IUD use can be evaluated are discussed.


Phosphatases in human seminal plasma.

Phosphatases in human seminal plasma. Alkaline and acid phosphatase activity was measured in the ejaculate of normo-, oligo-, and azoospermic males. Due to the considerable scattering no correlation was found between sperm density and enzyme activity. Phosphatase activities were very similar in the fertile and infertile groups. On the basis of this observation phosphatase activity measurements are not recommended in clinical practice.


Effect of indomethacin on prostaglandin and steroid synthesis by the marmoset ovary in vivo. Indomethacin decreased Prostaglandin F 2 alpha levels in the ovaries of marmoset monkeys which were treated with PMS/HCG. The production of estradiol and progesterone, however, was unaffected by the blockade in prostaglandin synthesis. The number of ovulations following PMS/HCG treatment was significantly reduced by indomethacin treatment. These observations suggest that prostaglandins are required for follicular rupture but not for progesterone and estradiol production by the ovary.