Endogenous substrates for epidermal transglutaminase. Potential in vivo substrates for epidermal transglutaminase have been isolated and partially characterized in human stratum corneum and new born rat epidermis. [14C]Putrescine and dansylcadaverine were incorporated into epidermal proteins in vitro. Two high molecular weight proteins incorporated the labels in both the rat ahd human homogenates. One of the proteins was too large to enter a 4% sodium dodecyl sulfate-polyacrylamide spacer gel; the other was seen at the interface between the spacer gel and a 10% sodium dodecyl sulphate-polyacrylamide running gel. These proteins were present in a buffer extract, sodium dodecyl sulphate-dithiothreitol extract and NaOH extract. The labels were also incorporated into protein in the insoluble pellet remaining after the afore-mentioned extractions. The incorporation of putrescine and dansylcadaverine was time dependent, and was inhibited by known inhibitors of epidermal transglutaminase. The two high molecular weight proteins had similar amino acid composition, characterized by high glycine, glutamic acid, serine and aspartic acid. The amino acid composition was similar to, although not identical with, the amino acid composition of alpha-keratin proteins. Epidermal homogenates incubated in the presence of transglutaminase showed progressive insolubilization of the protein. This cross-linking was inhibited by putrescine. [14C]Glycine, [14C]histidine and [4C]proline were incorporated into epidermal proteins in newborn rats in vivo. The glycine-labelled protein became progressively more insoluble when incubated in vitro in the presence of transglutaminase. In vitro incubation with transglutaminase had no effect on the histidine-and proline-labelled proteins.


Studies on (Na+ + K+)-activated ATPase. XLII. Evidence for two classes of essential sulfhydryl groups. 1. Preincubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits the (Na+ + 5+)-ATPase and K+-stimulated 4-nitro-phenylphosphatase activities. Phosphorylation of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the (Na+ + K+)-ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. Titration with 5,5'-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule (Na+ + K+)-ATPase (Mr = 250 000). 3. Treatment with N-ethylmaleimide, resulting in complete inhibition of (Na+ + K+)-ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5'-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. The reaction of N-ethylmaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their different reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide.


Studies on sphingomyelinase of Bacillus cereus. I. Purification and properties. A sphingomyelinase was purified 980-fold with recovery of 25.6% from the culture broth of Bacillus cereus, by (NH4)2SO4 precipitation and chromatography on CM-Sephadex, DEAE-cellulose and Sephadex G-75. The purified preparation was free of lipase, protease and other phospholipases. The enzyme specifically hydrolyzed sphingomyelin to ceramide and phosphorylcholine. Lysophosphatidylcholine was also attacked by the enzyme. The enzyme (Mr = 24 000) was maximally active at pH 6-7. Other properties of the enzyme, including hemolytic activity and activation/inhibition studies, are reported.


Indole to phenolate energy transfer, the alkaline quenching mechanism of beta-trypsin fluorescence. Evidence that indole to phenolate energy transfer causes the decrease in beta-trypsin fluorescence with increasing alkalinity is obtained indirectly from viscosity, lifetime, wavelength dependence and chemical modification studies which either exclude or minimize the involvement of other possible mechanisms.


Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy. The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.


The effect of evolution on homologous proteins: a comparison between the chromophore microenvironments of Italian water buffalo (Bos bubalus, L.) and sperm whale apomyoglobin. The perturbing effect of guanidium hydrochloride and pH on the molecular structure of water buffalo apomyoglobin has been investigated by circular dichroism in the far and near ultraviolet and by fluorescence. In the wavelength region between 320 and 260 nm the circular dichroic spectrum of the globin is highly structured and the contributions of the aromatic chromophores have been resolved. Buffalo apomyoglobin undergoes a structural transition at neutral pH which involves elements of the secondary and tertiary structure, as indicated by changes of dichroic activity of the peptide and aromatic chromophores and the fluorescence of the two tryptophanyl residues. The possibility of charge-transfer complex between indole and imidazole is discussed. A major structural transition with abrupt unfolding takes place in the pH region between 5.6 and 4.3. Below pH 4.3 the peptide helical residues, which survive the acid transition, appear to be resistent to further acidification to pH 2.0 while tryptophanyl emission is quenched and shifted to longer wavelengths. A structural transition occurs also in alkali above pH 10, which has been detected by the same techniques. The relationships between buffalo and sperm whale apomyoglobin are discussed.


Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium. In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH+4 is in equiblibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in gamma-glutamyl transferase assays containing 60 mM Mg2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of gamma-glutamyl transferase activities without and with addition of 60 mM Mg2+. Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH+4, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH+4 produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.


Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates. When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H+). Although its acid-base properties were indistinguishable from those of epsilon-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the epsilon-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0. It is apparent that the assay of epsilon-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236--250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.


Microcalorimetric method to determine competitive binding. Action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan . human serum albumin complex. A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated. The association constants and the enthalpy variations are equal to 14 000 +/- 2000 M-1 and --6.6 +/- 0.2 kcal/mol for human serum albumin . tryptophan complex and 13 000 +/- 1000 M-1 and --10.0 +/- 0.2 kcal/mol for human serum albumin . chlorazepate complex. In all cases the stoichiometry is equal to one. The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 +/- 200 M-1 and --2.6 +/- 0.3 kcal/mol.


Interactions between chondroitin sulfate and concanavalin A. Chondroitin sulfate, the major extracellular matrix glycosaminoglycan, formed an insoluble complex with concanavalin A at pH 5.4 or below. Concanavalin A (500 microgram/ml) reacted only within a relatively narrow concentration range of chondroitin sulfate (optimally between 5 and 50 microgram/ml) at pH 5.4 in 0.05 M buffer. Similar precipitin-like interactions were seen between concanavalin A and hyaluronic acid or heparin. No precipitating complexes formed between concanavalin A and the glycosaminoglycans at these concentrations in physiological salt solutions (approx. 0.15 M) unless the pH was below 4.5. Precipitating self-aggregates of concanavalin A appeared to be promoted by chondroitin sulfate at pH 7.3, but no significant precipitation occurred between the reactants at this pH even at very high concentrations, nor did soluble complexes form as determined by affinity chromatography on Sephadex G-200 or fractionation on Bio-Gel P-200. Thus, binding between the lectin and glycosaminoglycans appeared to depend upon reversible non-specific electrostatic interactions observed only at low Ph and low ionic strength. Stable interactions were not seen in experiments using physiologically balanced salts at near neutral pH.


Regulation of thyroid hormone metabolism in rat liver fractions. The nature of the conversion of thyroxine (T4) to triiodothyronine (T3) and reverse triiodothyronine (rT3) was investigated in rat liver homogenate and microsomes. A 6-fold rise of T3 and 2.5-fold rise of rT3 levels determined by specific radioimmunoassays was observed over 6 h after the addition of T4. An enzymic process is suggested that converts T4 to T3 and rT3. For T3 the optimal pH is 6 and for rT3, 9.5. The converting activity for both T3 and rT3 is temperature dependent and can be suppressed by heat, H2O2, merthiolate and by 5-propyl-2-thiouracil. rT3 and to a lesser degree iodide, were able to inhibit the production of T3 in a dose related fashion. Therefore the pH dependency, rT3 and iodide may regulate the availability of T3 or rT3 depending on the metabolic requirements of thyroid hormones.


Differences between human and rabbit transferrins. Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic. The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.


The interactions of gallium with various buffers and chelating agents in aqueous solution: gallium-71 and hydrogen-1 NMR studies. The interactions of gallium (Ga) with the ligands, EDTA, NTA, phosphate, lactate, MOPS, TRIS and HEPES are investigated using both 71Ga and 1H nmr measurements. Both EDTA and NTA form strong complexes with gallium, which have a 1:1 stoichiometry. In alkaline solution the tetrahedral Ga(OD)4- competes strongly with EDTA in complex formation. In the lactate complex, there are probably three lactates per gallium present. The phosphate complexes of gallium are difficult to characterize on the basis of this investigation. The buffers, MOPS, TRIS, and HEPES, do not interact with gallium significantly. The ability of the ligands to bind gallium correlates well with their ability to inhibit gallium incorporation by L1210 leukemic cells.


Formation and spectral characterization of Cu(II)-poly(L-ornithine) complexes. Cu(II)-Poly-(1-ornithine) complexes in aqueous solution have been studied using potentiometric titration and absorption and circular dichroism spectra. As in the case of Cu(II)-poly(L-arginine) complexes studied previously, two types of compounds have been detected, labeled complexes I and II. Complex I contains two amine nitrogens and two water molecules coordinated to the copper. Complex II, two amine and two amide nitrogens. Amide nitrogen coordination confers optical activity to the copper d-d transitions. Furthermore, amine and amide nitrogen coordination to the copper are characterized by charge transfer transitions at 250 and 320 nm respectively which were already identified in Cu(II)-poly(L-arginine) systems.


[Activation parameters for the cleavage reaction of cytidine-2',3'-monophosphate catalyzed by Penicillium brevicompactum and Aspergillus clavatus RNAses]. On the basis of coincidence of all activation parameters (E, deltaH*, deltaF* and deltaS*) for the reaction of cleavage of cytidine-2';3'-monophosphate catalyzed by "acid" (pH-optimum 4.7) nonspecific RNAses from Aspergillus clavatus and Penicillium brevicompactum (EC 3. 1. 4. 23) it has been proposed that the mechanism of action on this reaction stage (hydrolysis) for both enzymes is equal.


[Kinetics of N-dealkylation of amines with participation of microsomal cytochrome P-450]. Within the temperature range of 20-37 degrees C the kinetics of the demethylation reactions of a variety of amines with participation of hepatic microsomal cytochrome P-450, NADPH and O2 has been studied. Catalytical rate constants for all the substrates have been determined in generalized forms. Activation parameters deltaH* and deltaS* are also estimated. There is a linear relationship between deltaH* and deltaS*: deltaH*=20.7 kcal+366 degrees K deltaS*. Compensation relationship is characterized by a coefficient alpha=366 degrees/Taverage=1.21. The nature of the limiting step of the enzymatic amine demethylation involving cytochrome P-450 of hepatic microsomes is discussed.


[Catalytic properties of three isoenzymes possessing pyrophosphatase activity, isolated from baker's yeast]. A kinetic study of inorganic pyrophosphatase isolated from brewer's yeast was done. It was shown that all three isoenzymes have the same pH-optimum and specificity with respect to substrate and metal activator. Statistical treatment of the kinetic data yielded equilibrium and catalytical constants, describing enzyme interaction with the metal activator and substrate. The catalytic properties of all three isoenzymes are similar to those of the baker's yeast pyrophosphatase. The fluoride inhibition pattern for inorganic pyrophosphatase from brewer's yeast is similar to that for the baker's yeast enzyme.


Preliminary studies with L-asparaginase bound to implantable bovine collagen heterografts: a potential long-term, sustained dosage, antitumor enzyme therapy system. In this study, L-Asparaginase has been bound to collagen heterografts derived from carotid bovine arteries. The immobilization procedure utilizes both non-covalent and covalent interactions to fix the enzyme. Binding of the enzyme to the graft material was shown to the pH dependent, with optimum binding occurring at pH 6.0 and pH 8.5. Amidohydrolysis by the bound enzyme exhibited zero-order kinetic behavior at substrate saturating conditions. Total apparent asparaginase activity expressed by the grafts as a function of the number of repeated in vitro assay trials demonstrated that over a span of 3 months of intermittent storage and use, the enzyme-grafts retained as much as 62% of their initial activities. Implantation of 4 asparaginase-collagen grafts in various locations of the thoracic and abdominal aorta resulting in prolonged reductions of plasma asparagine levels in 3 of the 4 implants. Presence of plasma asparaginase was checked in one of the four implants and determined to be less than 2 X 10(-4) I.U./ml. Removal of grafts from 3 of the 4 animal subjects showed reductions in the apparent asparaginase activity expressed by the grafts of 7 to 70 percent after in vivo contact times which varied from 6 to 15 days.


Relation of the acid concentration of the parietal component of the gastric juice to the maximal acid output in man. In two populations of patients tested with S.C. histamine and with I.V. pentagastrin the values of the theoretical acid [H+]O and chloride [Cl-]O concentration of the parietal secretion for a zero value of sodium from the relationships H+ = f ([Na+]) and Cl- = f ([Na+]) were calculated. In both populations a decrease of [H+]O and [Cl-]o for low acid outputs was observed. The parallel decrease of the calculated H+ and Cl- ion concentration of the parietal component for low acid outputs gives indirect evidence that parietal secretion could become hypotonic in patients whose acid output is low.


Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde. High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20-50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, PH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60 degrees C, pH 10 for alkaline protease and 50 degrees C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the Km values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60 degrees C. Other peptide hydrolases, beta-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.


A new method for cell immobilization.

A new method for cell immobilization. A new method for producing particles and membranes containing immobilized bacteria is presented. These immobilized bacteria display good stability over time making them well suited for use in a packed-bed reactor. Such a reactor is tested as a function of the different parameters of the system. The results are qualitatively similar to those obtained with purified enzyme reactors, but some discrepancies with the plug-flow model are noted. It is necessary to use a more sophisticated model in order to fit the experimental data.


Isolation of lysozyme on chitosan.

Isolation of lysozyme on chitosan. Lysozyme has been immobilized on chitosan, a polyaminosaccharide, without using any intermediate reagent. The best pH conditions for operating the chitosan columns have been determined and the best eluting agent was found to be a 2% solution of propylamine. The lysozyme activity was determined after reacting lysozyme with the product of glycolchitin and Remazol Brilliant Blue R. The recovery of lysozyme from chicken egg white yields lysozyme with 55% activity.


[Effect of cholinolytic and adrenergic blocking preparations on rat erythrocyte resistance to hypo-osmotic hemolysis]. The influence of central cholinolytics and adrenoblocking drugs on the hemolysis of rat erythrocytes in the hypoosmotic buffer was studied in vitro. At pH 7.4 in a concentration of 10(-4) M central cholinolytics ethyl-dipracil, diphacil, pediphen, tropacin, and beta-adrenoblocking agent propranolol protected the erythrocytes from hemolysis most intensively. The central M-cholinlytics amizyl, glypin, and alpha-adrenoblocking agents purroxan, sympatholytin, phentolamin were less active. The antihemolytic effect of drugs reached the maximum in the course of 30 minutes, and was maintained for several hours. The protection of erythrocytes from hemolysis by drugs containing tertiary nitrogen was greater. Prevention of the hypoosmotic hemolysis pointed to the stabilization of the erythrocyte membrane by the preparations examined. In the mechanism of action of the central N-cholinolytics and beta-adrenoblocking drugs it is necessary to consider the possibility of stabilization of the membrane formations containing no synaptic contacts.


[Graft-versus-host reaction following granulocyte transfusion from a normal donor (author's transl)]. Graft-versus-Host disease following the transfusion of leukocytes from normal donor occured during remission-reinduction therapy in a 5 years old girl with acute lymphocytic leukemia. The clinical picture consisting of rash, diarrhea and somnolence was congruent with the diagnosis. Graft-versus-Host disease was confirmed by histology of skin biopsy and exclusion of other causes. The child survived the disease. We recommend the irradiation of leukocyte concentrates before transfusion.


Responses of rabbit portal vein to histamine. 1 Histamine produced a dose-dependent contraction of the isolated portal vein of the rabbit. This contraction was not antagonized by atropine, methysergide, indomethacin, cocaine or 6-hydroxy-dopamine, nor by pretreatment of the rabbit with reserpine. 2 The response to histamine was blocked by H1-receptor antagonists only when the blocking agent was used in very high concentrations, and was not antagonized by the H2-receptor blocking agent, metiamide, H1-receptor antagonists did not block the effects of 5-hydroxytryptamine. 3 The contractions elicited by histamine, 5-hydroxytryptamine and noradrenaline were blocked by phentolamine. 4 Desensitization to high doses of 5-hydroxytryptamine caused a concomitant depression in the response to histamine but not to noradrenaline or acetylcholine. 5 The results suggest that the contractions of rabbit portal vein elicited by histamine are not mediated by receptors of the H1- or the H2-type, but may involve an action of histamine at a receptor which is also involved in the action of 5-hydroxytryptamine.


Release of noradrenaline from cat spleen slices by potassium. 1 When cat spleen slices were exposed to a potassium-enriched (140 mM) Krebs solution, 367 +/- 31 ng g-1 5 min-1 of noradrenaline (NA) was released into the bathing medium. 2 Phenylephrine and clonidine (10(-7) to 10(-3) M) did not significantly modify the potassium-evoked NA release; acetylcholine decreased it in a dose-dependent manner. 3 Phenoxybenzamine increased NA release by 50% but phentolamine did not alter it; high concentrations of this drug greatly decreased NA release. Cocaine increased the NA release by about 30%. 4 It is suggested that the failure of sympathomimetic amines to depress, and of alpha-adrenoceptor blocking agents to enhance the release of NA by high potassium concentrations may be related to prolonged depolarization of the nerve terminals, which may desensitize presynaptic alpha-receptors. The fact that the same drugs are able to modify NA release during electrical nerve stimulation may be ascribed to the much shorter periods of depolarization occurring under these conditions.


An in vitro comparison of beta-adrenoceptor stimulants on potassium-depolarized uterine preparations from guinea-pigs. 1 A comparison of six beta-adrenoceptor stimulants has been carried out on in vitro preparations of guinea-pig uterus which were depolarized in K(+)-Krebs solution. Results have also been obtained on uterine preparations in which contractions to acetylcholine were inhibited. The establishment of the conditions for the K(+)-depolarized preparations are described.2 There was no significant difference between potency values (mean neg log EC(50) values) for any of the drugs on the two types of uterine preparation i.e. the preparations had the same sensitivity to the drugs.3 There was a less than two-fold difference between the relative potency values for the beta-adrenoceptor stimulants on the two types of uterine preparation. The relative potency values (isoprenaline = 100) on the K(+)-depolarized preparation were fenoterol 74.1, salbutamol 15.1, rimiterol 13.5, terbutaline 8.2 and orciprenaline 5.6.4 The relative potency values obtained on uterine preparations were less than three-fold different from those previously found for guinea-pig trachea (after inhibition of extraneuronal uptake).5 The pA(2) value for propranolol on the K(+)-depolarized uterine preparations was 9.13.6 It is concluded that the K(+)-depolarized guinea-pig uterine preparation can be used for quantitative studies on beta-adrenoceptor stimulant drugs. It lacks spontaneous activity, drugs can be added cumulatively and several drugs can be compared on a single preparation. In addition, the results obtained support the classification of the beta-adrenoceptors in guinea-pig uterus and trachea in the same sub-group (beta(2)).


In vivo release of gastrin into human gastric juice. The release of antral gastrin has been examined in human subjects by infusing saline and albumin into their stomachs. Immunoreactive gastrin (IRG) was present in fasting serum but not in fasting gastric juice. Intragastric infusions of saline and albumin induced small but significant (P less than 0.05) rises in the serum concentrations of IRG. There was no significant difference between the magnitude of the serum gastrin responses to these two stimuli. The stimuli induced a rise in the luminal concentration of IRG which was sixfold greater than that in the serum concentration of IRG. The response to albumin was significantly greater than the response to saline (P less than 0.05). The IRG in gastric juice was chromatographically similar to heptadecapeptide gastrin. The stimuli also released IRG in patients with duodenal ulcers. The magnitude of the release into serum and gastric juice in these patients was significantly reduced by antrectomy (P less than 0.05).


Studies on the avian shell gland during egg formation: mucosal intracellular pH. 1. The intracellular pH and the extracellular fluid volume of the shell gland mucosa were determined. 2. The pH value varied little during egg formation, the overall mean being 7.11 +/- 0.02 (n = 34). 3. The lowest pH, 7.01, was found 10 h after oviposition and the highest, 7.22, 18 h after oviposition.


Gastric volume and pH in out-patients.

Gastric volume and pH in out-patients. We measured the volume and pH of the gastric content of 21 out-patients and 21 in-patients under general anaesthesia. Gastric tubes were inserted after induction of anaesthesia, and gastric fluids were withdrawn for pH determinations. Gastric volumes were measured by a dilution technique using polyethylene glycol as the indicator and also by measurement of the volume aspirated through a gastrict tube. Out-patients had a mean gastric volume of 69 +/- 17 ml while in-patients had a mean volume of 33 +/- 4 ml. The average gastric pH for the out-patients was 1.8 +/- 0.2 and for the in-patients 2.0 +/- 0.3. Four out-patients had more than 75 ml of gastric fluid of pH less than 2.0. Aspiration through a gastrict tube did not empty the stomach completely and the volume thus obtained gave a falsely low estimate of the gastric volume.


Histopathology of veins after intravenous lorazepam and RO 21-3981. A previously established rat model has been utilized to demonstrate that an acute inflammatory response occurs after high intravenous doses of lorazepam. This occurs only with high concentrations of drug equivalent to 20 times the normal clinical dosage in man. In contract, water soluble RO 21-3981 produces no vascular pathology in any dosage evaluated. It appears that propylene glycol may play a role in the pathogenesis of the intravascular injury observed.


Detection and prevalence of pneumococci with increased resistance to penicillin. Susceptibility to penicillin was determined for 6000 strains of pneumococci isolated during 1974--76 from patients in Alberta and the adjacent region of the Northwest Territories. Strains were considered to be relatively resistant if the minimum inhibitory concentration (MIC) of penicillin was 0.16 microgram (0.26 U)/mL or more, which is eight or more times greater than the MIC for fully susceptible strains. Resistance was detected in 143 strains (2.4%) isolated from 122 patients and belonging to four capsular types. The MIC of the most resistant strains was 0.32 microgram (0.53 U/mL. Penicillin-resistant strains were highly resistant to oxacillin, the MIC being at least 30 times greater than that for penicillin-susceptible strains. Pneumococci resistant to penicillin may readily be detected by the narrowness or absence of a zone of inhibition around a 1-microgram oxacillin disc in susceptibility tests on blood agar. The degree of resistance reported here is relative and does not necessarily preclude successful treatment with full therapeutic doses of penicillin G, but penicillin preparations that give low blood concentrations may not be suitable for treating infections caused by these strains.


Gamma-glutamyltransferase in putative premalignant liver cell populations during hepatocarcinogenesis. The activity of gamma-glutamyltransferase, as measured quantitatively and by histochemical staining, was studied in different cell populations during the induction of liver cancer with 2-acetylaminofluorene (2-AAF) or diethylnitrosamine and compared with findings in fetal and in intact and regenerating adult liver. The enzyme activity is 20-fold higher in 12-week nodules than in control livers and 30-fold higher in 20-week nodules than in controls. A similar 30-fold increase in activity relative to control is present in hepatomas, induced by either 2-AAF or diethylnitrosamine, and in fetal hepatocytes. The enzyme shows increases in activity in foci of very early putative preneoplastic hepatocytes induced by a single dose of diethylnitrosamine and selected by low doses of 2-AAF plus partial hepatectomy. By 7 days, the foci show a 4-fold increase in enzyme activity, and by 3 weeks they are 40-fold higher than in the control liver. Histochemically, the foci are strongly positive for gamma-glutamyltransferase, especially in the bile canaliculi. By 21 days, the ductular (oval) cells induced by 2-AAF have disappeared. When stained for the enzyme activity, the foci stand out clearly against the negative background of the liver, allowing easy quantitation. It appears that gamma-glutamyltransferase is a useful marker for preneoplastic hepatocytes.


Psychotropic drugs as potential antitumor agents: a selective screening study. Compounds with known psychotropic properties were tested for activity in murine ip L1210 leukemia and B 16 melanoma in a protocol designed to obtain leads for new antitumor agents which might also possess central nervous system (CNS) antitumor properties. Barbiturates and hallucinogenic compounds were the only compound types deliberately excluded. Representatives from most of the other known CNS agent classes were included among the 297 psychotropic drugs evaluated. Sixteen of these agents were reproducibly active against the L1210 tumor system with T/C values of 125%. Phenothiazines such as fluphenazine and butyrophenones such as triperidol were prominent among the confirmed active structural types. Dopamine, a beta-phenethylamine neurotrasmitter, was active. While reproducible B16 melanoma activity was not observed among the psychotropic drugs, most of the L1210 confirmed active agents were effective against the ip P388 tumor model and also were active in vitro against KB cells. Ic L1210 activity was not observed among the few compounds chosen for testing in that tumor system. The yield of ip L1210 confirmed actives from this group of psychotropic agents was 18 times that which would have been expected from the random screening of compounds.


Lack of effect of hypercapnic acidosis on elasticity of cat papillary muscles. Chages in external pH from 7.40 to 6.95 obtained by changing the pCO2 of the medium at constant bicarbonate concentration produced in cat papillary muscles a significant decrease in isometric tension with no changes in time to peak tension. Active and resting stiffness, as determined by two different methods, did not change under conditions of hypercapnic acidosis.


Species-specific aggregation factor in sponges. VII. Its effect on cyclic amp and cyclic gmp metabolism in cells of Geodia cydonium. In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA-initiation phase the cyclic AMP- and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10(6) cells to 0.3 pmol/10(6) cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10(6) cells. The activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. The soluble as well as the particulate enzyme activities were checked in vitro. The cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.


Enteroendocrine cells in the digestive tract of Barbus conchonius (teleostei, cyprinidae). Just as in other cyprinids, three zones can be distinguished in the digestive tract of Barbus conchonius. A fat absorptive zone (65--75%), including the intestinal bulb, is followed by a protein absorptive zone (25--35%) and a small ion and water absorptive zone (less than 5%). The main characteristics of these zones are described. Four types of enteroendocrine cells can be distinguished between the intestinal epithelial cells. The number decreases in the caudal direction, and there are very few in the protein absorptive zone. All the enteroendocrine cells are argyrophilic and differ mainly in the size and shape of their secretory granules. Serotonin producing and hence argentaffin cells have not been found. Amine precursor uptake and decarboxylation (APUD) by the enteroendocrine cells of adult fishes has not been observed. The possible functions of the enteroendocrine cells are discussed. (Auto-)phagosomes, common in epithelial cells of the gut of B. conchonius, show similar staining characteristics as the enteroendocrine cells; their function is discussed.


Ultrastructural appearance of neurosecretory granules in the sinus gland of the crab after different fixation procedures. The appearance of neurosecretory granules in the crab sinus gland was studied after fixation at different pHs. Whereas at pH 7.0 the neurosecretory granules were pleomorphic with respect to electron density, at pH 5.0 or 6.0 all the granules remained electron dense. The possible role of maturation as an explanation of this observation is discussed.


A radioautographic study of the neuroepithelial bodies of the lungs in fetal and neonatal rabbits. The neuroepithelial bodies (NEB's) of the lung of 29-day-old fetuses and 1-day-old rabbits, under the conditions of this study, neither take up 3H-thymidine nor undergo mitosis. Also the NEB's are not derived at these times from proliferations of other kinds of epithelial cells in the intrapulmonary airways. It is, therefore, suggested that the difference in numbers of NEB's previously observed by us, between the 29-day fetus and the 1-day-old rabbit, is due either to regranulation or acquisition of argyrophilic material by the NEB's or differentiation of other epithelial types. It is concluded that the NEB's are composed of well differentiated cells, which have a greatly reduced capacity to undergo mitosis.


P-Nitrophenol-alpha-D-glucopyranoside as substrate for measurement of maltase activity in human semen. Hitherto, seminal plasma maltase has been measured with maltose as substrate; this method is time consuming and lacks specificity. The use of a synthetic substrate, p-nitrophenol-alpha-D-glucopyranoside, allows accurate and rapid determination of this activity. When maltase is added to the incubation medium (the substrate and reduced glutathione in potassium phosphate buffer, pH 6.8), maintained at 37 degrees C, hydrolysis of the original substrate to p-nitrophenol goes at a constant rate during 4 h. Under optimal conditions of incubation, the Michaelis constant of the reaction, calculated by the Hanes method, was 2.92 +/- 0.84 (SD) X 10(-3) for six different semen samples. Isomaltase appeared to be absent from seminal plasma. The enzyme is stable to freezing and slow thawing and can be stored for at least 26 days at -80 degrees C. Its molecular weight is 259 000. Tris(hydroxymethyl)aminomethane (pH 6.8) exerts a noncompetitive inhibition on the enzyme activity. In 68 men 23 to 45 years old, whose semen analyses were normal, the seminal plasma maltase activity was 467 +/- 135 (SD) mU/g of protein. It was generally decreased in patients with infertility disorders.


Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate at 25, 30, and 37 degrees C. Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate were determined for isoenzymes 1 and 5 at 25, 30, and 37 degrees C. Three of the nine different buffers examined--imidazole, triethanolamine, and N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid--are satisfactory. Beta-NADH, pyruvate, and hydrogen ion concentrations were chosen to measure both isoenzymes with maximal-equal-sustainable efficiency at the lowest substrate concentrations. Approximately 95% of each isoenzyme is measured, for activities up to threefold the upper normal limit, if the measurements are made immediately after the reaction is initiated. The Arrhenius relationship for each isoenzyme is unique. Interconversion of results from one temperature to another is practical only with reservations. Results at 37 degrees C are not as reliable as those at 25 degrees C.


D(-)-N-Methylglucamine buffer for pH 8.5 to 10.5. A new amine buffer for the pH range 8.5 to 10.5 based on D(-)-N-methylglucamine and its hydrochloride is described. Important physical and chemical constants characterizing this buffer system have been determined: thermodynamic pKa = 9.52 (25 degrees C), buffer capacity = 0.055, dilution value pH 1/2 = -0.02, temperature coefficient dpKa/dT = 0.023, the heat of ionization delta H degrees = 36.93 kj mol-1. D(-)-N-Methylglucamine, which is available in highly purified form, is highly soluble in water. Thus one can prepare either buffer solutions or stable solid mixtures when combined with D(-)-N-methylglucaminium chloride. D(-)-N-Methylglucamine buffers are suitable for both biochemistry and pH control.


Synthetic substrate beta-glucosidase activity in leukocytes: a reproducible method for the identification of patients and carriers of Gaucher's disease. A method is described for the identification of patients and carriers of Gaucher's disease, using leukocytes from a small volume of blood. The fluorogenic substrate, 4-methylumbelliferyl-beta-D-glucopyranoside, was assayed in the presence of pure sodium taurocholate (2.5 mg/ml) and Triton X-100 (2.0 mg/ml). Some commercial brands of pure sodium taurocholate were satisfactory for this purpose. The pH optimum for controls, Gaucher disease carriers and Gaucher disease patients was 5.4 using citrate-phosphate buffer. Although leukocytes prepared from only a small amount of blood (2-8 ml) are required, there is sufficient quantity for measuring other lysosomal enzymes as controls. Using this method, 12 patients with all types of Gaucher's disease and 12 obligate heterozygotes were identified. Carrier status was predicted in six other family members and ruled out in six others. Eight unaffected people married to Gaucher carriers or Gaucher patients were predicted to be non-carriers of Gaucher's disease, thereby ruling out children affected with Gaucher's disease in that mating.


Cystic fibrosis liver sialyltransferase. The activity of sialytransferase with regard to the glycoprotein substrates asialofetuin and asialo-ovine submaxillary mucin was determined in normal, pathological control, and cystic fibrosis liver homogenates. Cystic fibrosis and pathological livers have about 40% of the average normal specific activity for sialytransferase. Several properties of cystic fibrosis sialytransferase were investigated and compared to those of the normal liver enzyme (Alhadeff et al. 1977). The pH optima curves were similar, but cystic fibrosis sialyltransferase appears to be more thermolabile than the normal liver enzyme. Isoelectric focusing studies revealed that the three most basic forms of sialyltransferase which are found in normal livers are deficient or absent in most cystic fibrosis liver. The data suggest that altered glycoprotein-sialyltransferases may be present in cystic fibrosis livers, probably a secondary effect due to general liver pathology.


[Standardisation of obtaining blood samples: influence of tourniquet application on 33 constituents of blood and serum (author's transl)]. The extent and dynamics of changes by short (1 min) or prolonged (6 min) tourniquet application while obtaining venous blood samples were analysed with respect ot 33 frequently measured constituents of blood and serum. After 6-minute tourniquet application the values for red cells, haemoglobin, packed cell volume, total protein, albumen, gamma-glutamyl transferase, alkaline phosphatase, lactate dehydrogenase, creatinekinase, bilirubin, cholesterol, total glycerol and calcium increased by an average of 4-9%. One-minute tourniquet application did not have a significant effect. Levels of sodium, potassium, carbon dioxide, creatinine, uric acid, ratio of electrophoretic fractions and the MCV, MCH and MCHC indices were not affected even by 6-minute tourniquet applications. The introduction of blood sampling under standardised conditions is proposed.


Drugs and depression.

Drugs and depression. Moderate or severe depression is now one of the most common diseases of our time with a prevalence of nearly 3%. It seems likely that this prevalence has increased as a result of the wider use of drugs which have an effect on the neurotransmitters. Changes in the levels of these neurotransmitters in the central nervous system are thought to be the biochemical basis for the development of at least some depressive illnesses. Drug-induced depressions are more likely to occur in those individuals who are genetically predisposed to depression or who have had a previous depressive illness. Other groups who are particularly susceptible to these effects are the elderly. Many groups of drugs have a primary or secondary action on the central nervous system neurotransmitter function. Some 200 drugs have been claimed to cause depression in certain patients, but only a relatively small number precipitate depressive symptoms with any frequency. Those most commonly implicated are the long-acting antipsychotics, barbiturates, ethanol, oral contraceptives and antihypertensive agents. It is important to remember that some drugs, such as reserpine, cause depression as a side-effect during their therapeutic use whereas others, such as fenfluramine, cause depression mainly when they are withdrawn too rapidly. In those patients presenting with depression, it is important to review the current drug therapy in order to assess the part played by these drugs in the development of the depression. Following this assessment, drug therapy should be adjusted appropriately. However, a distinction must be made between the symptoms of depression, those physiological changes which occur during treatment with a variety of drugs, and the patient's reaction to the disease for which they are being treated.


Drug interactions with antihypertensive drugs. Drug interactions with antihypertensive drugs can be either beneficial or hazardous. The hazardous interactions are relatively infrequent but must be shown so they can be avoided. Those of most importance involve interaction with guanethidine-type agents and tricyclic antidepressants, amphetamine-type anorexiants or phenolpropanolamine-type common cold remedies; combined use of potassium retaining diuretics with potassium supplements; and incautious use of diuretics with cardiac glycosides. The beneficial interactions are the basis for modern antihypertensive therapy and can be of major help if logically applied to therapeutic problems.


The molluscicidal properties of natural products from Ambrosia maritima. The molluscicidal properties of Damsin "I", Ambrosin "II", and tribromodamsin "III" were investigated against the intermediate hosts of schistosomiasis Biomphalaria alexandrina and Bulinus truncatus. Although Damsin was to some extent more toxic against B. alexandrina than the other two compounds, it was less toxic than them against B. truncatus after 24 hr exposure periods. There is a flex relationship between concentration of Damsin and exposure period to prodce 100% mortality. The molluscicidal potency of Damsin remained stable over a wide range of pH values, but was affected by storage, sunlight and river bed-mud. Structure-activity relationship is included.


Arylsulfatases in bilharziasis.

Arylsulfatases in bilharziasis. Arylsulfatase A and B in urine have been estimated in 18 normal subjects and 50 bilharziasis patients. The bilharziasis patients were divided into two groups according to the type of infeciton. Those with bilharziasis haematobian type of infection and those with the bilharziasis mansoni type. Each group was further subdivided into subgroups according to the severity and progress of the disease. The activities of arylsulfatase A and B were significantly elevated in all the groups of patients studied and it is evident that there is a progressive increase with the progress of the disease in both types of bilharziasis infections (the haematobian and mansoni types). Liver dysfunction consequent of bilharzial infestation appears to take part in the mechanism of induction of the bilharzial bladder cancer.


Laboratory model ecosystem evaluation of the chemical and biological behavior of radiolabeled micropollutants. A standardized laboratory model ecosystem has been used to evaluate the comparative behavior of radiolabeled micropollutants including organochlorine, organophosphorus, carbamate, and hormone-mimic insecticides; herbicides; important industrial organic compounds including phthalate esters and PCB's; and specific pollutants such as TCBD and hexachlorobenzene. The compounds have been studies for ecological effects over a terrestrial-aquatic interface and through several food chains, especially with regard to the development of quantitative information on ecological magnification and biodegradability index. The pathways of chemical degradation in the various organisms have been evaluated and the ecological effects of degradation products investigated. Illustrations are given of the use of the model ecosystem technology for screening new candidate pesticides for effects on environmental quality; for evaluating the hazards of environmental pollution by industrial waste effluents; and for fundamental studies of the principles fo biodegradability of organic chemicals in a variety of organisms.


Influence of denervation and reinnervation on autolytic activity and on protein composition of skeletal muscle in rat. Changes in the proteolytic activity and in the relative content of protein in soluble, myofibrillar and insoluble fractions were investigated following denervation and reinnervation of the soleus and tibialis anterior muscles of the rat. After denervation an increase of autolysis in the acid and neutral pH range, but not in the alkaline one, was found in both muscles. An increased autolysis at the acid and neutral pH range was also observed in both muscles after reinnervation, when the weight of the muscles increased. The results indicate the lack of inverse relationship between the changes of proteolytic activity and the decrease or increase of the amount of muscle protein in the course of muscle atrophy and regeneration.


Glucokinase and hexokinase in pig erythrocytes. We have shown different enzymatic activities responsible for the phosphorylation of glucose in pig erythrocytes. These activities were observed after partial purification from hemolyzed red cells. One of the enzymes involved is the hexokinase which is present in all tissues; the other is similar to hepatic glucokinase. We have determined the kinetic properties of these activities in hemolysates and in partially purified preparations. Their electrophoretic-migration characteristics were studied too.


Electrostatic interactions at charged lipid membranes. Measurement of surface pH with fluorescent lipoid pH indicators. The 5-dimethylaminonapthalene-1-sulfonyl (dansyl) chromophore attached to the polar head groups of lipids has been used as a fluorescent lipoid pH indicator to evaluate the interfacial pH in lipid-water lamellar systems prepared from negatively charged lipids. The pH in the vicinity of the charged lipid bilayers is different from the pH of the bulk aqueous phase and the difference is a function of the electrolyte concentration in the aqueous phase and of the lipid packing in the bilayer. At a fixed electrolyte concentration in the aqueous phase, the observed interfacial pH is 0.6 to 0.7 pH units lower above the thermal phase transition of the lipid than it is below this temperature. A quantitative interpretation of the results is given on the basis of the Gouy-Chapman theory. The results indicate that the dansyl chromophore is located in front of the charged surface and its distance from this surface increases with a decrease in lipid packing.


Kinetics of carbon monoxide binding to fully and partially reduced human hemoglobin valency hybrids. The kinetics of carbon monoxide binding following fast reduction of the valency hybrids alpha2+betaCO2 and alphaCO2beta+2 by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 10(6) M-1S-1). At pH 6.0 the intermediates alphaCO2beta2 and alpha2betaCO2 bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 alphaCO2beta2 is for the greater part in the T state, while in the case of alpha2betaCO2 the R and the T state are about equally populated.


Mg2+ translocation across the thylakoid membrane: studies using the ionophore A 23187. 1. The degree of the light-dependent Mg2+ efflux across the thylakoid membrane is a function of pH. There is a considerable efflux at pH 7.2 which decreases to a negligible amount at pH 8.6. This conclusion is derived from studies using the divalent cation-specific ionophore A23187. The ionophore is active as an uncoupler at pH 7.2 even in the absence of added Mg2+, and completely inactive at pH 8.6 unless Mg2+ is added. The activity was assayed as the effect on both the rate of electron transport (stimulation at pH 7.2 and inhibition at pH 8.6) and deltapH. 2. Under conditions of maximal Mg2+ efflux, pH 7.2, and in the presence of EDTA, it is shown that Mg2+ is transported across the thylakoid membrane, but it is not diluted in the medium. 3. At high pH, light-induced proton influx is not compensated by Mg2+ efflux in the presence of a relatively permeable anion. However, in the presence of an impermeable anion, where cation efflux is necessary, Mg2+ efflux occurs, indicating its preference to K+ efflux. It is suggested that although light-induced Mg2+ efflux across the thylakoid membrane is evident, its magnitude is small and it is not diluted in the stroma. Thus, it seems hard to visualize how this transport is supposed to play an important role in CO2 fixation.


Therapeutic and collateral effects of 25-hydroxycholecalciferol in vitamin D deficiency. The clinical and biochemical response to 25-hydroxycholecalciferol (25-HCC) and vitamin D3, 150 microgram/day for 20 days has been compared in infants aged 3--18 months with nutritional rickets. The infants were allocated at random to Group I (11 infants) treated with 25HCC and Group II (9 infants) treated with vitamin D3. In addition 15 matched control children without rickets were allocated to Group III and received 25-HCC 75 microgram/day for 20 days. Preliminary studies showed that plasma calcium, phosphorus, alkaline phosphatase and urine pH all differed significantly between the rachitic and control groups. The biochemical parameters in both groups of rachitic children became normal after treatment with the exception of plasma alkaline phosphatase which remained elevated. The control group showed a significant increase in plasma and urine calcium values in spite of the low dose of 25-HCC. The findings suggest that 25-HCC is as effective as vitamin D3 in the treatment of rickets but did not demonstrate any therapeutic advantage.


The contribution of intestinal endotoxin to mortality in hosts with compromised resistance: a review. Sepsis, particularly with endotoxin-containing Gram-negative bacilli, is a serious complication in hosts whose defenses are compromised. This review examines work from our laboratory and others concerning infectious processes which may be critical to the survival of compromised individuals. Several avenues for control of sepsis are proposed. Gram-negative bacteria and their endotoxins can escape from the intestines of compromised animals to contaminate normally sterile host tissues. Endotoxins are especially toxic to compromised hosts because essential components of their inflammatory responses are missing (i.e., leukocytes and platelets in irradiated animals). Therefore, regulation of host responses to endotoxin is no longer possible. It is recommended that sepsis be controlled in compromised individuals through elimination of endogenous microbial agents. Should infection occur in these individuals, they should be transfused with blood cells necessary for clearance of bacteria and endotoxin and restoration of homeostasis.


Simultaneous monitoring by optical techniques of respiratory chain and intracellular pH in toad ventricle strip. Intracellular pH and oxidative metabolism can be measured in toad ventricle strips simultaneously by the use of the pH indicator dye, neutral red, and a rapid scanning spectrophotometer. The effects of hypoxia and acidification on mechanical function are approximately additive. The decrease in tension due to slight acidification is probably through an effect on the portion of the twitch tension supported by anaerobic metabolism.


[Nature of the melipramine inhibition of the K+-n-nitrophenyl phosphatase activity of a synaptosomal fraction]. The influence of antidepressant melipramine (imipramine) on the K+-paranitrophenyl-phosphatase (K+-n-NFP-ase) activity of the synaptosomal fraction in the bull's brain cortex, treated with sodium iodide, was studied in in vitro experiments. Melipramine (5.10(-5)--10(-3)M) is shown to inhibit the K+-n-NFP-ase activity, competing with potassium ions. With pH changing from acid and neutral to alkaline values the inhibitory action of melipramine gains in intensity, whereas the protective effect of potassium ions declines. It is suggested that one of the mechanisms responsible for the inhibition of the K+-p-NFP-ase activity by melipramine is interaction of the inhibitor in the anionic sections of the enzyme complex.


[Effect of pharmacological substances on the development of hemorrhagic erosions and on the noradrenaline level in the stomach wall in rats]. Hemorrhagic erosions of the gastric mucosa were produced through electric stimulation of immobilized rats via electrodes stuck into the muscles of the fore-paws. In the experiment there was applied square pulsed current of 5--7 v (per 10 rats) with frequency of 50 Hz and the pulse time of 10 ms. Prior to stimulation the rats received intraperitoneally one of the following drugs: clonidin (0.1 mg/kg), benactyzine (3 mg/kg), etherophen (20 mg/kg), benzohexonium (10 mg/kg), iprazid (100 mg/kg), amphethamine (4 mg/kg), atropine (1 mg/kg), tyrosine (300 mg/kg), aethimizol (10 mg/kg) and orotic acid (10 mg/kg). The animals were sacrificed directly after stimulation and the norepinephrine level in the gastric wall was determined after E. Sh. Matlina and T. B. Rakhmanova (1967). Introduction of clonidin, benacyzine, etherophen, benzohexonium and iprazid to immobilized rats after electric stimulation prevented the development of hemorrhagic erosions and a drop of the norepinephrine level in the gastric wall, whereas amphethamine and tyrosine intensified the process of the erosions development in the gastric mucosa and did not prevent a decline in the norepinephrine level. Atropine and aethimizol had no effect on the findings under study.


[Rat placental barrier permeability for the new antihistaminic preparation, fenkarol]. As established, following a single introduction of phencarol tagged with tritium in a quinuclidine nucleus perorally to rats on the 13th day of gestation its insignificant amounts gain access to the fetus through the placenta. The maximum concentration of the drug in the maternal blood was recorded 3 hours after its introduction, following 6 hours--in the placenta, with the radioactivity continuing to be constant during the first 6 hours in the embryonal tissues. In 48 hours after introduction the radioactive tag is still demonstrable in the study tissues. It is concluded that the drug is largely retained in the placenta, when introduced on the 13th day of pregnancy; on the 21st day of gestation the barrier functions of the placenta declines. The presence of phencarol and its transformation products in the embryonal tissues is confirmed by the method of thin-layer chromotography.


[Effect of the structure of liposomal membrane surface layer on the binding of phenothiazines]. By using a fluorescent 3-metoxybenzatrone probe the binding of the phenothiazine series drugs (chlorpromazine, triftazine, aethaperazine) with phospholipid vesicles--liposomes, was investigated. Cholesterol is shown not to affect the binding of these drugs with liposomes. The surface charge of liposomes influences only the combination with the chlorpromazine membranes. With a higher positive charge of the membranes the binding of chlorpromazine diminishes in force.


Acetylcholine--inhibition of transmitter release from adrenergic nerve terminals mediated by muscarinic receptors. The evidence is reviewed for the presence of muscarinic receptors on the sympathetic nerves to blood vessels. Activation of these receptors by acetylcholine in doses that are too small to affect the smooth muscle cells directly inhibits the release of norepinephrine evoked by electric impulses or potassium ions. This inhibitory action of acetylcholine is prevented by muscarinic blocking agents and is probably due to hyperpolarization of the adrenergic nerve terminals.


Histamine and 5-hydroxytryptamine-inhibition of transmitter release mediated by H2- and 5-hydroxytryptamine receptors. The vasodilatation caused by histamine and 5-hydroxytryptamine may be due, at least in part, to their inhibitory action on adrenergic neurotransmission. The evidence for this is as follows: 1) contractions of isolated canine vascular strips caused by sympathetic nerve stimulation are depressed by these substances whereas contractions caused by norepinephrine are either unchanged or augmented; 2) histamine and 5-hydroxytryptamine inhibit the release of norepinephrine evoked by sympathetic nerve stimulation of isolated vascular strips previously incubated with the labeled transmitter. This inhibitory effect can be demonstrated using concentrations of the substinces less than those required to affect the smooth muscle cells directly. By contrast, neither histamine nor 5-hydroxytryptamine inhibits the displacement of neuronal norepinephrine by tyramine, suggesting that these substances interfere with the exocytotic process. Additional studies have identified the histamine-H2 receptor as the probable mediator of this prejunctional action of histamine, whereas the nature of the receptor for 5-hydroxytryptamine remains to be clarified.


Actions of angiotensin on adrenergic nerve endings. In the perfused vascular bed, vasoconstrictor responses to adrenergic nerve stimulation are augmented to a greater degree by angiotensin II than are the responses to injected norepinephrine. Overflow of adrenergic transmitter is also greater during nerve stimulation in the presence of angiotensin than in its absence. The evidence indicates that facilitation of adrenergic transmitter release rather than uptake blockade accounts for these results. In addition, an increased responsiveness of isolated arterial strips to norepinephrine as well as other agonists appears to contribute to the adrenergic potentiating effect of angiotensin II as well as angiotensin III. This action, which appears to be a cell membrane effect, seems to participate in adrenergic potentiation mainly in the arterial segment of the intact vascular bed. Both of these effects of angiotensin, i.e., facilitation of release and increased smooth muscle responsiveness, appear to be mediated by angiotensin receptors.


Prostaglandins--modulation of adrenergic nervous system. Prostaglandins (PGs) affect vascular tone by a direct action on the vascular smooth muscle and by influencing vascular reactivity to adrenergic simuli and several vasoactive substances. Thus, in the isolated Tyrode's perfused rabbit renal, mesenteric and splenic vasculature PGE2 inhibited adrenergically induced vasoconstriction. Since the vasoconstrictor responses to renal nerve stimulation were enhanced by the blockade of PG synthesis and were reduced by stimulation of PG synthesis with arachidonic acid, this suggests that PGE2 functions as an inhibitory modulator of the adrenergic nervous system. However, our demonstration that PGE2 enhanced adrenergically induced vasoconstriction in the renal and mesenteric vasculature of the rat, but had opposite effects in the rat splenic vasculature indicates that the modulatory-effect of PGE-compounds on the adrenergic neuromuscular junction is species dependent and varies in different vascular beds within the same species. Prostaglandins, the release of which is evoked by several vasoactive substances including angiotensins, kinins, and adenine nucleotides, may also contribute to the regulation of vascular tone by either opposing or amplifying the vascular actions of vasoactive substances.


Metabolic modulation of neurotransmitter release--adenosine, adenine nucleotides, potassium, hyperosmolarity, and hydrogen ion. Evidence has accumulated that several factors, which have been proposed as mediators of exercise hyperemia, can modulate adrenergic neurotransmission in blood vessels. Adenosine and the adenine nucleotides depress the response of isolated blood vessels of the dog to nerve stimulation more than that to exogenous norepinephrine; this difference is explained by a decreased release of the neurotransmitter. Potassium, hyperosmolarity, and acidosis also depress adrenergic neurotransmission in isolated veins. These results are consistent with the hypothesis that metabolic changes in the vicinity of the adrenergic neuroeffector junction are capable of decreasing the output of neurotransmitter to the blood vessels in the exercising muscle.


Oxypertine in combination with imipramine: a controlled trial. A method for the controlled assessment of two agents for possible interaction in the treatment of endogenous depression is described. This consisted of dose-ranging the supplementary agent (oxypertine 30 mg and 60 mg daily or matching placebo) during Week 2 to Week 6 having established all patients on a therapeutic dose of imipramine during Week 1. The reported enhancement of imipramine by oxypertine was not confirmed.


Studies of the androgen binding protein in the rete testis fluid of the ram and its relation to sexual season. An androgen binding protein (ABP) with an electrophoretic mobility (Rf) of 0.56 is present in the rete testis fluid of adult rams. Its steroid specificity was found to be in the following order: 5alpha-DHT, testosterone, oestradiol-17 beta, dehydroepiandrosterone 5beta-DHT, androstenedione, cyproterone, cyproterone acetate, cortisol and progesterone. The characteristics of the ABP are similar to those found for the ABP of the testis and the epididymis of the rat and the rabbit. The concentration of ABP, determined by the dextran-coated charcoal method and sometimes confirmed by the steady-state polyacrylamide gel electrophoresis method, was significantly higher in the breeding season than in the non-breeding season (4.40 +/- 0.98 X 10(-9) M vs. 2.60 +/- 0.62 X 10(-9) M; P less than 0.037). The affinity constant of the ABP was independent of the season (2.45 +/- 0.21 X 10(9) M-1 vs. 2.66 +/- 0.1 X 10(9) M-1; NS). In addition, ABP was positively correlated with 5alpha-DHT (r = 0.506; P  less than 0.0009), testosterone (r = 0.445; P less than 0.0003), total protein (r = 0.329; P less than 0.02) and spermatozoa (r = 0.406; P less than 0.006) in the RTF and with blood plasma testosterone (r = 0.584; P less than 0.0001). Furthermore, testosterone and 5alpha-DHT in RTF were positively correlated (r = 0.582; P less than 0.0001). These androgens were also correlated with plasma testosterone (r = 0.262, P less than 0.052 for testosterone in RTF; r = 0.341, P less than 0.018 for 5 alpha-DHT). Total proteins and spermatozoa were found to be positively correlated in the RTF (r = 0.789; P less than 0.0001).


Animal influenza virus neuraminidase: studies on dependence of some of its properties on its association with hemagglutinin. Neuraminidase (Nase) thermostability and sensitivity to pH treatment as well as specific enzymatic activity (Nase activity per 1 HA unit) were determined in two groups of animal influenza virus strains containing equine 1 and equine 2 Nase subtypes, respectively (A/equine/Prague/56 (Heq1 Neq1), A/equine/Cambridge/63 (Heq1 Neq1), A/FPV/Dutch/34 (Hav1 Neq1), A/chicken/Germany "N" (Hav2 Neq1), in one group, and A/equine/Miami/1/63 (Heq2 Neq2), A/turkey/Canada/63 (Hav6 Neq2), A/duck/Ukraine/1/63 (Hav7 Neq2), in the other group). Nase of all the strains used was thermotabile when heated at pH 4.5. Nase of Neq1 subtype irrespective of strain containing it was thermolabilt when heated both at pH 6.5 and 8.1 and sensitive to pH 4.5 treatment as such (without heating). Inversely, Nase of Neq2 antigenic subtype irrespective of the strain containing it, was thermostable when heated at pH 6.5 AND 8.1 and resistant to the treatment of pH 4.5. Specific enzymatic activity was considerably higher in all the strains containing Neq2 as compared to Neq1-containing strains (4-6 times as much). The results suggest that thermostability and pH sensitivity of equine Nases of both antigenic subtypes, as well as their specific activities, do not depend on the sort of HA which is coupled with enzyme subunits at viral envelope, but attributed rather to properties of the subunits themselves, such as glycoprotein entities. The data concerning specific activities may suggest that in the case of various combinations of Nase subunits with different HA subunits the amount of enzyme per virion is of the same order.


Assessment of the plasma disappearance of cholyl'l14C-glycine as a test of hepatocellular disease. The plasma disappearance of a tracer dose of cholyl-l14C-glycine has been examined in 12 control subjects and in 32 patients with hepatocellular dysfunction. Simple analysis of the data did not detect hepatic dysfunction except in severe hepatocellular disease. The greatest degree of discrimination between normal subjects and patients with mild liver disease was obtained by taking the ratio of the plasma retention at 60 minutes to that at 10 minutes; it was similar to that obtained with serum gamma-glutamyl transferase. The two hour post-prandial plasma "total" bile acid concentration gave complete separation between the control subjects and patients with liver disease.


Postprandial gastro-oesophageal reflux in healthy people. Distal oesophageal pH was monitored for three hours after a standard meal in 10 young healthy subjects without symptoms of gastro-oesophageal reflux. Episodes of reflux occurred in nine of these subjects; and, in five, oesophageal pH was less than 5 for between 11 and 75% of the first postprandial hour. Intermittent incompetence of the lower oesophageal sphincter after food must, therefore, be regarded as a normal phenomenon. The method described would be suitable for the evaluation of agents believed to weaken or to strengthen the lower oesophageal sphincter.


Transport kinetics of 6-deoxy-D-glucose in Candida parapsilosis. The strictly aerobic yeast Candida parapsilosis transports the nonmetabolizable monosaccharide 6-deoxy-D-glucose by an active process (inhibition by 2.4-dinitrophenol and other uncouplers but not by iodoacetamide), the accumulation ratio decreasing with increasing substrate concentration. Measured accumulation ratios are in agreement with those predicted from kinetic constants for influx and efflux. Energy for transport is probably required in the translocation step. The maximum rate is temperature-dependent with a transition point at 21 degrees C. the accumulation ratio is not. The uptake is most active at pH 4.5--8.5. It appears not to involve stoichiometric proton symport. The transport system is shared by D-glucose, D-mannose, D-galactose and possibly maltose but not by fructose, sucrose or pentoses. The apparent half-life of the transport system was 3.5--4 h.


Production of amylase by a submerged culture of Aspergillus wentii. Soluble starch was hydrolysed to maltose by Aspergillus wentii Wehmer (IMI 17295). Studies on nutritional requirements of Aspergillus wentii for production of amylase revealed that the optimum conditions were achieved in fermentation culture medium containing 1% starch, and incubated at 20 degrees C for 3 days at pH 6.0. Tryptophan was the best nitrogen source. The amylase activity was completely inhibited when 1 mM sodium iodoacetate was incorporated into the medium. With 10 mM sodium citrate the amylase activity was increased from 3.51 to 6.0 mg/ml.


Parallel occurrence of oxidant-sensitivity and decreased inhibition by NADPH in G-6-PD Lublin and G-6-PD Poxnań. In two studied variants of G-6-PD without chronic hemolysis in probands, sensitivity of enzymes to inhibition by NADPH was decreased. Ki for NADPH was 28 micronM in Gd Lublin and 19 micronM in Gd Poznań. Susceptibility to the oxidant-induced hemolysis was described in probands, as well as in patients hemizygous for two other variants of G-6-PD with increased Ki for NADPH. It is suggested that in these cases, the oxidant-induced hemolysis is aggravated by their inability to counteract the drop in NADPH concentration with an increase in G-6-PD activity.


Studies of cyclic AMP action using mutant tissue culture cells. S49 mouse lymphoma cell mutants, each with a specific defect in its ability to generate or respond to cyclic AMP, have been isolated. Analysis of the properties of these cells has begun to provide information on complex and significant biologic problems related to the cyclic AMP system.


[Correlation of growth-phase and streptodornase-production of beta-hemolytic streptococci group A (author's transl)]. 77 M- and 48 T-typed strains of A-streptococci were compared with respect to their extinction, acidification and nuclease-production as a function of the growth-time. Strains of the same serotype (M 12) showed a change in the distribution of the isoenzymes of nucleases with respect to the growth-time. Half of the strains produced nuclease activity after 4 h. Streptodornase B was produced by all strains. With the growth the number of the nucleases found at the same time diminished, after 96 h in 70% of the strains the nuclease activity was represented by streptodornase B only.


Depression of cell-mediated immunity following inoculation of Trichinella spiralis extract in the mouse. Mice pretreated with Trichinella spiralis extract (TsE), or infected with the parasite, rejected primary skin allografts in 18-23 days and secondary allografts in 12-16 days. Mice pretreated with saline or with bovine serum albumin (BSA) rejected the primary allografts in 12-18 days and did not accept the secondary grafts. Inoculation of increasing doses of parental spleen cells from mice pretreated with saline or with BSA in F1 hybrids produced proportionately stronger graft-versus-host reactions (GvHR) whereas increasing doses of cells from TsE pretreated mice reduced proportionately the capacity of the inoculum to induce a GvHR. Immunodepression of the parental cells was obtained with 7 and with 4, but not with 2, daily injections of TsE. The depression waned rapidly after the treatment with TsE but a significant degree still remained after 3 days. Immunodepression by TsE cannot be solely explained by antigenic competition and although our results are consistent with the induction of suppressor cells, it is probable that other mechanisms are also involved.


Responses to polyvinyl pyrrolidone and pneumococcal polysaccharide in protein-deficient mice. Indirect haemagglutination titres induced by polyvinyl pyrrolidone (PVP) or pneumococcal polysaccharide Type III (S111) were determined in mice maintained on a 4% albumin diet from weaning and normally-fed littermates. Responses to PVP, given intravenously (i.v.) or intraperitoneally (i.p.), were elevated by protein-deficiency at low antigen doses and increasingly depressed at high doses. Increases in the duration of protein-deficiency generally improved these responses. The persistence of tolerance was reduced by protein-deficiency and priming was evident in both groups when tolerance was broken. The low protein diet depressed responses to moderate doses of S111 given i.p. to C57Bl mice, but such responses were normal in BALB/c mice and in C57Bl mice injected i.v. High doses of S111 (i.p., i.v.) elicited poor responses in deficient mice. These findings are discussed in relation to previous studies using other antigens, with a view to elucidating mechanisms responsible for the effects of protein-deficiency.


Restoration of suppressor function in athymic mice. Antibody responses of normal and congenitally athymic (nu/nu) mice were measured to the phosphorylcholine determinant of a pneumococcal C-polysaccharide. The plaque-forming cell responses of athymic mice were approximately tenfold higher than for intact controls. This enhancement was equivalent to that obtained by treatment of controls with antithymocyte serum. In both cases, the enhanced responses presumably result from a lack of suppressor T cells. Reconstitution of athymic mice with normal thymocytes restored both suppressor and helper T cell functions. Whereas suppressor activity did not appear until 6 days after transplantation of T cells, helper activity was fully restored within 24 h and had diminished by 6 days.


Bactericidal activity of specific and azurophil granules from human neutrophils: studies with outer-membrane mutants of Salmonella typhimurium LT-2. Extracts of specific granules and azurophil granules from human neutrophils were tested for their bactericidal activity against various lipopolysaccharide mutants of Salmonella typhimurium LT-2. Three purified granule populations, one specific and two azurophil, were obtained by isopycnic centrifugation of homogenized neutrophils. Each was extracted with 0.2 M acetate buffer (pH 4), and the extracts were dialyzed against phosphate-buffered saline (pH 7) to remove acetate. These extracts contained >/=84% of the lysozyme, lactoferrin, or myeloperoxidase initially present in the whole granules. The S. typhimurium mutants possessed Ra, Rc, Rd(1), Rd(2), or Re lipopolysaccharide. As the carbohydrate content of the lipopolysaccharide decreased, the bacteria became increasingly more susceptible to the bactericidal activity of all granule extracts. Bactericidal activity of the extracts was in the order: mixed (azurophil + specific) >/= azurophil >> specific. Specific granules were bacteriostatic for S through Rd(2) bacteria. They were bactericidal only for the Re mutant. Both azurophil granule populations were equally bactericidal. Extracts boiled for 30 min retained none of their bactericidal activity for any of the bacteria; however, they remained bacteriostatic for the deep rough (Rd(2), Re) mutants. Bactericidal activity was dependent upon pH, in that mixed and azurophil granule contents killed the smooth parent and Ra mutant best at pH 5, the Rc and Rd(1) mutants to the same degree at pH 5 to 8, and the deep rough mutants (Rd(2) and Re) best at pH 8. Specific granule contents were most bacteriostatic for S through Rd(2) bacteria at pH 5 and killed the Re mutant only at pH 8. Thus, as the S. typhimurium lipopolysaccharide content decreased, the bactericidal pH optimum increased. Killing by all extracts was dependent upon incubation temperature, with almost no bactericidal or bacteriostatic activity observed when bacteria and granule fractions were incubated on ice (2 degrees C) and plated immediately. Intermediate killing was observed at 22 degrees C. If bacteria were incubated with granule extracts at 2 degrees C, washed free of extract, suspended in medium without extract, and reincubated at 37 degrees C, killing was observed. This suggested that a component(s) of the extracts was sticking to the bacteria at 2 degrees C but killing only at 37 degrees C.


Role of complement in lethal bacterial lipopolysaccharide-induced hypotensive and coagulative changes. The effect of C3 depletion on the multiple pathophysiological changes produced by a lethal dose of Serratia marcescens lipopolysaccharide (LPS) was evaluated. The injection of this LPS into rabbits resulted in biphasic hypotensive changes and thrombocytopenia. These changes were characterized by an acute, transient decrease occurring within minutes after injection followed by a second more gradual decrease beginning 30 to 60 min post-LPS. Prior depletion of C3, with the anticomplementary protein from cobra venom (CoF), did not alter the extent of either the gradual hypotensive and platelet changes or the coagulative and metabolic changes when normal and C3-depleted rabbits were compared. Importantly, the lethal effects of S. marcescens LPS were not reduced by prior depletion of C3. Only the immediate, reversible thrombocytopenia and hypotension were abrogated by C3 depletion.


Attachment of Bacteroides melaninogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets. This investigation examined the ability of cells of Bacteroides melaninogenicus subsp. asaccharolyticus 381 to adhere to surfaces that might be important for its initial colonization of the mouth and its subsequent colonization in periodontal pockets. Of 48 asaccharolytic strains of B. melaninogenicus, 47 agglutinated human erythrocytes, whereas none of 20 fermentative strains, which included reference cultures of the subspecies intermedius and melaninogenicus, were active. Electron microscopy indicated that both asaccharolytic and fermentative strains possessed pili; hence, the presence of pili did not correlate with the hemagglutinating activities of B. melaninogenicus strains. Both asaccharolytic and fermentative B. melaninogenicus strains suspended in phosphate-buffered saline adhered in high numbers to buccal epithelial cells and to the surfaces of several gram-positive bacteria tested, including Actinomyces viscosus, A. naeslundii, A. israelii, Streptococcus sanguis, and S. mitis. B. melaninogenicus subsp. asaccharolyticus 381 also attached, but in comparatively low numbers, to untreated and to saliva-treated hydroxyapatite. Addition of clarified whole saliva to suspensions of strain 381 almost completely eliminated adherence to buccal epithelial cells and to hydroxyapatite surfaces, but saliva had no detectable effect on attachment to gram-positive plaque bacteria. Both fermentative and nonfermentative strains of B. melaninogenicus also attached in high numbers to crevicular epithelial cells derived from human periodontal pockets, but normal human serum strongly inhibited attachment. Serum also inhibited attachment of strain 381 to saliva- and serum-treated hydroxyapatite, but it had little effect upon attachment to gram-positive bacteria. These observations suggested that salivary and serum components would strongly inhibit the attachment of B. melaninogenicus cells to several oral surfaces, but not to the surfaces of certain gram-positive bacteria commonly present in human dental plaque. This was confirmed by an in vivo experiment in which streptomycin-labeled cells of B. melaninogenicus 381-R were introduced into the mouths of two volunteers. After 10 min, several hundred-fold higher numbers of the organism were recovered from preformed bacterial plaque present on teeth than from clean tooth surfaces or from the buccal mucosa and tongue dorsum. High numbers of B. melaninogenicus cells were also recovered from preformed plaque after 150 min, but virtually no cells of the organism were recovered from the other surfaces studied. These data suggest that the presence of dental plaque containing Actinomyces and other gram-positive bacteria may be essential for the attachment and colonization of B. melaninogenicus cells after their initial introduction into the mouth. Similarly, the presence of subgingival plaque containing gram-positive bacteria may be necessary for its secondary colonization in periodontal pockets.


Adoptive immunochemotherapy of a transplantable AKR leukemia (K36). Adoptive immunotherapy of a transplantable AKR leukemia (K36) was carried out as an adjunct to cytoxan chemotherapy using normal allogeneic H-2-incompatible spleen cells as well as sensitized H-2-matched allogeneic spleen cells. A significant therapeutic effect was obtained with cytoxan and allogeneic C57BL/6 splenocytes, demonstrating the potential use of the graft-versus-host reaction. Utilizing specific adoptive immunochemotherapy, a maximum effect was found with splenocytes from allogeneic but H-2-compatible CBA/J mice immunized against an allogeneic Gross-virus-induced lymphoma (E female G2). This therapeutic effect was most likely the result of prior sensitization of donor lymphocytes to common virus-associated tumor antigens.


Sperm-freezing and donor insemination.

Sperm-freezing and donor insemination. A comparison between freezing of human sperm using glycerol or glycerol egg-yolk citrate is presented. Freezing was performed with a gradual lowering of the temperature using alchol-liquid nitrogen or by immersion into liquid nitrogen. Great variations were observed within the same donor sperm sample when the sperm was frozen according to different techniques and marked post-thaw, motility variations were also found between spermatozoa from different donors. No statistically significant differences were found in the number of pregnancies obtained following inseminations with sperm frozen according to the two methods.


Electrochemical effects of metals on IUDs. Electrochemical effects within the uterus are being studied, particularly as they pertain to IUDs made in total or in part of metals. The intrauterine potential as measured with either metal or nonpolarizable salt electrodes is positive at the fundal with respect to the cervical end of the uterus. Placement of dissimilar metal electrodes within the uterus will induce voltage gradients that are roughly predictable from their cell potential in blood as an electrolyte. When copper and zinc electrodes are placed at opposite ends of the uterus, they induce voltage gradients that are typically greater than 100 mv per cm. External copper and zinc wires connected at one end, and with the other ends in contact with various positions on the skin of rats, were found to influence potential gradients within the uterus. When a length of zinc wire was wound around the upper portion of the stem of a small Cu-7 200 IUD, it took about 1 year from insertion for the zinc to disappear. When the zinc wire was wound around the lower portion of the stem, it disappeared in 1 month. The zinc reduced intrauterine corrosion of the copper and it was more effective at reducing copper corrosion when in the lower stem position.


Antigens from human seminal plasma. Part III: studies on two new antigens from the tricholoracetic acid soluble fraction. Two new antigens were isolated from the trichloroacetic acid soluble fraction of human seminal plasma: a polysaccharide (G-3) and a glycopeptide (G-4). Both were homogeneous, and hand sedimentation constants at infinite dilution of 2.9 X 10(-13) and 2.0 X 10(-13) resectively. Chemical and physicochemical studies suggest that both are branched molecules composed mainly of alpha-D-glucopyranosidic residues. The peptide component of G-R contains a high proporiton of the basic amino acids arginine and lysine. Since both antigens are polydispered, the low percentage of sialic acids or of some amino acids determined might have antigenic significance.


Augmentative effect of ascorbic acid upon induction of human ovulation in clomiphene-ineffective anovulatory women. The effect of the administration of 1-ascorbic acid, either alone or combined with clomiphene, upon induction of human ovulation was investigated in clomiphene-inffective anovulatory women. Oral administration of daily 400 mg of ascorbic acid induced ovulation in two out of five habitually anovulatory cycles and in one out of eight first-grade amenorrhea cases, and was ineffective in all six second-grade hypothalamic amenorrhea cases. Combined administration of ascorbic acid with 5 days of clomiphene induced ovulation in five out of five habitually anovulatory cycles, in 10 out of 17 first-grade hypothalamic amenorrhea cases, and in two out of nine second-grade hypothalamic amenorrhea cases. Pregnancy was established in eight out of 18 sterile, habitually-anovulatory or first-grade amenorrheic women with the combined ascorbic acid-clomiphene therapy, and in one out of five sterile, habitually anovulatory women with ascorbic acid therapy alone. Since administration of ascorbic acid induced no changed in blood FSH, LH, and amount of cervical mucus, and it is well established that LH decreases dose-dependency of the ascorbic acid content in the rat ovaries, the possible site of action of ascorbic acid seems to be at the ovarian level.


Varicocele: relation between anoxia and hypospermatogenesis. Three groups of patients were analysed: varicocele patients with abnormal seminal fluid (17 cases), variococele and normal sperm (seven cases) and normal males (17 cases). Blood gas was secured in the internal spermatic vein, perpheric vein and femural artery, in an attempt to evaluate the possibility of altered gas content as a cause of spermatogenic depression. No statistical differences were found when we compared the three groups. We concluded that anoxia was not the cause of hypospermatogenesis in varicocele patients.


A morphological and physiological study of mesotubarium ovarica in humans. In order to clarify the mechanisms by which the egg is transported from the ruptured follicle into the fimbrial end of the Fallopian tube in the human being, the mesotubarium ovarica (MTO), the unique anatomical structure which connects the tubal fimbriae and the ovary, was studied in seven human adnexal specimens histochemically and electron microscopically. The results demonstrated clearly the presence of smooth muscle cells in the MTO, and failed to demonstrate the presence of cilia in the lining epithelial cells of the MTO. Based on these morphological results, contractility of the MTO was studied in vitro by using a muscle chamber and a pressure transducer with 26 human adnexal specimens. Spontaneous contractile activites of regular frequency and moderate intensity were observed in the MTOs of all specimens examined. A possible role of the MTO in the mechanisms of ovum pickup at the time of ovulation is discussed.


Postnidatory effects of luteinizing hormone releasing hormone (LHRH) in hamsters. Whereas the administration of LHRH to pregnant hamsters has no effect during the prenidatory period, the hormone is effective in terminating pregnancy when given after implantation (days 6-10). The ED50 for pregnancy termination over this period approximates a dose of 0.35-0.4 mg b.i.d. When given to pregnant females in a second study, the effects of LHRH at this dose were completely reverse by minute doses of progesterone (30 microgram and above). Finally, administration of LHRH at 1.5 mg b.i.d., from days 6-10 was followed by daily sacrifice through day 12; bloods were sampled at autopsy for progesterone evaluation. Autopsies on days 7 and 8 showed few differences between controls and LHRH-treated hamsters, although decreased weights of the uterine/conceptus units signaled the initation of resorption. Significant LHRH-induced decreases in circulating progesterone were seen by day 9. Fetal resorption continued and was essentially complete by day 11, while progesterone levels continued depressed through the end of the study.


Vascular changes in traumatic amenorrhea and hypomenorrhea. Pelvic angiography was performed in 12 cases of amenorrhea and hypomenorrhea which developed following curettage of abortion and in the puerperium. Six cases of similar age and obstetric history with normal menstrual cycles served as control. Pelvic angiography revealed widespread vascular occlusion of myometrial arteries, in seven of the twelve cases. These findings account for the small amount of endometrium removed on diagnostic curettage in these cases as well as the greatly reduced menstrual loss. The poor obstetric history of the cases studied may well be due to this excessive vascular damage.


Quenching of tryptophan fluorescence in human antithrombin III by iodide ion. Iodide is an efficient quencher of antithrombin III intrinsic tryptophan fluorescence. The quenching pattern indicates that about 60% of the tryptophyl fluorescence originates from exposed residues in the multitryptophan-containing protein. In denaturing media all of the tryptophyls are solvent-exposed. The binding of heparin to antithrombin III influences the number of solvent-exposed tryptophan residues. By studying the dependence of the quenching on pH, information regarding the presence of charged residues adjacent to tryptophyls was obtained.


Photochemical evidence for the presence of histidyl residue in the active site of alkaline mesentericopeptidase. The photosensitized oxidation of alkaline mesentericopeptidase in the presence of methylene blue results in a first-order rate of inactivation. The loss of enzymatic activity towards casein and N-acetyl-L-tyrosine ethyl ester closely correlates with the destruction of one histidyl residue. A pK value of 6.8 is determined from the sigmoid pH-dependence of the photoinactivation rate. This suggests the involvement of a normal titrating imidazole group in the active site of mesentericopeptidase. The competitive inhibitor Na-benzoyl-L-arginine protects the enzyme from photoinactivation. A conclusion is made that the active site histidyl residue is modified. Circular dichroism spectra show no change in the protein conformation during the photodynamic treatment.


The peripheral and central neural actions of clonidine in normal and glaucomatous eyes. The peripheral and central neural actions of clonidine on normal and glaucomatous eyes have been investigated. Threshold doses of clonidine applied topically induced a monotonic decrease of intraocular pressure in the treated eye and had no effect on the contralateral eye. With increased clonidine dose, a decrease of intraocular pressure occurred in the untreated eye, and there was a concomitant decrease of systemic arterial blood pressure. Analysis of aqueous humor dynamics showed that the ocular response to the peripheral and the central neural actions of clonidine were without effect on the tonographic coefficient of outflow facility. The episcleral venous pressure decreased in both the treated and the untreated eyes, but the changes were too small to account for the observed decrease of intraocular pressure. The results are consistent with the concept that both the peripheral and central ocular hypotensive actions of clonidine are mediated by an inhibition of adrenergic neurogenic vasoconstriction in the eye.


Reappraisal of the sympathetic role in the sphincteric urethra. Denervation supersensitivity of the urethra of the chronic neurogenic bladder to alpha-adrenergic drugs. The response of urethral pressure to administration of an alpha-stimulant was compared between a group of eight patients with chronic neurogenic bladders as evidenced by positive denervation supersensitivity to parasympathomimetic bethanechol chloride and a group of ten control patients. A supersensitive response to administration of an alpha-stimulant with a rise of maximum urethral pressure, 10 mm Hg or more above the control urethral pressure, was uniformly observed in the urethra of patients with chronically denervated bladders. Our results appear to add pharmacologic evidence of alpha-adrenergic predominance over the parasympathetic in the urethra which is believed to be innervated dually in the recently envolving new concept.


[Endocrine active pancreatic neoplasms]. The tumor-forming endocrine cells of the pancreas belong to the APUD system. These cells are of neuroectodermal origin. The tumor can be diagnosed in most cases by a distinct clinical picture, and the diagnosis can be veryfied by direct hormone determination or/and by the biochemical disorders caused by the hormones. For localisation angiography, szintigrams, endoscopic pancreatography, and sonograms were used up to now without convincing results in many cases; computerized tomography promises to be the decisive examination in the future. Three hormones can, up to now, not yet be correlated with a distinct clinical picture specific for a pancreatic tumor. On the other hand, four tumors are responsible for a very typical clinical entity, the insulinoma, the glucagonoma, the gastrinoma, and the vipoma, as illustrated by our own cases. The surgical therapy consists mainly in enucleation of an adenoma or in partial pancreatic resection. Total pancreatectomy is indicated only in few cases. The Zollinger-Ellison syndrome is treated best by total gastrectomy. Malignant tumors are sensible to streptozotozin.


Hemoglobin Lufkin: beta 29 (B11) Gly replaced by Asp. An unstable hemoglobin variant involving an internal amino acid residue. Hemoglobin Lufkin was found in a Black-American family. Structural analysis of the abnormal hemoglobin indicates a substitution of aspartic acid for glycine at position 29 in the beta chain. Marked instability of the variant hemoglobin is demonstrated by the rapid formation of inclusion bodies upon exposure of the red cells to redox dyes and by the large percentage of precipitated hemoglobin at 65 degrees C. The oxygen affinity, the Bohr effect, and the degree of cooperativity of Hb Lufkin and Hb A are similar over the physiologic pH range. However, at acid pH the oxygen affinity of the variant is increased. Unlike several other reported variants in the B helix, Hb Lufkin is not associated with methemoglobinemia.