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Neurobiology of Polyorchis. II. Structure of effector systems.

The gross and fine morphology of the major effector systems in the anthomedusan, Polyorchis penicillatus, is described and discussed in relation to the known physiological and behavioral properties of these systems. Swimming is controlled by an anastomosing network of giant neurons within the inner nerve ring and radial nerves. Although these neurons may be coupled by gap junctions it is likely that they form a syncytium. The photosensitivity of the "giants" is attributed to reflexive membranes within the cytoplasm. Giant neurons act as both the pre- and postsynaptic cell when forming synapses with other neurons of the inner nerve ring. Neuromuscular synapses between "giants" and the striated swimming muscle are found around the margin and along the radii. Swimming muscle cells are connected laterally by gap junctions and end-to-end by desmosomes which are sometimes elaborated with extra-thick filaments. Unstriated sphincter and radial muscles, the major muscles associated with crumpling, are both greatly folded over mesogloeal ridges and have processes that cross the mesogloea to contact the ring and radial canals, respectively. Synapses or other sites that might be responsible for exciting these muscles during crumpling have not been found. The ability of the endodermal lamella and canals to propagate action potentials can be accounted for by the numerous gap junctions that are seen in these tissues. The precise location where excitation is transferred to the nervous system to initiate crumpling is not known but epithelial bridges crossing the mesogloea are likely routes. Synapses between neurons originating in the outer nerve ring and tentacle longitudinal muscle can account for the control of tentacle length. Neurons of the outer nerve ring also synapse onto velar, radial fibers and the sphincter muscle. The inner and outer nerve rings have nervous connections. The organisation of the outer nerve ring and the arrangement of nerves within the endodermal plexus is described. A diagram showing the major connections and interactions of components of the effector systems is presented.

Selective influences of age and thyroid hormones on type A monoamine oxidase of the rat heart.

The specific actiivty of rat heart MAO, towards both tyramine and benzylamine as substrates, was found to increase with the age of the animal, and also after administration of (-)-thyroxine to young male rats. Conversely, enzyme activity was decreased in animals made hypothyroid by including 2-thiouracil in their diet. However, with both age and altered thyroid status, relatively greater changes in the deamination of tyramine rather than in that of benzylamine, were obtained. Clorgyline and deprenyl, used as inhibitors of rat heart MAO, indicated that tyramine is metabolized solely by MAO-A, whereas benzylamine is a substrate for both MAO-A and -B, and also a clorgyline- and deprenyl-resistant enzymic activity. The proportional contribution of MAO-A, -B and the clorgyline-resistant enzyme towards the total benzylamine deamination in the rat heart was found to vary with the age and with altered thyroid status of the animal in such a way that selective changes in the activity of MAO-A appear to be largely responsible for the overall changes in the specific activity of rat heart MAO which occur in response to these developmental factors.

Renal excretion of pseudoephedrine in the rat.

Pseudoephedrine is an organic base used in the treatment of upper respiratory tract disorders. Surgical techniques and experimental procedures were developed to study the renal elimination mechanisms for this drug in the rat. The ability to measure renal clearance accurately and to demonstrate renal secretion by a carrier-mediated transport system was verified by comparing results from N'-methylnicotinamide (NMN) excretion studies with literature results. Renal tubular secretion of NMN was shown to be saturable and was inhibited by mepiperphenidol to the same extent as that reported in the literature. Pseudoephedrine was cleared by the kidney at a rate in excess of inulin and close to or possibly greater than renal plasma flow. In addition to filtration and secretion, pseudoephedrine appeared to be subject to pH dependent passive reabsorption. When the secretion of pseudoephedrine was studied in detail, it was found to be nonsaturable for plasma levels of pseudoephedrine ranging from 0.16 to 1.5 microgram/ml. Secretion, however, was inhibited by mepiperphenidol suggesting a carrier-mediated process.

Effects of hyperglycemia and hyperthermia on the pH, glycolysis, and respiration of the Yoshida sarcoma in vivo.

Tissue (extracellular) pH (pHe) and intracellular pH (pHi) were measured together in vivo in the solid Yoshida sarcoma and normal organs (liver, gastrocnemius muscle) of noninbred Wistar rats. pHe was monitored by insertion of a miniature capillary glass electrode, and pHi was measured indirectly by equilibrium partitioning of the weak organic acid 5,5-dimethyloxazolidine-2,4-dione across the cell membrane. Under normal conditions, tumor, liver, and gastrocnemius had a similar pHe of 7.05--7.30; tumor pHi was consistently higher (7.2) than that of the normal tissues (6.8--7.1). Curative hyperthermia (42 degrees C for 1 hr) did not significantly change tumor pHe or pHi. After ip glucose injection [6 g/kg body wt; blood glucose level greater than 400 mg/100 ml (22 mmoles/liter) for 4 hr], tumor pHe decreased markedly to 6.6 within 4 hours and did not return to normal for a further 12--14 hours, whereas tumor pHi was hardly affected. No marked change was noted in pHe or pHi of the normal organs following glucose loading of the host. In tumor slices removed from hyperglycemic hosts, marked reduction of both respiration and glycolysis was observed. Hyperglycemia (4 hr) plus hyperthermia at 40 degrees C (1 hr) had a synergistic inhibitory effect on metabolism that was equivalent to heat alone at 42 degrees C, and respiration and glycolysis almost ceased after 3--4 hours. However, tumor heating at 40 degrees C in hyperglycemic hosts was not equivalent to hyperthermia at 42 degrees C: With the former treatment, tumor regression did not occur, and animal survival did not differ from that of control untreated rats. The data do not support the postulate that the effects of heat on tumor cells are mediated via low pHi or that hyperglycemia leads to a lowered pHi which sensitizes the tumor to destruction at 40 degrees C instead of 42 degrees C.

Divalent cation-induced aggregation of chromaffin granule membranes.

Divalent cations induce the aggregation of chromaffin granule ghosts (CG membranes) at millimolar concentrations. Monovalent cations produce the same effect at 100-fold higher concentrations. The kinetics of the dimerization phase were followed by light-scattering changes observed in stopped-flow rapid mixing experiments. The rate constant for Ca2+-induced dimerization (kapp) is 0.86-1.0 x 10(9) M-1sec-1, based on the "molar" vesicle concentration. This value is close to the values predicted by theory for the case of diffusion-controlled reaction (7.02 x 10(9) M-1sec-1), indicating that there is no energy barrier to dimerization. Arrhenius plots between 10 degrees and 42 degrees C support this; the activation energy observed, +4.4 Kcal, is close to the value (4.6-4.8 Kcal) predicted for diffusion control according to theory. Artificial vesicles prepared from CG lipids were also found to have cation-induced aggregation, but the rates (values of kapp) were less than 1/100 as large as those with native CG membranes. Also, significant differences were found with respect to cation specificity. It is concluded that the slow rates are due to the low probability that the segments of membrane which approach will be matched in polar head group composition and disposition. Thus large numbers of approaches are necessary before matched segments come into aposition. The salient features of the chromaffin granule membrane aggregation mechanism are as follows: (a) In the absence of cations capable of shielding and binding, the membranes are held apart by electrostatic repulsion of their negatively charged surfaces. (b) The divalent and monovalent cation effects on aggregation are due to their ability to shield these charges, allowing a closer approach of the membrane surfaces. (c) The major determinants of the aggregation rates of CG membranes are proteins which protrude from the (phospholipid) surface of the membrane and serve as points of primary contact. Transmembrane contact between these proteins does not require full neutralization of the surface charge and surface potential arising from the negatively charged phospholipids. (d) After contact between proteins is established, the interaction between membranes can be strengthened through transmembrane hydrogen bonding of phosphatidyl ethanolamine polar head groups, divalent cation-mediated salt bridging, and segregation of phosphatidylcholine out of the region of contact.

The evolution of genetic diversity.

The existence within natural populations of large amounts of genetic variation in molecules and morphology presents an evolutionary problem. The 'neutralist' solution to this problem, that the variation is usually unimportant to the organism displaying it, has now lost much of its strength. Interpretations that assume widespread heterozygous advantage also face serious difficulties. A resolution is possible in terms of frequency-dependent selection by predators, parasites and competitors. The evidence for pervasive frequency-dependent selection is now very strong. It appears to follow naturally from the behaviour of predators, from the evolutionary lability of parasites, from the ecology of competition and, at the molecular level, from the phenomena of enzyme kinetics. Such selection can explain the maintenance not only of conventional polymorphism but also of continuous variation in both molecular and morphological characters. It can account for the occurrence of diversity within groups of haploid and self-fertilizing organisms, and for the evolution of differences between individuals in their systems of genetic control.

Synaptic transmission: ion concentration changes in the synaptic cleft.

Currents flowing through the postsynaptic membrane of an active synapse will tend to change the concentrations of ions in the synaptic cleft. Published experimental data are used to predict (a) the sodium and potassium concentration changes in the cleft at the frog neuromuscular junction, and (b) the sodium depletion in the cleft under a Ia synaptic bouton on a cat motoneuron. Significant concentration changes are predicted at both synapses. These changes will contribute to the time dependence of the observed current and will cause the reversal potential of the current to be time dependent. At the frog neuromuscular junction, the time course of the endplate current has been shown previously to depend on the magnitude of the current flowing (at a given potential). We attribute this to changes of the cleft ion concentration. The time dependent changes of the endplate current reversal potential that we predict for the neuromuscular junction are probably too small to be detected. This is because the effects of sodium depletion and potassium accumulation on the reversal potential almost cancel. We predict that near the reversal potential small currents of complex time course will remain, i.e. no true reversl potential exists. Such currents have previously been experimentally. At the cat Ia synapse, the synaptic current is predicted to deplete a significant fraction of the available extracellular sodium ions. Consequently, the magnitude of the synaptic current should be relatively independent of the number of postsynaptic channels activated, and of the membrane potental, as has previously been found experimentally.

[Results of bone marrow transplantation in severe aplastic anemia. Bibliographic review].

A review of the present results of bone-marrow transplantation (B.M.T.) in severe aplastic anaemia is presented. Nowadays, there is little doubt that for patients with severe aplastic anaemia the treatment of choice is the B.M.T. provided always that a suitable donor exists. If the patient is fortunate enough to have a normal identical twin, the syngeneic B.M.T. without immunosuppresive conditioning must be performed. This is usually successful, though in some patients failures can be observed due to probable immunological interference. This can be overcome by a new syngeneic B.M.T. preceded by immunosuppression. The usual type of suitable donors is an HLA-identical (including locus D) sibling (allogeneic B.M.T.). Approximately 50% of patients treated in this way can become long-term survivors. The chief complications causing mortality from the allogeneic B.M.T. are graft rejection and graft-versus-host-disease (G.V.H.D.). In order to reduce the graft rejection rate, transfusions from marrow-donor and relatives prior to the transplantation should be avoided. Other probable factor influencing the final outcome of the allogeneic B.M.T. are the interval diagnosis-transplantation, age of the patient, marrow cell dose, the difference of sex between the donor and the recipients and others. Semi-incompatible and incompatible allogeneic B.M.T. are briefly considered.

Renal functional response to the mushroom poison gyromitrin.

The poison gyromitrin, found in the edible false morel Gyromitra esculenta Fr. ex Pers., caused in rats an increased diuresis in which urine was produced with a weak alkaline pH, a high excretion of sodium (530%), and potassium (210%). The observed increase lasted for about 12 h and was followed by a rentention with regard to the volume and the Na-excretion for about 72 h. On the basis of [3H] inulin excretion an increased glomerular filtration was determined followed by a decrease 12 h after application of gyromitrin. An increase of creatinine and urea in the serum could not be established during the retention phase. The diuresis as well as the excretion of sodium could be antagonized by injection of an equimolar dose of pyridoxine. The hydrazine derivative N-methyl-N-formylhydrazine (MFH), which is formed rapidly from gyromitrin by hydrolysis, was without any effect on the renal function. In order to explain the effectiveness of the relatively lipophilic gyromitrin, the non-effectiveness of the more hydrophilic hydrazine MFH and the blockade of the gyromitrin effect by pyridoxine, a mechanism involving the central nervous system is discussed.

An autoradiographic analysis of the mode of proliferation in the buccal mucosa of rats incubated up to 5 hours.

This study was concerned with the course of DNA synthetic activity (3H-TdR-LI-method) in the buccal mucosa of adult male. Sprague Dawley rats over an incubation period of 5 h. Interest was focussed on the influences of different media and, in particular, on temoporary changes in the proliferative activity. 3H-LI were compared in specimens (a) kept in active (= i.e., 3H-thymidine containing) medium throughout their respective incubation period and (b) pre-incubated in inactive medium for varying, but clearly defined periods before being transferred into active medium for 30 min (actual 3H-LI). Independent of the medium used the rate of DNA synthesis was markedly lowered at 60 min, the reduction being more or less significant in the different media. This was due to a temporary inhibition of DNA synthesis, which was restored after 120-180 min. In contrast, the blockade of G2-phase and/or mitosis persisted up to the end of incubation, as indicated by the unchanged number of labelled mitotic figures after 120 min. Addition of glutamine to Mc Coy's 5A markedly enhanced the activity of 3H-thymidine incorporation, but could not prevent the temporary inhibition of DNA synthesis. The biochemical mechanisms relevant for cell proliferation have been reviewed and correlated to the present results.

Intermembrane inclusions induced by anoxia in heart and skeletal muscle mitochondria.

Heart and skeletal muscle from rats of different ages were incubated in vitro in an oxygen-free medium supplied with substrates in order to investigate the effect of anoxia on muscle fine structure, particulary on the mitochondria. In skeletal muscle fibers anoxia has been found to induce changes similar to those previously described in ischemic muscles in vivo namely giant mitochondria, apparently derived by mitochondrial fusion, and intermembrane inclusions with a paracrystalline structure. The plate-like inclusions are mostly located in the intracristal spaces and are closely associated to cristal membranes even in markedly swollen mitochondria. Identical inclusions have been observed in cardiac muscle cells following anoxic injury, whereas they are never found in non-muscle cells such as endothelia, fibroblasts and nerve fibers. Cardiac and skeletal muscle fibers from newborn rats maintained in an oxygen-free medium show mitochondrial swelling but no intermembrane inclusions. The different response of mitochondria from developing vs adult striated muscle to anoxia may be due to changes during postnatal development in the quality or quantity of the protein component(s) involved in paracrystal formation.

[Participation of vaccinia virus in the pathogenesis of different clinical forms of postvaccinal complications. I. Frequency of vaccinia virus detection in the vaccinted who have usual and complicated reactions to vaccination].

The virological examination of 1365 samples taken from 469 children vaccinated against smallpox revealed considerable differences in the frequency and the time of vaccinia virus detection in different clinical forms of postvaccinal pathology as compared with uncomplicated vaccinal process. During the postvaccinal period taking its normal course vaccinia virus was isolated from 7.3% of children only from the pharynx till day 8 following vaccination. In generalized and creeping vaccinia the virus was isolated from 71.4% of children, in postvaccinal encephalitis from 57.1% of children, in vaccinal angina frove-mentioned complications vaccinia virus was detected in the samples obtained from the patients till days 24, 35, 15 and 24 respectively. The etiopathogenetic role of vaccinia virus in a number of postvaccinal complications is discussed.

Kinetics of reduction of purified liver microsomal cytochrome P-450 in the reconstituted enzyme system studied by stopped flow spectrophotometry.

Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar ratio of reductase to P-450 LM of 1.0. Benzphetamine enhanced the oxidation of NADPH by P-450 LM2 but had no effect on the activity of P-450 LM4. Rates of NADPH oxidation in the presence of P-450 LM2 and LM4 decreased by 80 and 40%, respectively, when phospholipid was omitted from the reconstituted enzyme system. These studies provide evidence for the formation of a catalytically functional 1:1 complex between the reductase and P-450 LM4, and indicate that P-450 LM2 and LM4 differ in their dependence on phospholipid.

Effects of GABA-analogues on the high-affinity uptake of GABA in astrocytes in primary cultures.

Employing primary cultures of astrocytes which seem to constitute a valid model of their in vivo counterparts, it has been demonstrated that this cell type is likely to be of importance in terminating the transmitter activity of GABA. It has been shown that the transport carrier in astrocytes is stereospecific for the C-4 hydrogens of the GABA molecule and that its structural requirements are distinct from those exhibited by the neuronal GABA carrier. beta-Proline was a selective inhibitor of GABA transport in astrocytes, whereas R-trans-4-methyl-4-aminocrotonic acid and S-nipecotic acid seemed to be selective inhibitors of neuronal GABA transport, as studied using very thin slices ("prisms") of brain cortex. These findings may be important for studies on the relative significance of the two transport systems in GABA-mediated neurotransmission, and thus for future pharmacological manipulations of the GABA system. This may eventually be beneficial for the treatment of neurological disorders such as epilepsy, Huntington's chorea and Parkinson's disease in which the GABA system seems to be disturbed (34,60,62).

Hemoglobin Cowtown (beta 146 HC3 His-Leu): a mutant with high oxygen affinity and erythrocytosis.

A new mutant, hemoglobin Cowtown, has been found in a white man and his father, both of whom have erythrocytosis. The father had previously been treated with 32P for polycythemia vera. The abnormal hemoglobin is not detectable on electrophoresis in alkaline buffers, but it resolves distinctively on electrophoresis in citrate agar, pH 6.0; similarly, the abnormal beta-globin chain does not separate from beta-A in urea 2-mercaptoethanol buffers of pH 8.9, but it moves anodically to beta-A at pH 6.0. Peptide chromatography and amino acid analysis of the beta chain reveal that the C-terminal histidine residue (beta 146) has been replaced by leucine. Like several other hemoglobins substituted at this residue, Hb Cowtown has a high oxygen affinity and a diminished Bohr effect.

Pneumococcal infections after human bone-marrow transplantation.

Seven of 26 long-term survivors (greater than 7 months post-transplant) of bone-marrow transplantation developed penicillin-sensitive pneumococcal infections more than 7 months after transplantation. One patient had two infections. Six of eight infections were associated with pneumococcal bacteremia, and Streptococcus pneumoniae type 6A was isolated in three cases. Two infections were fatal. All patients had normal nematopoietic function, and none was receiving immunosuppressive therapy. The development of pneumococcal infection was significantly associated with males and with abnormally low or high serum IGG and IgM levels but not with graft-versus-host disease. Serum opsonic activity for S. pneumoniae type 6A was decreased in six of the seven patients when compared to normal pooled serum in an in-vitro bactericidal assay. Four of the six patients with impaired opsonic activity had low serum antibody levels for S. pneumoniae type 6A capsular polysaccharide, while the other two patients had low serum CH100 complement activity. Bone-marrow transplant recipients have an increased susceptibility to pneumococcal infections and should be evaluated for prophylactic penicillin or pneumococcal vaccination.

Hyperproduction of tryptophan by Escherichia coli: genetic manipulation of the pathways leading to tryptophan formation.

Conversion of glucose and ammonium salts into tryptophan by mutants of Escherichia coli was examined as part of a feasibility study on the manufacture of tryptophan. This involved construction, largely by transduction, or a variety of multiple-mutation strains with defined genotypes. By comparing the properties of these strains, we were able to define in biochemical terms several changes that significantly enhance process productivity, namely (i) release of the first enzyme of the common pathway of aromatic biosynthesis and the first enzyme of the tryptophan pathway (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and the anthranilate aggregate, respectively) from inhibition by end products, (ii) blockage of the diversion of chorismate to phenylalanine and tyrosine biosynthesis, and (iii) presence of highly elevated tryptophan pathway enzyme levels, such as result from interference with both repression and attenuation, combined with gene amplification. By using strains carrying appropriate mutations to effect all of these changes, high values of specific productivity were obtained in bath culture (approximately 80 mg/g [dry weight] per h). Furthermore, a pronounced decay in the level of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase activity was implicated as a cause of declining process producitivity during stationary phase, emphasizing the value of having derepressed levels of this enzyme.

In vitro studies on Borna virus. II. Properties of the virus.

Successful cultivation and titration of Borna disease virus in cell cultures enabled detailed studies of the virus properties. Borna virus is labile towards treatment with heat, pH 3.0 and lipid solvents. It is relatively stable at low temperatures and in frozen state. It is easily inactivated by ultraviolet light as e.g. vesicular stomatitis virus. After ultrafiltration studies, the size of the infectious virus unit is between 80 and 100 nm. Its buoyant density in cesium chloride is 1.165 g per ml. The one step multiplication curve shows that Borna virus has a replication cycle of about 2 days in BSC 1 cells. In growth experiments using antimetabilites it behaves like certain RNA containing viruses. As its multiplication is not inhibited by bromo- and iododeoxyuridine and actinomycin D, no DNA step seems to be involved in virus synthesis. Regarding these properties and the intracellular antigen distribution as shown by fluorescent antibodies, it is not possible to attribute Borna virus to any of the established virus groups.

Biosynthesis of ethylene from methionine. Isolation of the putative intermediate 4-methylthio-2-oxobutanoate from culture fluids of bacteria and fungi.

Methods are described for identifying the 2,4-dinitrophenylhydrazones of 4-methylthio-2-oxobutanoate by means of t.l.c., n.m.r. and mass spectroscopy. By using these methods 4-methylthio-2-oxobutanoate, a putative intermediate in the biosynthesis of ethylene from methionine, has been identified in culture fluids of Aeromonas hydrophila B12E and a coryneform bacterium D7F grown in the presence of methionine. Relative to 4-methylthio-2-oxobutanoate, the yield of 3-(methylthio)propanal (methional) from the same cultures was less than 1%. Because 4-[2H]methylthio-2-oxobutanoate was obtained from cultures grown on [Me-2H]methionine, the 4-methylthio-2-oxobutanoate must be derived from methionine. By means of t.l.c. alone, 4-methylthio-2-oxobutanoate was identified in the culture fluids of a range of bacteria, the yeast Saccharomyces cerevisiae and the fungus Penicillium digitatum. A photochemical assay developed for 4-methylthio-2-oxobutanoate shows it to be a product of the metabolism of methionine by Escherichia, Pseudomonas, Bacillus, Acinetobacter, Aeromonas, Rhizobium and Corynebacterium species.

The effect of enzyme induction on diazepam metabolism in man.

1 The elimination and metabolism of diazepam in man was investigated following the induction of the liver microsomal enzyme system by antipyrine. 2 Seven healthy volunteers were given 1200 mg antipyrine as an inducing agent for a period of 14 days. Before and after the induction period the elimination of diazepam and desmethyldiazepam was measured in the plasma by gaschromatography. As parameters of liver microsomal enzyme activity, antipyrine elimination and gamma-glutamyl-transpeptidase in the plasma, D-glucaric acid and 6-beta-hydroxycortisol urinary excretion were measured on both occasions. 3 Following the induction period most parameters of microsomal enzyme activity measured were significantly changed indicating an increase of the microsomal enzyme system. The elimination of diazepam was significantly altered having a half-life of 37 h before and 18 h afterwards combined with a significant increase in total body clearance after the induction period, although the volume of distribution remained unaltered. The formation of the main metabolite N-desmethyldiazepam was not changed, but its elimination was increased having a half-life of 139 or 58 h respectively. 4 The elimination of unchanged diazepam and desmethyldiazepam is significantly increased by the induction of the liver microsomal enzyme system using antipyrine as an inducing agent in healthy volunteers, which might be important under certain clinical conditions.

Studies on the mechanism of iron release from transferrin.

Iron release from human, rabbit, rat and sheep transferrin, chicken conalbumin and human lactoferrin was measured by the change in absorbance of solutions of the iron-protein complexes or by the release of 59Fe from the protein conjugated to agarose. Several phosphatic compounds and iron chelators were able to mediate the process (ATP, GTP, 2,3-diphosphoglycerate, inositol hexaphosphate, pyridoxal 5-phosphate, cytidine 5-triphosphate, pyrophosphate, inorganic phosphate, citrate, EDTA, oxalate, nitrilotriacetate). The greatest rate of iron release was found with pyrophosphate and the least with inorganic phosphate. Different rates of iron release were obtained with the different proteins, greatest with human transferrin and least with lactoferrin. With each of the proteins and the mediators there was a linera relationship between the H+ concentration and the rate of iron release. At any given pH the rate of iron release increased to a maximal rate as the mediator concentration was raised. It is concluded that iron release from transferrin under the conditions of these experiments involves an initial interaction between H+ and the iron-transferrin complex followed by release of the iron under the action of the mediator.

Influence of steric factors on oxygen binding. I. Studies on 2,4-diisopropyldeuteroheme-myoglobin.

Sperm whale apomyoglobin was recombined with 2,4-diisopropyldeuterohemin to form 2,4-diisopropyldeuteroheme-myoglobin and its various physico-chemical properties were investigated to get an insight into the structural and functional role of the peripheral vinyl groups. 2,4-Diisopropyldeuteroheme-myoglobin showed a four times lower oxygen affinity at 25 degrees C and larger enthalpy and entropy changes of oxygenation than the corresponding values of native myoglobin. 2,4-Diisopropyldeuteroheme-metmyoglobin shows a pKa value of 9.68 which is higher than those of native metmyoglobin and mesoheme-metmyoglobin. The rate of autooxidation of oxy-form was about seven times larger in 2,4-diisopropyldeuteroheme-myoglobin than in native myoglobin. The electron-donating effect of isopropyl groups does not give straightforward explanation for these anomalous properties of 2,4-diisopropyldeuteroheme-myoglobin. It is proposed that site and stereospecific van der Waals' interaction between the polypeptide side chains and the peripheral 2,4-diisopropyl groups may weaken the interaction between the bound oxygen molecule and the distal His, resulting in the decrease in the stability of oxyform.

Amino acids as neurotransmitters of corticofugal neurones in the rat: a comparison of glutamate and aspartate.

1 The relative sensitivities to aspartate and glutamate of neurones receiving a corticofugal innervation were examined by microiontophoresis, and compared with the relative sensitivities of neurones not appearing to receive such an input.2 On all the cells tested, glutamate appeared to be a more potent excitant than aspartate in terms of neuronal response size or effective dose.3 DL-alpha-Aminoadipate (alphaAA) reduced the excitatory amino acid responses on all the neurones tested. On many of these cells a control excitation could be produced, by acetylcholine or hydrogen ions, which was in most cases unaffected by doses of alphaAA producing antagonism of amino acid excitation.4 On 70% of the cells, aminoadipate showed no selectivity for aspartate compared with glutamate but a differential action, involving blockade of aspartate but not glutamate, was apparent on the other 30%.5 Doses of alphaAA which selectively reduced responses to aspartate had no effect on short latency evoked spikes, but doses which also reduced responses to glutamate reduced the short-latency synaptic excitation induced by electrical stimulation of either the surface of the cerebral cortex, or of the pyramidal tracts in the medulla.6 These findings suggest that corticofugal neurones having an excitatory action on cells in various parts of the brain may use an amino acid, probably glutamate, as a common neurotransmitter.7 As no significant difference could be demonstrated in the potency ratios of glutamate:aspartate on monosynaptically activated cells compared with other cells, doubt is cast on the validity of drawing conclusions about transmitter identity from potency ratios alone, without the support of antagonist studies.

Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.

Late in the terminal differentiation of epidermis and cultured epidermal cells, a protein envelope located beneath the plasma membrane becomes cross-linked by cellular transglutaminase. The process of cross-linking can be initiated in cultured epidermal cells by agents affecting cell membrane permeability--nonionic detergents, high salt concentrations and ionophores. These agents initiate the cross-linking process by making calcium ions available to the transglutaminase. A soluble precursor of the cross-linked envelope has been identified in crude extracts of cultured epidermal cells by its ability to incorporate labeled amines through the action of transglutaminase. The protein has been purified to homogeneity by gel filtration and chromatography on columns of DEAE-cellulose and hydroxyapatite. Comprising an estimated 5--10% of the soluble cell proteins, it has a molecular weight of about 92,000, is isoelectric at pH 4.5 +/- 0.3 and has an unusual amino acid composition (46% Glx residues). It is chemically and immunochemically unrelated to keratins. The following evidence confirms that the protein becomes incorporated into cross-linked envelopes: first, washed cross-linked envelopes bind antibody to the purified protein, as shown by indirect immunofluorescence; second, absorption of the antiserum with washed envelopes removes all detectable antibodies to the purified protein; and third, the protein cannot be extracted from keratinocytes after their envelopes have become cross-linked. Examination of sections of epidermis by immunofluorescence, using antiserum to the purified protein, reveals that in addition to the stratum corneum, the living cells of the outer half of the spinous layer react strongly. The envelope precursor is present in the cytoplasm, but becomes concentrated at the cell periphery, where it will be cross-linked later, when the cells have passed through the granular layer. The protein is also concentrated in a peripheral location in cultured epidermal cells.

The in vivo examination of the irreversible beta-adrenoceptor antagonist Ro 03-7894 on cardiac rate and contractility.

1. Two benzofuran-2-ethanolamines Ro 03-5255 (1-(5-acetylamino-benzofuran-2-yl)-1-hydroxy-2-isopropylaminoethane) and Ro 03-7894 (1-(5-chloracetyl aminobenzofuran-2-yl)-1-hydroxy-2-isopropylaminoethane) which had previously been shown to exhibit respectively competitive and irreversible beta-adrenoceptor antagonism in guinea-pig isolated atria, were compared in vivo using isoprenaline-induced tachycardia of anaesthetized guinea-pigs and heart rate and contractility (dp/dtmax) of open-chest anaesthetized guinea-pigs and of conscious cats. 2. In urethane-anaesthetized guinea-pigs doses of 3 mg/kg, s.c. of both antagonists produced significant blockade of the rate response to an 80% of maximum dose of isoprenaline after 4 h. In other experiments, guinea-pigs were pretreated with the antagonists and the responses to isoprenaline were then monitored. The slopes of the dose-response curves to isoprenaline were depressed for up to 24 h by Ro 03-7894 but this was not so with Ro 03-5255. 3. In conscious cats the course of blockade by Ro 03-7894 was followed in the same animals and was still evident after 48 h. In contrast, the beta-adrenoceptor blockage produced by Ro 03-5255 was not evident 24 h after administration. 4. The persistence of blockade by Ro 03-7894 was consistent with the irreversible mode of action demonstrated in vitro.

The control of lipogenesis by dietary linoleic acid and its influence on the deposition of fat.

The replacement of dietary starch by sucrose results in an increase in hepatic lipogenesis in the rat. When corn oil (4% by weight or 9% of the energy content of the diet) was included with the sucrose (20% by weight, 20% of the energy content) the lipogenic effect of the sucrose was completely suppressed. In contrast, when beef tallow replaced the corn oil, the induced activity caused by the sucrose was reduced by only approximately 20%. No significant differences were observed between males and females. These diets containing sucrose supplemented with either 4% (w/w) corn oil or 4% (w/w) beef tallow, were then used to ascertain whether or not the effects on hepatic lipogenesis were reflected in changes in the amount of fat deposited during growth from 4--24 weeks of age. It was shown that the percentage body fat was only statistically different (P less than 0.05) when animals fed sucrose-supplemented diets were compared with animals fed diets supplemented with sucrose and beef tallow. However, there were no significant differences in total carcass weight of these rats. The results are discussed in terms of the relative contribution of liver and adipose tissue to total lipogenesis and the factors which control the lipogenic activity in the two tissues.

Presynaptic noradrenergic alpha-receptors and modulation of 3H-noradrenaline release from rat brain synaptosomes.

The depolarization (15 mM K+)-induced release of 3H-NA from superfused rat brain synaptosomes and the effects of alpha-noradrenergic drugs thereon were studied. Noradrenaline (NA; in the presence of the uptake inhibitor desipramine) reduced synaptosomal 3H-NA release. Reduction of the concentration of calcium ions in the medium during K+ stimulation greatly enhanced the sensitivity of 3H-NA release to alpha-receptor-mediated inhibition. Under these conditions NA dose-dependently inhibited 3H-NA release from synaptosomes obtained from cortex or hypothalamus, but did not affect 3H-NA release from striatal (i.e dopaminergic) synaptosomes. Adrenaline, clonidine and oxymetazoline potently inhibited 3H-NA release from cortex synaptosomes at concentrations in the nanomolar range. Phentolamine by itself did not affect synaptosomal 3H-NA release, but antagonized the inhibitory effects of both noradrenaline and adrenaline. The data obtained further substantiate the hypothesis that the alpha-receptors mediating a local negative feedback control of NA release are localized on the varicosities of central noradrenergic neurons, Furthermore, noradrenergic nerve terminals in the hypothalamus appear to be less senstive to alpha-receptor-mediated presynaptic inhibition than those in the cortex.

The history of pregnancies that occur following female sterilization.

Data on 208 pregnancies occurring among 20,749 women following sterilization were collected by the International Fertility Research Program and the histories of these pregnancies from conception to termination were analyzed. In the laparoscopic series, the operator's failure to interrupt the tube by electrocoagulation or a tubal occlusion device was the major reason reported for sterilization failure. In the culdoscopic series, operator error or device deficiency were the major reasons for failure. About three quarters of pregnancies in this study were conceived within the first year following sterilization and were confirmed during the first trimester. The rate of ectopic pregnancy occurring in this series was higher than the rate reported for nonsterilized women and was especially high when electrocoagulation was used.

Trypsin and chymotrypsin inhibitor from chick peas. Selective chemical modifications of the inhibitor and isolation of two isoinhibitors.

The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 M -- 0.01 M and in KOH 0.01 M -- 0.05 M even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleyation and succinylation of the inhibitor resulted in almost full loss of its trypsin-inhibitory activity but had no effect on the chymotrypsin-inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin-inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin-inhibitory activity but also the trypsin-inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin-inhibitory activity indicating that the only methionine is involved in the chymotrypsin-inhibitory activity. When CI-fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP-Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.

Separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri.

The separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri have been achieved. The two enzymes had different elution properties on diethylaminoethyl (DEAE) cellulose and differed in their sensitivity to N-ethylmaleimide (NEM), preference for different template-primers, and sedimentation velocity in linear glycerol gradients. The first enzyme activity, ScA, was retained on DEAE-cellulose and was not inhibited by NEM. Activated deoxyribonucleic acid and poly(dA)-oligo(dT12) were the preferred template-primers. Arabinosyl-cytidine triphosphate had no effect. The sedimentation coefficient of ScA was 6.3s. The second activity, ScB, was not retained on DEAE-cellulose and was inhibited by NEM. Poly(dA)-oligo(dT12) was the preferred template-primer, whereas activated DNA was only poorly utilized. ScB was not affected by arabinosyl-cytidine triphosphate, and its sedimentation coefficient was 4.4s. The polymerization activities of the two enzymes were maximum at 37 to 40 degrees C.

Phosphoenolpyruvate carboxylase of Escherichia coli. Affinity labeling with bromopyruvate.

Phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli W was alkylated by incubation with bromopyruvate, substrate analog, leading to irreversible inactivation. The reaction followed pseudo-first-order kinetics. Mg2+, an essential cofactor for catalysis, enhanced the inactivation, and the enhancing effect increased as the pH increased. The inactivation rate showed a tendency to saturate with increasing concentrations of bromopyruvate, indicating that an enzyme-bromopyruvate complex was formed prior to the alkylation. DL-Phospholactate, a potent competitive inhibitor with respect to phosphoenolpyruvate, protected the enzyme from inactivation in a competitive manner. Examination of the acid hydrolysate of the enzyme modified with [14C]bromopyruvate by paper chromatography showed that radioactivity was solely incorporated into carboxyhydroxyethyl cysteine. In addition, determination of sulfhydryl groups of the native and modified enzymes with 5,5'-dithiobis(2-nitrobenzoate) showed that inactivation occurred concomitant with the modification of one cysteinyl residue per subunit. The results indicate that bromopyruvate reacted with the enzyme as an active-site-directed reagent.

Electron microscopic analysis of tropomyosin paracrystals.

Negatively stained images of divalent cation-induced tropomyosin paracrystals show polymorphic patterns which are almost bipolar. Although these bipolar forms are naturally due to antiparallel arrays of molecules, the precise molecular arrangements have not been clarified yet except in the case of one type of these polymorphic paracrystals by Stewart and McLachlan [(1976) J. Mol. Biol. 103, 251--269]. In the previous paper we showed that the lead-induced polar paracrystal is a parallel and in-register array of tropomyosin molecules. Moreover, we have made it possible to locate a given residue on the staining pattern. By overlapping two photographic transparencies of the polar paracrystal antiparallel, directly observed images of polymorphic bipolar paracrystals could be synthesized photographically with fidelity. The overlap length between N-terminals of antiparallel pairs of molecules could be easily determined without any assumptions. Next, we considered the stabilizing forces involved in the morphogenesis of such polymorphic paracrystals. The cation-bridged attractive forces already proposed by some groups were insufficient to account for the stability of some specific forms of tropomyosin paracrystals. From the primary amino acid sequence of tropomyosin, we calculated the changes of repulsive forces between the basic residues with changes of molecular overlap length between the N-terminals of antiparallel pairs. By setting the values of charge appropriately, we could account well for the stability of the polymorphic structures observed by electron microscopy.

Reversal of innate aversions: attempts to induce a preference for chili peppers in rats.

Although humans frequently develop preferences for innately unpalatable bitter or irritant substances, such preferences are extremely rare in animals. An attempt was made to understand the nature of this difference by systematic experiments with laboratory rats, with chili pepper as the unpalatable substance. In parallel with major aspects of the human experience with chili pepper, rats were exposed to it as a flavoring in all their food for periods up to 11 mo from birth, without significant preference enhancement. Gradual introduction of chili into the diet also had no effect, nor did a series of poisoning and safety experiences designed to teach the rats that only chili-flavored foods were safe to eat. A sequence of seven pairings of chili-flavored diet with prompt recovery from thiamine deficiency did significantly attenuate the innate aversion and may have induced a chili preference in at least one case. Extensive experience with chili did not reliably make rats much less sensitive to its oral effects. The only reliable way to eliminate chili aversion in rats is to destroy their chemical irritant sense, which was accomplished in one group of rats. It is concluded that in contrast to humans, it is extremely difficult to reverse innate aversions in rats.

Nigericin-induced death of an acidophilic bacterium.

At an external pH of 3.5, nigericin (which catalyses an electroneutral H+/K+ exchange) abolished the transmembrane proton gradient (delta pH) of Bacillus acidocaldarius, causing a rapid acidification of the cytoplasm from approximately pH 6.0 to pH 3.5. A pronounced loss of viability and fine-structural changes rapidly followed treatment with nigericin. A marked decline in respiration and an even more rapid decrease in cytoplasmic ATP were observed. Activity of at least one cytoplasmic enzyme decreased more slowly. There was no generalized loss in the integrity of the cytoplasmic membrane, as assayed by permeability to inulin or Na+ or by release of ultraviolet light-absorbing compounds. The loss of viability upon treatment with carbonyl cyanide m-chlorophenylhydrazone was similar to what observed with nigericin, so proton influx alone, rather than together with K+ efflux, was probably involved in the death of the organism. Moreover, acidification of the cytoplasm rather than abolition of the delta pH was the lethal event, since no loss of viability was observed when the delta pH was abolished by elevation of the external pH.

Inter-relationship of sodium, chloride, bicarbonate and acetate transport by the colon of the pig.

1. Net transport of Na, Cl, HCO3 and acetate was examined in the temporarily isolated colon of conscious pigs weighing 46 +/- 8 kg. 2. The entire colon absorbs 4.1 ml. H2O, 0.8 m-equiv Na, 1.3 m-equiv acetate and secretes 0.5 m-equiv HCO3/min with a solution comparable to the normal contents. The absorptive capacity of the proximal and distal halves of the colon was comparable per unit dry weight of mucosa when each segment was presented with the same solution. 3. A series of studies using ion replacement solutions showed that net Na absorption and net HCO3 accumulation in the lumen solution were both increased in the presence of acetate. Cl absorption was independent of Na absorption and was accompanied by an equivalent net secretion of HCO3 in the absence of Na. When NaCl in the perfusion solution was replaced with Na2SO4, Na and HCO3 were absorbed at equal rates. 4. Final pCO2 values observed in NaCl and Na2SO4 solutions were greater than those observed in plasma while the pCO2 of the Na acetate solution after perfusion was reduced to values below plasma concentrations. 5. Results are consistent with the hypothesis that hydration of CO2 in the lumen solution or mucosal cell provides a continuous source of H ions for absorption of the more permeable undissociated acid. The evidence also suggests an additional source of H ions may be provided by a Na-H exchange process located in one of the limiting cell membranes.

Smectite clays in Mars soil: evidence for their presence and role in Viking biology experimental results.

Various chemical, physical and geological observations indicate that smectite clays are probably the major components of the Martian soil. Satisfactory ground-based chemical simulation of the Viking biology experimental results was obtained with the smectite clays nontronite and montmorillonite when they contained iron and hydrogen as adsorbed ions. Radioactive gas was released from the medium solution used in the Viking Labeled Release (LR) experiment when interacted with the clays, at rates and quantities similar to those measured by Viking on Mars. Heating of the active clay (mixed with soluble salts) to 160 degrees C in CO2 atmosphere reduced the decomposition activity considerably, again, as was observed on Mars. The decomposition reaction in LR experiment is postulated to be iron-catalyzed formate decomposition on the clay surface. The main features of the Viking Pyrolytic Release (PR) experiment were also simulated recently (Hubbard, 1979) which the iron clays, including a relatively low '1st peak' and significant '2nd peak'. The accumulated observations on various Martian soil properties and the results of simulation experiments, thus indicate that smectite clays are major and active components of the Martian soil. It now appears that many of the results of the Viking biology experiments can be explained on the basis of their surface activity in catalysis and adsorption.

Effects of cadmium on the contractility of a frog cardiac muscle in relation to pH of external solution.

In order to examine the pH dependency of Cd effects on cardiac muscle, the electrical and mechanical activities in the bullfrog heart were investigated in relation to various external pHs and concentrations of Cd. The amplitude and duration of the action potential or the spontaneously beating frequency in the atrium were not significantly affected during the 3-min administration of various concentrations of Cd in a range from pH 6 to 10, but the isotonic contraction at the end of the same period was greatly altered: the lower the external pH the more Cd decreased the contractility of the atrium. This pH dependency of Cd effects was also observed in SO4-Ringer's solution whose anions, SO4, were more impermeable than Cl. Decrease in contractility in Cd-Ringer's solution was counteracted by excess Ca. The lower the pH of the Cd-Ringer's solution, the more Ca was necessary to counteract an equal amount of Cd. The amount of Cd-uptake into the atrium was analyzed after soaking the atrium in various concentrations of Cd-Ringer's solution. The higher the concentration of external Cd, the larger the Cd-uptake into the atrium. No pH dependency, however, was observed in this relationship. This suggests that probably only a small fraction of total Cd-uptake interacts with Ca-binding sites which is specific to contraction, and causes the Cd-induced decrease in contractility. This process is considered to be pH-dependent. However, most Cd-uptake into cardiac muscle is pH-independent and may be nonspecific to contraction.

Iodine labelling of sea anemone toxin II, and binding to normal and denervated diaphragm.

1. Sea anemone toxin II (ATX II) which keeps the activated sodium channels open, can be labelled at its histidine residues with 125I up to a specific radioactivity of 500 Ci/mmole. Upon intraventricular injection in mice, ATX II causes acute, short-lasting hyperexcitation and convulsions. Its LD50 in mice is between 25 and 50 ng of the native peptide, and between 50 and 100 ng of the radioactive material per animal. 2. The labelled peptide is bound to mouse diaphragm from where it can be displaced by ATX II and, even better, by scorpion neurotoxin but not by other basic peptides, e.g., histone or aprotinin. Binding is not significantly influenced by 50 mM potassium, by replacing sodium with choline, by veratridine or tetrodotoxin. In contrast to binding of alpha-bungarotoxin, binding of ATX II is not changed by denervation of the diaphragm. ATX II binds not only to the muscular but also to the tendinous moiety of the mouse diaphragm. 3. ATX II lowers the surface tension of water. Further experiments are needed to establish the usefulness of 125I-ATX for labelling sodium channels in excitable membranes.

Effect of an increase in HbO2 affinity on the calculated capillary recruitment of an isolated rat heart.

The effect of an increase in hemoglobin O2 affinity on myocardial O2 delivery was studied in a blood perfused working rat heart preparation. In a first series of experiments P50 (PO2 for which saturation is 50%) was lowered by use of carbon monoxide. The heart was alternatively perfused with the blood sample of P50 = 32 mm Hg and the blood sample of P50 = 17 mm Hg. O2 capacity of both samples was kept the same by appropriate hemodilution. In a second serie of experiments change of P50 was obtained by the use of adult human erythrocytes containing hemoglobin creteil with a P50 of 13.6 mm Hg. As P50 decreased from 25 to 10 mm Hg, coronary sinus PO2 (PcsO2) diminished from 26 +/- 2 to 18 +/- 2 mm Hg (-29 +/- 2%), coronary sinus O2 content (CcsO2) increased by 15 +/- 3%, myocardial oxygen consumption did not change significantly. The percentage of increase of coronary flow was 23 +/- 4%. Analysis of these results with a simple mathematical model of O2 delivery suggest that increase in HbO2 affinity is corrected by a simultaneous increase in coronary flow and capillary recruitment.

[Influences of different anaesthetic techniques on serum levels of hepatic enzymes and of bilirubin (author's transl)].

The influence of four different types of anaesthesia -- halothane and enflurane inhalation anaestheis, neurolept analgesia and epidural analgesia with single shot technique -- on the serum levels of GOT, GPT, GLDH, LDH, AP, LAP, gamma-GT, total protein and bilirubin was examined in 104 comparable cases undergoing gynaecological surgery. A significant increase in GOT, GPT, GLDH, AP, and gamma-GT levels was observed between the 6th and 9th postoperative day with all types of anaesthesia. The serum levels remained elevated until the 15th postoperative day. There was no significant difference between the four types of anaesthesia as regarded their effects on the serum constituents investigated. The same applied to the serum CHE activity which reached a minimum on the 3rd postoperative day followed by a steady rise. The results indicate that the postoperative changes of the liver enzyme pattern are not related to the type of anaesthesia used. Bilirubin was elevated on the first postoperative day, then dropped rapidly and stayed below the pre-operative values until the 12th postoperative day. There was no difference between the four types of anaesthesia. Glucuronisation of bilirubin may be inhibited initially by the anaesthetic agents. The subsequent rapid fall of the bilirubin levels may be the result of enzyme induction.

Initiation of plasma prorenin activation by Hageman factor-dependent conversion of plasma prekallikrein to kallikrein.

Plasma prorenin is an inactive form of renin (EC 3.4.99.19) that can be converted to active renin in acid-treated plasma by an endogenous serine protease that is active at alkaline pH (alkaline phase activation). To identify this enzyme we first tested the ability of Hageman factor fragments, plasma kallikrein (EC 3.4.21.8), and plasmin (EC 3.4.21.7) to activate prorenin in acid-treated plasma. All three enzymes initiated prorenin activation; 50% activation was achieved with Hageman factor fragments at 1 microgram/ml, plasma kallikrein at 2-4 microgram/ml, or plasmin at 5-10 microgram/ml. We then showed that the alkaline phase of acid activation occurred normally in plasminogen-free plasma but was almost completely absent in plasmas deficient in either Hageman factor or prekallikrein; alkaline phase activation was restored to these latter plasmas when equal parts were mixed together. Therefore, both Hageman factor and prekallikrein were required for alkaline phase activation to occur. We then found that, although plasma kallikrein could activate prorenin in plasma deficient in either Hageman factor or prekallikrein, Hageman factor fragments were unable to activate prorenin in prekallikrein-deficient plasma. These studies demonstrate that alkaline phase prorenin activation is initiated by Hageman factor-dependent conversion of prekallikrein to kallikrein which, in turn, leads to activation of prorenin. In this fashion, we have revealed a possible link between the coagulation-kinin pathway and the renin-angiotensin system.

13C nuclear magnetic resonance studies of anaerobic glycolysis in suspensions of yeast cells.

Anaerobic glycolysis in Saccharomyces cerevisiae has been studied by 13C NMR at 90.5 MHz. [1-13c]Glucose and [6-13C]glucose were fed to suspensions of yeast cells. Time courses for concentration changes of the starting material, of courses for concentration changes of the starting material, of the intermediate fructose 1,6-bisphosphate (Fru-P2), and of the end products, ethanol and glycerol, have been followed with 1-min time resolution. The glucose uptake was well fitted by a Michaelis-Menten model, assuming competition of alpha- and beta-glucose for the same site. The Km for the uptake was found to be 10 mM for beta-glucose and 5 mM for alpha-glucose. The concentration of Fru-P2 showed an initial oscillation before it reached a co,stant level. The 13C label, introduced only as [-13C]- or [6-13C]glucose, was observed in Fru-P2 in both the C1 and C6 positions, simultaneously. From the relative intensities of the C1 Fru-P2 and C6 Fru-P2 peaks in the presence of [1-13C]- and [6-13C]glucose, in vivo kinetic information was obtained about the aldolase-triosephosphate isomerase triangle. We found that under the conditions of these experiments the ratios of backward to forward velocities through aldolase and triosephosphate isomerase were 0.9 +/- 0.1 and 0.8 +/- 1, respectively, indicating they were close to equilibrium.

Quantitative relationship between beta-adrenergic receptor number and physiologic responses as studied with a long-lasting beta-adrenergic antagonist.

The aminobenzyl analog of propranolol, 1- (p-amino-alpha,alpha-dimethylphenethylamino)-3-(1-naphthoxy)-2- propanol, was synthesized and found to be a potent beta-adrenergic blocking agent. The beta-adrenergic receptors of cultured rat C6 glioma cells (2B clone) as assessed by [(125)I]iodohydroxybenzylpindolol binding were decreased 50 and >95% after pretreatment with 8 nM and 1 muM aminobenzylpropranolol, respectively. Unlike propranolol, aminobenzylpropranolol displayed a prolonged blockade of receptors that was maintained during several hours of washing. [(125)I]Iodohydroxybenzylpindolol saturation binding experiments in cells exposed to aminobenzylpropranolol and subsequently washed indicated that the compound effectively diminished receptor number with no change in the affinity of the remaining receptors for iodohydroxybenzylpindolol. Aminobenzylpropranolol inhibited catecholamine-stimulated intracellular cyclic AMP accumulation; with increasing blockade, isoproterenol dose-response curves became progressively shifted to the right but the maximal response was unaltered. Aminobenzylpropranolol inhibited the beta-adrenergic contractile response in atria isolated from rats and guinea pigs. Treatment with 0.1 and 10 muM aminobenzylpropranolol produced decreases of 0.5 and 2 orders of magnitude in the contractile potency of isoproterenol. As in glioma cells, aminobenzylpropranolol failed to decrease the maximal response to isoproterenol. The effects of aminobenzylpropranolol persisted during extensive washing of atria (up to 17 hr). Repeated exposures to isoproterenol at concentrations sufficient to produce maximal tension development also failed to alleviate the blockade. The inotropic potency of histamine in guinea pig atria was not affected by aminobenzylpropranolol. These data suggest that catecholamines are capable of eliciting full biological responses in glioma cells and isolated atria even though the great majority of beta-adrenergic receptors are persistently blocked.

The annual testicular cycle in an equatorial colony of lesser rock hyrax, Heterohyrax brucei.

Adult males from a colony of lesser rock hyrax found near the equator in Kenya exhibited an annual cycle of testicular activity characterized by intense spermatogenesis and elevated androgen status from May to July. Average masses of testes and seminal vesicles taken in these months were almost fourfold greater than those from September to January. During the months of peak testicular activity average diameters of Leydig cells and seminiferous tubules were increased by approximately one half and total tubule length was doubled, compared with values for the quiescent months. Variable testicular development occurred during transitional intervals preceding and following peak testicular activity. From February to Aril thickening of the seminiferous epithelium and appearance of spermatozoa in the caput epididymidis signalled re-establishment of sperm production. In August shedding of germinal cells from the epithelium heralded impending failure of spermatogenesis. Evidence of an annual testicular cycle contradicted the prevalent belief that equatorial hyrax breed all year and suggested that the testicular cycle is a conservative element of hyracoid reproductive strategy.

Carbohydrate metabolism in the fetal pig during late gestation.

In acute experiments on pregnant sows under sodium pentobarbitone anaesthesia, acid base balance, oxygenation and plasma metabolite concentrations were well maintained in the dam and all fetuses which remained undisturbed in utero, irrespective of the duration of the experiment. Fetal liver glycogen concentrations were also unaffected by the time of removal of the fetus. By contrast, intravascular catheterization and withdrawal of blood led to fetal hyperglycaemia and depletion of hepatic glycogen although blood gas and pH values were not changed by these procedures. In the 1 1/2--2 h sampling period following catheterization the normal positive umbilical venous-arterial differences in plasma glucose and lactate generally became reversed. These changes were prevented by the administration of hexamethonium (10--15 mg . kg-1 i.v.) but the drug did not block the fall in hepatic glycogen in catheterized fetuses. Both adrenaline and noradrenaline, which were each infused intravenously at 2.7--3.9 or 0.6--0.9 microgram . kg-1 . min-1, resulted in fetal hyperglycaemia and lacticacidemia together with a fall in arterial blood pH; hepatic glycogen concentrations in these fetuses were also reduced. The apparent sensitivity of the glycogenolytic mechanism to surgical trauma and haemorrhage in the fetal piglet is discussed in relation to findings in other species.

Cardiac performance: optimal heart rate for maximal cardiac output.

To determine optimal heart rate for the maximal cardiac output at various levels of inotropy and blood volume, the relationship between heart rate (HR) and stroke volume (SV) was examined in anaesthetized dogs during right atrial pacing. Myocardial inotropy was raised by intravenous infusion of isoproterenol, a stimulator of adrenergic beta-receptors, and reduced by propranolol, an inhibitor of adrenergic beta-receptors. Circulating blood volume was increased by saline infusion. Within the range of optimal heart rate, SV and HR were inversely related: SV = k (HR0-HR), where k indicates the relationship between changes in SV and HR. The intercept with the HR axis is HR0. At constant HR a rise in inotropy increased SV and a fall in inotropy reduced SV. These changes in SV were eual at every HR, and k was therefore constant. In contrast, blood volume expansion increased SV more at low than at high HR (k increased), but HR0 was not significantly changed. Calculated maximal cardiac output: k.HR02/4, and optimal heart/rate: HR0/2, agreed with observations when maximal cardiac output was raised from 1900 to 4500 ml/min by increasing blood volume and inotropy. Optimal HR was not influenced by changes in blood volume, but was increased from 160 to 200 beats/min by increasing inotropy. We conclude that the optimal heart rate and the maximal cardiac output can be predicted from the linear relationship between SV and HR during right atrial pacing.

Immunospecific depletion of graft-versus-host-reactive lymphocytes using sensitized syngeneic initiator T lymphocytes.

We investigated a model of a lethal graft-versus-host (GVH) reaction with the aim of depleting donor spleen cells of immunospecific GVH-reactive lymphocytes. In previous studies of the recruitment of effector T lymphocytes by sensitized syngeneic initiator T lymphocytes (ITLs) we found, using a local GVH reaction, that precursors of specific GVH-reactive lymphocytes were recruited to a draining lymph node. In this study, adult F1 hybrid mice were lethally irradiated and reconstituted with 2 x 10(6) syngeneic bone marrow cells and varying numbers of spleen cells from parental strain mice. To deplete donor spleen cells of GVH-reactive lymphocytes, parental strain mice were given injections in the hind footpads 6 days earlier of syngeneic ITLs that had been sensitized in vitro against allogeneic fibroblasts. We found that injection of ITLs sensitized against the relevant allogeneic antigens led to a marked decrease in the specific GVH potential of donor spleen cells. These findings show that GVH-reactive lymphocytes can be depleted selectively by activating their recruitment to particular lymph nodes, using syngeneic ITLs.

Inhibited autophagic degradation of cytoplasm during compensatory growth of liver cells after partial hepatectomy.

The livers from 56 sham-operated and 56 partially hepatectomized male albino rats killed 4--81 h after operation were investigated by electron microscopic morphometry. Following partial hepatectomy, the principal changes in volume fractions in the hepatocellular cytoplasm were: decrease of glycogen and, to a lesser extent, of mitochondria together with considerable increase of fat droplets. The volume fraction of microbodies (= peroxisomes) showed no significant difference between control and regenerating liver. By evaluating large test fields of about 40,000 micrometers 2 sectioned cytoplasm per animal it could be demonstrated that the volume fraction and the numerical density of autophagic vacuoles (AV's) were significantly reduced after partial hepatectomy. The extent of this reduction depended on the postoperative time interval. AV's were reduced by 75% at day 0 (4--17 h p.o.), by 98% at day I (19--33 h p-o.), by 75% at day II (43--57 h p.o.), and still by 50% at day III (67--81 h p.o.). The different types of AV's, defined on the basis of the different cytoplasmic components enclosed, were reduced to similar extent during the respective time periods. The reduction of AV's seems to be specific for the regenerating organ since no significant differences in the volume fraction of AV's could be found in the proximal tubular cells of the kidney of partially hepatectomized animals when compared with those of sham-operated controls. The inhibition of intracellular autophagic degradation in regenerating liver has its biochemical equivalent, i.e. inhibited protein catabolism, and is interpreted as an important and adequate mechanism in effecting the shift from the physiological steady state between anabolism and catabolism to the positive balance which is required for the compensatory growth of the liver after partial hepatectomy.

Effect of a deproteinized blood extract on experimental granulation tissue.

The present work was undertaken to study the effects of a deproteinized extract of calves' blood (Solcoseryl) on developing granulation tissue in rats. Cylindrical hollow viscose cellulose sponge implants were used as an inductive matrix for the growth of granulation tissue. In the first, sham group the implants were treated daily by withdrawing 1 ml of wound fluid from the central dead space of the implant and then re-injecting the fluid. In the second, experimental group the aspirated wound fluid was replaced by a corresponding volume or Solcoseryl. Analyses of wound fluid and granulation tissue were carried out 4, 10 and 21 days after implantation. A statistically significant increase of granulation tissue hemoglobin (+21%) was observed at 10 days in the Solcoseryl group as compared with the sham-treated rats, indicating an enhanced capillary ingrowth. Concurrently, the mean amount of DNA in the Solcoseryl-treated tissues was elevated by 48% over the level of the sham-treated group, demonstrating an augmented cellularity of granulation tissue. At 21 days the mean amount of collagen hydroxyproline of the Solcoseryl group was 31% above the level measured in the sham-treated animals. PO2, PCO2 and pH in the wound fluid and the amounts of RNA and uronic acids showed no essential differences between the two groups. These data demonstrate a stimulatory effect of Solcoseryl on several aspects of granulation tissue formation: augmented vascularization, elevated cellularity and subsequent enhancement in the accumulation of collagen.

In vitro damage of cultured ookinetes of Plasmodium gallinaceum by digestive proteinases from susceptible Aedes aegypti.

After exposure to extracts from blood fed A. aegupti cultured ookinetes of P. gallinaceum were damaged to various, defined extents. Immature ookinetes were found to be more sensitive to damage than mature ones. The damage was dependent on the digestion time after which the Aedes extracts had been prepared and could be correlated with the proteolytic activity in the extracts. Control experiments demonstrated that the factors responsible for damage were neither present in unfed mosquitoes nor in blood alone and that the damage was not a result of osmotic stress. After the treatment of the Aedes extracts with lima bean trypsin inhibitor the ookinete damage was much less, indicating that the Aedes trypsin was the major agent of damage. These results were supported by experiments in which the tryptic activity of the extracts was eliminated by thermal denaturation. It is concluded that in the mosquito midgut most of the ookinetes are damaged by digestive enzymes and that this is one factor leading to the discrepancy between the number of ingested macrogametocytes and the number of oocysts which is usually found in nature. It seems that the only ookinetes which have a chance of surviving are those which develop in the centre of the blood clot, away from the site of enzyme action.

Lorazepam in status epilepticus.

Lorazepam, a dichloro-3-hydroxy-1,4-benzodiazepine, has been shown to be a potent anticonvulsant in animal models of epilsepsy and has minimal depressant effects on respiration and circulation in humans. The effects of this compound were studied in status epilepticus. Twenty-five patients were given intravenous lorazepam during status epilepticus of varying cause. Four or 8 mg of the drug controlled status in 22 of the 25 patients. Although single seizures recurred in 5 of the 22 patients, none experienced recurrence of status during a prolonged follow-up period. Transient respiratory arrest occurred in 1 patient, but no other significant complications were observed. Studies of plasma drug levels suggest that most patients attain good seizure control at concentrations between 30 and 100 ng per milliliter. Clinical observations indicate that repetitive injections are not required for continuing control of seizures in patients whose seizures are initially controlled. Lorazepam appears to be an effective and safe drug for treatment of status epilepticus, with a duration of control longer than that achieved with diazepam.

The endocrine cells of the rectum of adult ox.

In order to identify the endocrine cell types in various parts of the Ruminant gut, we have applied ultrastructural, both morphological and cytochemical, techniques, in parallel to the histochemical ones, to study the rectal mucosa of the adult Ox. In these studies we show that: "EC" cells, of the intestinal type, contain predominantly pleiomorphic granules, which are very electron dense and heavily reactive to "Masson" and "Grimelius" methods; "L" cells are recognizable by their numerous granules, which are fairly homogeneous in shape and osmiophilia. They do not react with "Masson" and are weak or negative to Grimelius s reaction. These granules occur near to others that are less dense, unreactive to "Masson", and that contain an argyrophilic matrix, with an eccentric electron dense core, which does not react with silver; "F-like" cells contain granules which are variable in shape, size and osmiophilia. They are unreactive to "Masson" and weak or unreactive to Grimelius silver; "H" cells contain few, small and uniformly osmiophilic granules. These are unreactive to "Masson" and uniformly reactive to "Grimelius". Our data suggest that the morphology, frequency and distribution of the cell types we have identified in the mucosa of the bovine rectum correspond with those reported in large intestine and rectum of Monogastrics, as by other authors described.

On the range of alpha-adrenergic regulation of coronary vascular resistance.

In order to obtain an estimate of the range of alpha-adrenergic resistance regulation in the coronary vascular system, the following studies were performed: in 15 anesthetized dogs the circumflex coronary artery was cannulated and perfused with above-normal constant pressure to that coronary venous oxygen tension never fell below 40 mmHg. Thus, it was possible to eliminate the influence of the metabolic factor regulating coronary resistance. Furthermore, 15 isolated isovolumetrically working guinea-pig hearts were perfused according to the Langendorff method. Stimulation, resp. blockade, of alpha-receptors was achieved by administering xylometazoline, resp. phentolamine. Xylometazoline increased coronary resistance in both the isolated and the in-situ heart. Administering maximum doses to the anesthetized dog led to an increase in resistance to about 200%. This is equivalent to a reduction of conductance to about 50%. Phentolamine produced no significant effects in the isolated heart. Maximum dosage administered to the heart in situ led to a resistance decrease to about 60%, equivalent to an elevation of conductance to about 170%. If we let control values of coronary resistance and conductance be equal to 100%, our experiments showed alpha-adrenergic regulation of coronary resistance to range from about 60% to 200% and conductance to range from about 50% to 170%.

Adrenaline and the regulation of acetyl-coenzyme A carboxylase in rat epididymal adipose tissue. Inactivation of the enzyme is associated with phosphorylation and can be reversed on dephosphorylation.

1. Exposure of rat epididymal fat-pads or isolated fat-cells to adrenaline results in a decrease in acetyl-CoA carboxylase activity measured both in initial extracts and in extracts incubated with potassium citrate; in addition the concentration of citrate required to give half-maximal activation may also be increased. 2. Incorporation of 32Pi into acetyl-CoA carboxylase within intact fat-cells was investigated and evidence is presented that adrenaline increases the extent of phosphorylation of the enzyme. 3. Dephosphorylation of 32P-labelled acetyl-CoA carboxylase was studied in cell extracts. The rate of release of 32P is increased by 5mM-MgCl2 plus 10--100 microM-Ca2+, whereas it is inhibited by the presence of bivalent metal ion chelators such as EDTA and citrate. 4. The effects of adrenaline on the kinetic properties of acetyl-CoA carboxylase disappear if pad or cell extracts are treated with Mg2+ and Ca2+ under conditions that also lead to dephosphorylation of the enzyme. 5. The results of this study represent convincing evidence that adrenaline inactivates acetyl-CoA carboxylase in adipose-tissue preparations by increasing the degree of phosphorylation of the enzyme.

[Evaluation of the pituitary reserve of gonadotropins and seminal function of the testis in subjects operated for cryptorchism].

76 patients (prepuberal, puberal and adults) who had undergone surgery for monolateral (35) or bilateral (41) cryptorchidism in childhood were studied. Testicular volume (76 cases), seminiferal function (18 cases) and pituitary gonadotropin reserve (51 cases) were evaluated. We obtained the following results: 1) the prepuberal patients had a normal testicular volume, while 70% of the puberal and adult patients had a mean testicular volume below normal levels. 2) 55.6% of the adults who underwent spermiogram had a pathological seminiferal function. 3) The number of patients whith exagerated gonadotropin response to GnRH-test increases with increasing puberal stage and reaches its highest significance after complete puberal development. These data confirm that: 1) the long permanence of one or both testis out of their natural position has a negative influence on their trophism; 2) the long-term prognosis of the tubular function of the testis after orchidopessis is poor in a high percentage of cases. 3) the endocrine anomalies which follow the early morphologic and functional changes of the cryptorchid testis are more easily detected during puberty as a reduced hypothalamic feedback of the gonadotropin secretion.

Arylsulphatase A and B in juvenile metachromatic leukodystrophy.

A series of five living patients with juvenile metachromatic leukodystrophy (MLD), ten first-degree relatives, and a number of controls were subjected to biochemical investigations including quantitative determination of arylsulphatase A (ASA) and B (ASB) activities in peripheral leukocytes and polyacrylamide disc gel elctrophoresis of arylsulphatases. Five relatives were family members of four previously deceased patients with juvenile MLD. The mean ASA activity of the patients was 1.3 nmol of p-nitrocatechol sulphate hydrolysed in 30 min per mg protein. It was 84 nmol in the relatives, 129 nmol in other neurological patients and 136 nmol in normal controls. The corresponding ASB activity was 38 nmol in the patients, 49 nmol in the relatives, and 99 nmol in normal controls. An extremely low ASB activity, 3.4 nmol, was found in one relative. No ASA band could be visualised in the enzyme electrophoretic patterns of the patients' leukocytes but the bands representing ASB appeared normal. Seven relatives showed ASA bands weaker than normal, and the relative with low ASB activity exhibited very weak ASB band. The low ASB activity in the patients and heterozygotes may be a characteristic feature of the slowly progressive juvenile type MLD diagnosed in the present series.

Biotransformation of furosemide in patients with acute pulmonary edema.

Furosemide (20-80 mg) was administered iv over 5 min to 16 patients with the diagnosis of acute pulmonary edema due to left heart failure. Serum and urine samples collected during the 24 hr after administration were assayed for furosemide and its biotransformation products by gas-liquid chromatography. A biexponential decay of serum furosemide concentrations vs. time was observed. Recovery of furosemide and its metabolites from urine in 24 hr varied between 30 and 98% of the administered dose. The excretion of unchanged drug accounted for 22.6-73.4% of the dose. The excretion of the glucuronide metabolite and 2-amino-4-chloro-5-sulfamoylanthranilic acid accounted for 3.3-40.4% and from 0.13-3.92% of the dose, respectively. Urinary excretion of furosemide was less in patients with, than in those without, myocardial infarction. Urinary excretion of the oxidative acidic metabolite was increased in patients with reduced creatinine clearance. The glucuronide metabolite of furosemide was the major biotransformation product in these patients with acute pulmonary edema.

Altered activation of tyrosine hydroxylation by gamma-butyrolactone following chronic ethanol treatment.

The results from the present study support the contention that chronic ethanol administration alters the activity of central dopamine neurons. Chronic ethanol administration was shown to disrupt normally occurring feedback mechanisms involved in the regulation of tyrosine hydroxylation.

Dopamine neurons: effect of lergotrile on unit activity and transmitter synthesis.

Intravenous administration of increasing doses of the ergoline, lergotrile mesylate, caused a rapid, dose-dependent and haloperidol-reversible inhibition of unit activity of dopamine cells in the pars compacta of the rat substantia nigra. 6 microgram/kg caused significant depression of dopamine cell firing rates; 50% inhibition was achieved with a cumulative dose of 100 microgram/kg. 60% of the cells were completely inhibited by increasing doses of the drug, the remainder became resistant to further inhibition after reaching rates 25--50% of baseline. Pretreatment with reserpine and alpha-methylparatyrosine did not significantly attenuate the lergotrile-induced inhibition. Lergotrile had no consistent effect on firing rates of cells in the pars reticulata of the substantia nigra. The ergoline reduced the apparent activation of striatal dopamine synthesis associated with complete cessation of impulse flow in nigral-striatal dopamine neurons induced by gamma-butyrolactone treatment. These effects are compared with those of apomorphine and amphetamine. The results are consistent with the idea that lergotrile is a direct acting dopamine agonist.

Pepsin 5 in gastric juice: determination and relationship to the alkali-stable peptic activity.

Pure human pepsins 1 and 3 are inactivated by incubation at pH 7.1-7.3 for 30 minutes, losing 90% or more of activity. Pepsin 5 is alkali-stable, retaining 100% of activity. Mixtures of pure pepsins 1 and/or 3 with pepsin 5 were found to have greater alkali-stable activity than predicted. Two published methods for determining the alkali-stable fraction of the peptic activity of gastric juice gave, respectively, in our hands values of 45.4-80.0% and 27.5-43.9% of the total activity. These values seemed too high to be attributable only to pepsin 5 in gastric juice, as agar gel electrophoresis shows pepsin 3 to have the principal activity. Electrophoretograms of alkaline incubated gastric juice revealed that large amounts of pepsin 3 retained activity as well as pepsin 5, and a proteolytic zone "4" appeared between them. Alkali inactivation thus does not allow the estimation of pepsin 5 individually in gastric juice. Pepstatin, at a final concentration of 100 to 170 pmol/ml, may be used to estimate pepsin 5 in gastric juice and gave values of 18.0 to 27.6% of the total peptic activity. Pepsin 5, in gastric juice and in mixtures of pepsins, appears to protect pepsin 3 from alkaline-inactivation, and to a lesser extent from pepstatin inhibition.

Phenotypic stability of the cell wall of Streptococcus mutans Ingbritt grown under various conditions.

Quantitative analyses of cell walls from Streptococcus mutans Ingbritt grown under carbohydrate limitation in the chemostat showed that growth conditions had no statistically significant effect on the composition of polysaccharide, peptidoglycan, or the proportion of polysaccharide in the cell wall. Lysis of cell wall preparations with a muramidase supported this conclusion and further indicated that there was little difference in their overall structure. In contrast, there was a consistent difference between the rates of lysis by this enzyme of organisms grown in 0.2% glucose and 0.5% glucose. Extremes of pH or dilution rate essentially did not influence the immunogenicity of type c antigen in whole organisms irrespective of whether the carbohydrate source was glucose or sucrose. However, differences were found in the immunogenicity of lipoteichoic acid under similar circumstances. The results indicated there was an inherent phenotypic stability in the cell walls of S. mutans Ingbritt despite changes in pH, generation time, and carbohydrate source, and that any changes that did occur were probably due to associated cell-surface components.

Effect of growth rate and glucose concentration on the biochemical properties of Streptococcus mutans Ingbritt in continuous culture.

A comparison was made of the properties of Streptococcus mutans Ingbritt grown in continuous culture under conditions of excess glucose (nitrogen limitation) and limiting glucose at mean generation times of 1.7 to 14 h. Only low levels of glucoamylase-specific glycogen were formed in cells from either culture, and the total carbohydrate content of the cells under excess glucose was only at most 1.6-fold higher than in the glucose-limited culture. A negligible amount of cell-free polysaccharide was formed in either culture, although a significant level of glucosyltransferase activity was observed in both, with the highest activity at D = 0.2 and 0.4 h(-1) with a glucose limitation. Other differences were observed. (i) Lactate was the main end product of the glucose-excess culture, whereas acetate, formate, and ethanol were the main products of the glucose-limited culture except at a mean generation time of 1.5, when lactate represented 30% of the products. (ii) The yield (in grams per mole of glucose) of the latter culture was 2.6- to 4.0- fold higher than the yield of the glucose-excess culture. (iii) Washed cells from the glucose-limited culture were much more acidogenic (1.7- to 6.2-fold) than the glucose-excess cells when incubated with glucose, sucrose, and fructose. Endogenous glycolytic activity by the latter cells was significant, being 31 to 92% of the exogenous glucose rate at the four dilution rates. (iv) Cells from the glucose-excess culture were more insensitive to fluoride than cells from the glucose-limited culture. The NaF 50% inhibition dose values for the effect of fluoride on the metabolism of glucose, sucrose, and fructose were calculated for the four dilution rates at four pH values. This analysis indicated that rapidly metabolizing cells were more sensitive to fluoride than cells that metabolized the sugars more slowly.

Acidic degredation of cephaloglycin and high performance liquid chromatographic determination of deacetylcephaloglycin in human urine.

In order to provide fundamental knowledge about the determination of deacetylcephaloglycin excreted in human urine as an active metabolite of cephaloglycin, the degradation of cephaloglycin in acidic media has been investigated with varying reaction temperature between 30 degrees and 50 degrees C and pH between 1.2 and 2.8. The degradation pathway observed under these conditions was the elimination of the 3-acetyl group yielding deacetylcephaloglycin followed by formation of deacetylcephaloglycin lactone. Estimation of first order rate constants revealed that deacetylation is the rate-determining step for the degradation of cephaloglycin to the lactone. It is found from the kinetic results that reproducible assays of deacetylcephaloglycin excreted in urine can be achieved by a quantitative conversion to deacetylcephaloglycin lactone in a medium of pH 1.4 at 37 degrees C for 2 hours, followed by a reversed-phase ion-pair high-performance liquid chromatography. The utility of the present method is demonstrated by determining the time course of urinary excretion of deacetylcephaloglycin after oral administration of cephaloglycin capsule.

Experimental otitis media in chinchillas following nasal colonization with type 7F Streptococcus pneumoniae: prevention after vaccination with pneumococcal capsular polysaccharide.

Chinchillas were colonized intranasally with type 7F Streptococcus pneumoniae, and pneumococcal otitis media developed in greater than 50% of the animals during the first week after negative middle ear pressure (-25 mm Hg) was briefly applied. Twenty-three chinchillas were vaccinated subcutaneously with the capsular polysaccharde of type 7F S. pneumoniae to determine whether vaccination could prevent the development of experimental otitis media. Following vaccination, 14 animals seroconverted with at least a twofold rise in serum antibody concentration; nine animals that were vaccinated did not seroconvert. All of 23 vaccinated animals and 42 of 42 unvaccinated control animals became colonized after intranasal inoculation with pneumococci. Only one (7%) of the vaccinated seroconverting animals developed pneumococcal otitis media, whereas 26 (62%) of the control animals developed middle ear infection with type 7F pneumococci. Four (44%) of nine vaccinated nonseroconverting animals developed pneumococcal otitis media. Protection was associated with high levels of serum antibody prior to intranasal inoculation. Higher antibody levels were found in sterile middle ear effusions than in S. pneumoniae-infected effusions. Vaccination with type 7F pneumococcal capsular polysaccharide significantly reduced the incidence of pneumococcal otitis media following intranasal inoculation of type 7F S. pneumoniae in chinchillas.

Release of [3H]noradrenaline induced by 5-hydroxytryptamine from cat pial arteries.

Pial arteries of cats were used to analyse the effects of 5-hydroxytryptamine (5-HT) on the release of [3H]noradrenaline. To achieve this the vessels were preincubated with [3H]noradrenaline and the effect of different concentrations of 5-HT (10(-6), 10(-5), 10(-4) M) on the release of tritium was studied. 5-HT elicited release of radioactivity in a dose-dependent manner. Removal of both superior cervical sympathetic ganglia 15 days before the experiment of pretreatment of the animals with reserpine (3 mg kg-1, total dose) produced a significant decrease in the outflow of tritium induced by 5-HT. In these arteries, the amount of radioactivity retained at the end of the experiment was much diminished. Cocaine (10(-6) M) caused a significant decrease in the tritium efflux induced by 5-HT (1"0(-5) M). These results show that 5-HT has an indirect adrenergic effect in the pial arteries of the cat only at high doses of 5-HT, and confirm that sympathetic innervation of these vessels mainly comes from the superior cervical ganglia.

Plasma levels and beta-blocking effect of alpha-hydroxymetoprolol--metabolite of metoprolol--in the dog.

The plasma levels and the beta-blocking effect of metoprolol and its active metabolite alpha-hydroxymetoprolol have been studied after i.v. bolus injections of the substances to dogs. For both substances the beta-blockade increased with the dose, and there was a linear relationship between percent reduction in exercise heart rate and the logarithm of plasma concentration. The dose of the metabolite, however, had to be 5 times higher than that of metoprolol to induce the same degree of beta-blockade. Because of differences in the volume of distribution, 2.0 liters/kg for alpha-OH-metoprolol and 3.5 liters/kg for metoprolol, the 5 times higher dose of alpha-OH-metoprolol resulted in 10 times higher plasma levels of the metabolite than of metoprolol. alpha-OH-Metoprolol was more slowly eliminated (t1/2 approximately 7.0 hr, total body clearance approximately 3.5 ml-kg-1-min-1) than metoprolol (t 1/2 approximately 2.0 hr, total body clearance approximately 20.0 ml-kg-1-min-1). Approximately 5% of an i.v. dose of metoprolol was metabolized to alpha-OH-metoprolol. The half-life of the endogenously formed metabolite was the same as after an i.v. dose of the compound.

[Choledochal obstruction due to Fasciola hepatica (author's transl)].

A 45-year-old woman was admitted in July, 1976 with an acute cholecystitis without jaundice. She had suffered from hepatic colic without fever, jaundice, diarrhea or allergic episodes for the past 8 years. The physical examination only revealed an elective pain on the cystic point. Laboratory data were unremarkable, except for a 12 percent eosinophils. The cholecystogram showed a cholelithiasis. The lithiasis was confirmed during the surgical operation and a fasciolasis was diagnosed after one and 10-12 parasites had been found into the cystic and common bile duct, respectively. A cholecistectomy and choledochoduodenostomy were performed. The patient was treated with 60 mg dehydroemetine during 10 days and 500 mg chloroquine during the other next 10 days. Eggs of Fasciola hepatica were found in the stool culture. The follow-up examinations 3 months and a year after surgery were completely normal. The national literature on this topic is reviewed and the clinical manifestations and therapy of this disease are commented on.

[Effect of cultivation conditions on growth of Candida lipolytica yeasts and alpha-ketoacid biosynthesis in the presence of thiamine deficiency].

The effect of pH and aeration on the growth of Candida lipolytica and the biosynthesis of alpha-keto acids on acetate and glucose was studied in batch cultures at thiamine deficiency. If the initial thamine concentration was the same, then, irrespective of the carbon source, the yeast biomass in a medium saturated with oxygen by 5--10% was 1.5--2.0 times higher than in a medium with 60--90% [O2]. The rate of alpha-keto acid biosynthesis, on the contrary, decreased in the conditions of low aeration. The biomass increased and the rate of acid production decreased when pH was changed from 6.0 to 8.0 in a medium with acetate. However, at all studied values of pH and [O2] in a medium with either acetate or glucose, the growth at the deceleration phase was of a linear character, and was accompanied with the accumulation of alpha-keto acids in the cultural broth. The rate of acid production was maximal when the specific growth rate decreased to 0.04--0.06 hr-1. The presence of a linear phase in the conditions of thiamine deficiency suggests that here the growth of yeasts is determined by the constant activity of one of the thiamine dependent enzymes per unit volume of the cultural broth. However, the value of this activity seems to change depending on the cultivation conditions which, in turn, causes changes in the rates of biosynthetic processes.

"Classical" and "atypical" antipsychotic drugs: differential antagonism of amphetamine- and apomorphine-induced alterations of spontaneous neuronal activity in the neostriatum and nucleus accumbens.

The ability of clozapine and haloperidol to antagonize the depression of firing rate produced by d-amphetamine and apomorphine in the neostriatum and nucleus accumbens was tested in immobilized, locally anesthetized rats. In the neostriatum, an intraperitoneal injection of 2.5 mg/kg d-amphetamine or 1.0 mg/kg apomorphine produced a prolonged inhibition of neuronal activity that was reversed by a subjsequent injection of either 20 mg/kg clozapine or 2.0 mg/kg haloperidol. An analysis of the onset and magnitude of the blockade revealed that clozapine was more effective than haloperidol in reversing the amphetamine response but that both antipsychotic drugs produced a comparable blockade of the apomorphine-induced depression. Similar results were obtained in the nucleus accumbens. The data indicate that although clozapine acts equieffectively in the neostriatum and nucleus accumbens, this atypical antipsychotic drug, aside from blocking postsynaptic dopamine receptors, may exert at least some of its effects by preventing dopamine release.

Epidemiology for geochemists.

Epidemiologists study the distribution and the determinants of disease in human populations. Geochemists may be more concerned to find diseases to fit the observed patterns of geochemistry. This paper is concerned with the application of epidemiological techniques to the interrelations between health, disease and geochemistry, with particular reference to the hazards of man-made chemicals in the environment. Descriptive studies of disease in terms of person, place and time allow for crude comparison with the results of geochemical mapping and for the development of hypotheses. Analytical studies allow for the exploration of these hypotheses but demand careful sampling techniques and vigorous quality control. The epidemiological approach should be directed towards the identification of national/regional problems and of high-risk groups, the definition of priorities and the opportunities for preventative measures. The problems and possibilities for epidemiological research are illustrated from recent and current studies.

Geochemistry and pollution.

During the past 7 years, the National Science Foundation-Research Applied to National Needs (R.A.N.N.) programme has supported extensive interdisciplinary research concerned with Pb, Cd and other hazardous trace metals. Various aspects of geochemistry and pollution research at the universities of Missouri, Illinois, Colorado State and Purdue are presented and summarized. The transport, pathways and distribution of Pb, Cd and other trace metals are discussed and the utilization of research findings by government and state agencies for the development of standards and by industries for pollution control are presented.

Trace elements in the atmosphere.

The distribution and behaviour of particulate trace elements in the atmosphere have been studied by continuous measurements for 5 years at seven non-urban sites in the United Kingdom. Samples have been taken regularly of airborne dust, rainwater and dry deposition: these have been analysed for up to 36 elements. Concentrations of trace elements vary considerably between sites but the relative concentrations are among uniform: this suggests similarity of origin or good atmospheric mixing. By comparing the relative concentrations with those in soil it is possible to differentiate between trace elements that are derived from soil and those that may be attributed to industrial activity. This classification is supported by estimates of the particle sizes in air. The deposition of trace elements can be related to the concentrations presnet in soil and to the annual removal by crops. Retrospective analyses of stored samples from one site describe the history of trace element concentrations in air since 1957. The sea surface is considered as a possible source of atmospheric trace elements.

Regional geochemical mapping.

One of the prime requirements for effective study of environmental geochemistry in relation to health is the production of multi-element atlases showing the distribution of the elements on the regional scale. The choice of method for compiling such atlases can vary according to a number of geological, environmental and other factors. The overriding consideration, however, is to assist (in conjunction with other relevant sources of information) in defining, quickly and cheaply, potential problem areas wherein to concentrate more detailed studies to ensure maximum return from the funds and scientific manpower available. Numerous sampling and analytical techniques have been employed. Each technique and approach has its own scope, limitation and problems of interpretation. Whatever method is chosen, the use of computer-based statistical data reduction, analysis and map compilation is mandatory. Although it was apparent more than 20 years ago that geochemical atlases would eventually become a national cartographic requirement, regional geochemical mapping is still in the experimental stage. This trend is now evident in activity in a number of countries. The methods being employed, however, are so diverse that there is an urgent need for international collaboration aimed at securing data that are as mutually compatible as possible, having regard to the conditions, needs and resources of the individual countries involved.

Single- and multiple-dose kinetics of estazolam, a triazolo benzodiazepine.

The pharmacokinetic properties of estazolam, a triazolo benzodiazepine hypnotic agent, were assessed in a series of healthy volunteers following single and multiple doses. After single oral doses of 2--16 mg, peak plasma concentrations were reached within 6 h. Values of elimination half-life ranged from 8.3--31.2 h (mean 17.0 h) and did not vary significantly with dose. During 3 weeks of therapy, steady-state plasma concentrations increased approximately in proportion to increasing doses. and accumulation was essentially complete within 3 days of each dose change. The mean observed accumulation ratio was 1.84, which was slightly larger than the predicted ratio of 1.53. Exposure to multiple-dose estazolam therapy had no significant influence on the kinetics of a single dose of antipyrine, suggesting that estazolam neither stimulates nor inhibits enzyme activity in humans. Thus the accumulation and elimination kinetics of estazolam can be classified as intermediate to those of the short-acting (such as oxazepam) and the long-acting (such as diazepam) benzodiazepine derivatives.

Removal of endogenous ligands from a high-affinity antiserum for radioimmunoassay.

A method for removal of endogenous ligands from high-affinity antisera (stripping) is described. A thyroxine antiserum of very high affinity was used to develop the method. The antiserum was incubated at 50 degrees C in a glutamate buffer at pH 4.4 together with some ethanol and methyl cellulose and a large amount of activated charcoal. After incubation for up to 2 days, the stripped antibodies were separated from the ligand adsorbed to the charcoal by centrifugation. The estimated titre increased several fold by the stripping, although the stripping method caused a loss of about 13% of the total number of binding sites over a period of 2 days. When stripped antiserum was used instead of unstripped antiserum in an assay system, the sensitivity was up to 3 times better, and the concentration, which could be measured with the best relative precision, was 3 times lower. The stripped antiserum showed a poorer specificity than the unstripped antiserum when a short incubation period was used. However, when a long incubation period was used, the specificity was nearly the same.

Effects of deferoxamine methanesulfonate on Trichophyton mentagrophytes.

Deferoxamine methanesulfonate (Desferal), an iron chelator, inhibited germ tube formation and growth of Trichophyton mentagrophytes in a microculture assay. A 50% reduction of germ tube formation required Desferal at 5 mg/ml and a 50% reduction of growth required 1.5 mg/ml. Growth was almost completely inhibited with 50 and 100 mg/ml. Also, Desferal at 100 mg/ml inhibited further elongation when added to short hyphae (II and 21 micrometer), but showed less inhibitory effects when added to long hyphae (64 micrometer). Iron (133 microgram/ml) reversed the inhibition of growth produced by incubating spores with Desferal at 5 mg/ml, providing iron was added before 72 h incubation. Desferal at 100 mg/ml decreased viability of activated spores incubated for 3 days at 30 degrees C, but did not decrease viability of spores incubated for 3 days at 4 degrees C. The growth inhibitory effect of Desferal and transferrin were compared. Transferrin was inhibitory at low molarities (0.001 to 1.0 mM), while Desferal was inhibitory only at higher molarities (greater than 1 mM). Desferal (0.05 mM) also reversed the inhibition expected with 0.05 mM transferrin. These findings indicate that Desferal and transferrin deprive T. mentagrophytes of nutritional iron and thus inhibit growth of the fungus. Low concentrations of Desferal can also promote growth in the presence of transferrin.

[The behavior of sensory and ganglia cells of the vestibular apparatus of the guinea pig in response to inadequate stimulations].

The behaviour of the sensory- and the ganglia cells in response to inadequate stimuli was studied at 12 adult guinea pigs. The sensory cells of the three Cristae ampullares dont respond remarkably to acoustic stimulation. At the range of 1000 Hz a recognizable increase in the size of the sensory cells at the upper ampulle was noticed. Of no account was the rest of the volume variations. At the range of 5000 Hz the sensory cells of the Maculae staticae responded with a considerable increase in the size of their sensory nuclei up to 38%. The cells of the Ganglion vestibulare react upon acoustic stimulation throughout with an increase in their nuclei up to between 16% and 54%. It was noticed that the volume enlargement increases from the lower to the higher frequencys. The cells of the Ganglion geniculi nervi facialis react upon acoustic impulses too with an increase in their nuclear volume. In low tone acoustic range and in high tone acoustic range the growth rates are more than 50% beyond the standard. Thos cells respond to frequency of 1000 Hz remarkably intensive. It was noticed that the maximum volume increase of their nuclei was in the average up to 103%. All the sensory and ganglia cells react upon rinsing the external auditory canal with 12 degrees C water, with a shrinkage of their nuclei. Besides that the sensory cells of the Cristae and the Maculae show a volume decrease between 8% and 19%. The cells of the Ganglion vestibulare et geniculi respond with a considerable volume decrease of their nuclei from 32 to 41% under the average.

[Possibilities and problems in the rehabilitation of aged patients with mental illness (author's transl)].

In the Federal Republic of Germany as in other Western Countries institutions suitable for the rehabilitation of aged patients with mental illness are lacking. As long as "psychiatry of the higher age" is synonymous to "psychiatry of demented patients" the term "rehabilitation" means only a slogan, not a maxime. Jurisdiction in the Supreme Court though, has shown the way of going on. It will depend also on the attitude of the doctors in charge of the patients to fulfill the expectations posed in that jurisdiction and to reach so the far set marks in the interest of the patients. Big institutional deficits have to be ameliorated as far as possible; of prime importance here is the necessity to place the opportunities for the employment of qualified employees. Rehabilitation instead of custodial care here is synonymous for efficiency and humanity. The change from an obsolete and nonorganized "system" of care to a modern network of rehabilitation should be obtained by voluntary cooperation and by voluntary coordination of medical and social institutions. So an integration of all possibilities of help to a network could be reached.

Effects of copper on the sabellid polychaete, Eudistylia vancouveri: I. Concentration limits for copper accumulation.

Three experiments with a sabellid polychaete (Eudistylia vancouveri) show the threshold concentration for increasing copper accumulation with time to lie between 3 and 6 micrograms/L total copper in seawater during winter conditions. The branchial crown, probably the major absorptive site, concentrated more copper than the body. Accumulation was influenced by size but not by sex. Our studies indicate that the body burden of copper will increase above natural levels in areas of industrial discharge where copper levels are above the threshold limit for accumulation.

Structural studies of a branchiopod crustacean (Lepidurus bilobatus) extracellular hemoglobin. Evidence for oxygen-binding domains.

The extracellular hemoglobin of the notostracan branchiopod Lepidurus bilobatus has an apparent molecular weight of 680,000 and may exist in a dissociation-association equilibrium dependent on pH and ligand state. The pigment contains one heme per 18,000 g protein. However, attempts to dissociate the hemoglobin by harsh denaturing conditions results in a 33-34,000 molecular weight polypeptide chain as well as traces of some 62-64,000 molecular weight material. Limited proteolysis of this hemoglobin with subtilisin produces 14,800 and 16,500 dalton heme-containing polypeptides (domains) which bind oxygen reversibly. These domains, isolated by column chromatography, have a heme content similar to the intact pigment. It is proposed that the intact 34,000 dalton subunit of Lepidurus hemoglobin consists of two linearly linked oxygen binding domains. Oxygen binding properties of the intact hemoglobin show a low oxygen affinity with a slight Bohr effect. In contrast, the isolated domains display a relatively high oxygen affinity and lack a Bohr effect between pH 7.0 and 8.0. It is apparent that the intact 34,000 dalton polypeptide is necessary for the expression of the heterotropic interactions of the native pigment.

Asparagine metabolism and asparaginase activity in a euryhaline Chlamydomonas species.

A Chlamydomonas species isolated from a marine environment possesses an L-asparaginase, an enzyme not yet reported in the microalgae. This enzyme enabled the organism to grow as well with asparagine as sole nitrogen source as with inorganic nitrogen sources (NO3-, NH4+). Only the amide nitrogen was used for growth since growth did not occur on aspartate and aspartate accumulated in the media when cells were either grown on asparagine or during short-term incubations with L-[U-14C]asparagine. Cells grown on NO3-, NH4+, or L-asparagine in batch culture possessed equivalent asparaginase activities. However, nitrogen-limited cells possessed four times the activity of cells grown with sufficient nitrogen for normal growth, regardless of the possessed the lowest activity per cell, while lag phase and stationary phase cells possessed greater activity. The enzyme behaved like a periplasmic space enzyme since (1) breaking the cells did not release into solution more activity than was shown by whole cells and (2) whole cells converted L-[U-14C]asparagine to [14C]aspartate with little intracellular accumulation of radioactivity. Cell-free preparations of the enzyme possessed a Km value for asparagine of 1.1 x 10-4 M, with no glutaminase activity.

Brain regional levels of neurotransmitter amines as neurochemical correlates of sex-specific ontogenesis in the rat.

Brain regional levels of three neurotransmitter amines - serotonin (5-HT), norepinephrine (NE), dopamine (DA) - were measured in young rats prior to weaning to determine the extent to which modifications in levels of amines might reflect alterations in the sex steroid hormonal environment during the first postnatal week in the life of the rat. Sex-related levels of DA, NE, and 5-HT were found in some brain regions of the 12-day-old rat. Male midbrain DA exceeded the corresponding female value while female hypothalamic NE levels were greater than those of the male. Levels of 5-HT in the corpus striatum and the midbrain of males were greater than those of the female. Castration of the male on day 1 or testosterone propionate (TP) administration to the newborn female resulted in modifications of levels of midbrain 5-HT which reflected feminization of the castrated males and masculinization of the TP-treated females. Castration on day 1, or diethylstilbestrol given on days 2, 4, and 6, resulted in apparent feminization of NE levels in the hypothalamus of 12-day-old male rats. Thus, it appears that regional levels of hypothalamic NE and midbrain 5-HT in the 12-day-old rat may reflect the course of brain organizational activity which becomes recognizable in the adult as sex-specific behavior.

[Pharmacological properties of procaterol, a newly synthetized, specific beta 2-adrenoceptor stimulant. Part I. Effects on the CNS (author's transl)].

Effects of procaterol (PRO) on the CNS were investigated in comparison with those of salbutamol (SAL) and isoproterenol (ISO). PRO, 15 to 50 mg/kg given subcutaneously suppressed spontaneous movement in mice, rats and rabbits and with a large dose, 1000 mg/kg, the animals became quiet and immobile. In dogs, PRO produced similar symptoms and in addition, there was nausea and vomiting. The animals recovered within 3--8 hours. ID50's in depressing spontaneous movement were 20.2 and 245 mg/kg for PRO, 51.1 and 133 mg/kg for SAL and 2.37 and 143 mg/kg for ISO, respectively, both by the subcutaneous and oral routes of administration. Methamphetamine induced increase in motility and fighting behavior was also suppressed by PRO when similar doses were given. PRO had no effect on coordinating movement, halothane anesthesia, drug and electric stimulation induced convulsions and body temperature, and there was no muscle relaxant action. However, PRO in large doses prolonged sleeping time with hexobarbital. The analgesic effect of PRO was not observed with Haffner's and Landall Selitto's methods but acetic acid induced writhing was suppressed by PRO. PRO had little effect on spontaneous EEGs either cortical or from deep structures, and EEG arousal responses. The effects of PRO on the CNS were slight and nonspecific, and similar to those of SAL and ISO.

Central chemical regulation of respiration in term newborn.

The role of the medullary H+-sensitive chemoreceptors on the drive of breathing was studied in 10 unanesthetized newborn animals (8 lambs and 2 kids). The experiment consisted of sequential measurements of ventilation (VE) during a progressive change in the arterial pH (pHa) and in the pH of the cisternal cerebrospinal fluid (pHCSF), induced by intravenous infusion of hydrochloric acid (HCl) followed after an 8-h steady state of acidosis by rapid bicarbonate [HCO3-] infusion. It is shown that a rapid change in [HCO3-]CSF occurs during the infusion of HCl or NaHCO3. As a consequence both CSF and arterial pH change in the the same direction and large changes in pHCSF (from 7.331 to 7.227) were observed. Such CSF acidosis did not contribute to further increase VE beyond the level by hyperventilation induced by the initial fall of pHa. The ventilatory response to the decrease in pHa was found to fall off with moderate to severe acidosis (pHa less than 7.20). In conclusion, this study demonstrates an instability of the pHCSF during neonatal metabolic acidosis and it suggests an immaturity of both the H+-sensitive medullary and peripheral chemoreceptors in the 8-day-old newborns.

Brain protease activity after experimental head injury.

In an experimental series on twelve cats, activity changes of brain cell proteolytic activity were measured two hours after a blunt head injury without hematoma or contusions. Protease activity was estimated in two different brain tissue homogenate supernatants containing total soluble and only cytoplasmic activity without proteases in cell organelles, respectively. Total activity was doubled two hours after injury in the acid and the neural pH-range, in comparison to control values. Free soluble activity was doubled in the acid and increased to the threefold value in the neutral range. From these data, it seems that two different changes appear in lysosomes, the enzyme-reservoir of the cell: (1) Enzyme-synthesis is increased after trauma, measured as augmentation of total soluble protease activity in our experiments; (2) Breakage or increased permeability of lysosomes lead to augmentation of especially neutral proteases in the cytoplasm followed by the well known autolytic areas of generalized traumatic brain edema.

Secretion of hexosaminidase isozymes by Tetrahymena.

Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A1 and B1, and 2 minor forms, A2 and B2, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B1 has a molecular weight of approximately 93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase B1 has a molecular weight of approximately 93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A1 has a molecular weight of approximately 170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-beta-D-galactosaminide than the B forms. Neither hexosaminidases A1 or B1 has any endo-beta-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete approximately 20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxybenzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.

Effects of several beta-blocking agents on the development of hypertension in spontaneously hypertensive rats.

Antihypertensive effects of chronic oral administration of adrenergic beta-blocking agents were assessed in SHR. Propranolol, pindolol, oxprenolol, atenolol and labetalol were used as beta-blockers and the effects of these compounds on the blood pressure and the heart rate were compared with those of hydralazine, a representative vasodilating antihypertensive agent. Propranolol, oxprenolol and atenolol produced a definite decrease in the heart rate; the development of hypertension was retarded. Pindolol produced antihypertensive effects only after a longer period of administration and such were associated with insignificant decrease in heart rate. With a shorter period of administration the drug produced only an insignificant fall of blood pressure with practically no change in the heart rate. With labetalol, a beta-blocker with alpha-blocking action, a fall of blood pressure appeared earlier and was of greater magnitude. Hydralazine produced a definite antihypertensive effect, which appeared immediately after administration and was associated with a tachycardia. In pithed rats, only pindolol produced a definite fall of blood pressure. On the basis of these findings, possible mechanisms of antihypertensive effects of beta-blockers were discussed.

Effects of dimorpholamine on frog sciatic nerve-sartorius preparation.

The present study describes the potentiating effect of dimorpholamine on twitch contraction of skeletal muscle and its mechanism on a cellular basis. Low concentrations (about 2 x 10(-5) g/ml) of dimorpholamine potentiate the twitch contraction of frog sartorius muscle. In relatively high concentrations of 10(-4)--10(-3) g/ml, however, the potentiation was followed by depression. Endplate potential was not affected by the drug. Dimorpholamine depolarized slightly the muscle membrane. The contracture tension vs. membrane potential relationship was hardly affected by the drug in the presence of 2 x 10(-7) M tetrodotoxin. Action potentials recorded from muscle fibers treated with ethylene glycol showed marked and progressive increases in duration during exposure to the drug, and were finally blocked. It was concluded that twitch potentiation caused by dimorpholamine is due to the prolongation of the action potential. A likely molecular mechanism of this drug is discussed in terms of the kinetic model proposed by Adrian et al. (J. Physiol. 208, 607--644, 1970) for the excitable membrane of frog sartorius muscle.

Unimportance of perivascular H+ AND K+ activities for the adjustment of pial arterial diameter during changes of arterial blood pressure in cats.

The role of perivascular H+ and K+ in the adjustment of pial arterial diameter during changes in arterial blood pressure was investigated in chloralose anesthetized cats. Blood pressure was reduced by i.v. mecamylamine or pentolinium and was increased by i.v. hypertensin. Pial arterioles and arteries with a control diameter ranging from 37--218 microns at a spontaneous mean arterial blood pressure of 128 +/- 16 (SD) mm Hg were studied. Vascular diameter as measured by TV image splitting showed the typical reactions, i.e. constriction during increase (up to 200 mm Hg) and dilation during decrease in blood pressure (down to 60 mm Hg). Perivascular H+ and K+ activities were measured using pH microelectrodes (Hinke type) and K+ ion exchanger microelectrodes, respectively. Under control conditions perivascular pH was 7.25 +/- 0.11 (SD) and K+ activity was 2.46 +/- 0.65 (SD) mM, respectively. During changes in blood pressure the vascular reactions of pial arteries were not accompanied by significant alterations in perivascular H+ or K+ activity. From these data it can be concluded that mechanisms other than those which are mediated by H+ or K+ are involved in the adjustment of pial arterial diameter during changes in arterial blood pressure.

Stroke due to vasculitis.

Vasculitis should be suspected as a cause of stroke in younger patients or older patients with an elevated erythrocyte sedimentation rate, especially in the presence of systemic disease and mononeuritis multiplex. Corticosteroids and cyclophosphamide show promise of producing remission.

[Scanning microscopical observations on the foregut structures o mosquitoes and their role for the ingestion of microfilariae (author's transl)].

Experiments on the transmission of Brugia malayi by various mosquitoes had shown that microfilariae ingested by some species were badly damaged when they reached the stomach, but were much less hurt in others. The structures of the foregut likely to cause these injuries, were investigated and documented by scanning microscope techniques. In Anopheles albimanus, A. arabiensis, A. stephensi and A. pharoensis which have well developed armatures the microfilariae showed a high rate of destruction. In A. stroparvus as well as in Aedes aegypti, Ae. togoi and Culex fatigans in which these structures are missing or poorly developed the larvae were much less affected. From the size, shape and position of the different papillae, spines, rods and cones observed it can be concluded and confirmed that the pharyngeal armature (buccopharyngeal bar) will be by far the most important structure responsible for the injuries of the microfilariae. However, it appears that the characteristics of different filaria species can play an important role in preventing such damages.

Immunization against Nippostrongylus brasiliensis in the rat. A study on the use of antigen extracted from adult parasites and the parameters which influence the level of protection.

It was found that protective immunity in excess of 90% reduction in worm burden could be stimulated against Nippostrongylus brasiliensis in rats by using an extract of adult Nippostrongylus worms. The level of protection achieved was influenced by several factors. Thus, the use of Bordetella pertussis as adjuvant significantly increased the level of protection which, in addition, was shown to be influenced by the amount of worm antigen used. Furthermore, antigen administered in multiple doses was more effective than a single inoculum and, when using such a regime, the interval between doses was also found to be critical. The route of antigen administration was important and, while protection was achieved by subcutaneous and oral administration, the intraperitoneal route was the most effective. Using the optimal immunization regime of 3 doses of 5 mg worm protein and 4 x 10(10( B. pertussis organisms, as adjuvant, levels of protective immunity in excess of 90% reduction in worm burden were shown to exist for at least 60 days after the last dose. It was found that adult worm extracts did not stimulate any obvious immunity against larval forms of N. brasiliensis.

[Pathology of endocrine polyneoplasms (polyadenomatoses) (author's transl)].

The term "endocrine polyadenomatoses" includes two types of pathological entities in which there are an association of at least two endocrine tumors having no physiological relationships, and hereditary familial characteristics: Wermer's syndrome (Type I): pancreatic endocrine tumor, pituitary adenoma, and hyperplasia or adenoma of the parathyroids. Sipple' syndrome (Type II): medullary thyroid cancer, one of two pheochromocytomas, and parathyroid hyperplasia. The multifocal character of the pancreatic D-cell lesions in the first type, and the bilateral nature of the thyroid and adrenal lesions in the second type are particular features of each of them. Apart from some parathyroid lesions, for which the origin is still debatable, these endocrine tumors enter into the framework of the apudomes and are derived therefore from the neural crest. The association of other tumoral varieties of the APUD (carcinoid) type with nervous tissue tumors, and with dysmorphic anomalies suggests that these syndromes are the expression of a dysgenesis affecting more or less completely, structures derived from the neural crest.

Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli.

Regulation of p-nitroanisole O-demethylation in perfused rat liver. Adenine nucleotide inhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase.

Perfusion of rat livers with 10 mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70 mg/kg) 6 h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and "malic" enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9 micrometer for "malic" enzyme to 1.85 mM for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (Ki 2.8 mM) and by ADP (Ki 0.9 mM), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes.

Stability of microtubule protein over the pH range: 6.9--9.5.

The pH stability range of a microtubule protein preparation has been investigated between 6.9 and 9.5. Microtubule protein was exposed to various pH values in this range and then returned to pH 6.9. The appearance of microtubules as verified by electron microscopy and sedimentation analysis under polymerizing conditions was taken as an indication of a conformationally stable protein. Between pH 6.9 and pH 8.0 the loss in the ability to form microtubules was found to be reversible, at pH 8.2 it was partially reversible, above pH 8.2 it was irreversible. Tubulin and the microtubule-associated protein fraction were separately exposed to high pH. It was observed that tubulin exposed to high pH can still form microtubules in the presence of untreated microtubule-associated protein. On the other hand, microtubule-associated protein exposed to high pH could not initiate microtubule assembly with untreated tubulin. It was concluded from these observations that the loss in the ability of a microtubule protein preparation to assemble at high pH is due to a change in the microtubule-associated protein fraction and that tubulin is conformationally stable even after exposure to pH 9.5.

The development of inducibility for glutamine synthetase in embryonic neural retina: inhibition by BrdU.

The hydrocortisone-mediated induction of glutamine synthetase (GS) in the neural retina of the chick embryo is a characteristic and unique feature of differentiation of this tissue. The induction involves genomic activity elicited by the inducer resulting in synthesis and accumulation of the enzyme. We describe correlations between the growth of embryonic retina tissue in vivo and in vitro and the development of its inducibility for GS, and demonstrate that this development proceeds through two phases: competence-acquisition phase (before the 7th day of development), and maturation phase. BrdU applied for 24 h to retinas of 5-day embryos irreversibly suppresses the development of induction-competence. However, BrdU does not affect the progressive maturation of inducibility when applied to retinas that already are fully induction-competent (8 days and older). The short treatment with BrdU of 5-day retinas also causes defective histogenesis resulting in drastic malformation of the tissue. The nature of the processes involved in competence-acquisition and in the maturation of inducibility for GS are examined. Possible mechanisms by which BrdU prevents the development of induction-competence for GS in the early embryonic retina and elicits defective histogenesis are discussed.

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

Statistico epidemiological study of changes in the vaginal flora of contraceptive pill users in Alexandria.

A stratified random sample of 1000 women with proportionate allocation according to district of residence was taken from normal females living in Alexandria, Egypt, and attending family planning centers in order to understand social-pathological changes in the vaginal flora of oral contraceptive (OC) users. Cases were examined over 18 months, and all cases were given a combined OC. Bacteriology and pH changes in vaginal flora were determined after 18 months. Results of the bacteriological examination revealed a positive correlation between those having a vaginal discharge and pH above 5, mixed infection, and illiteracy. As the duration of pill use increased, so did the incidence of monilla, staphylococcus aureus, anaerobic streptococci, gram negative bacilli, trichomonas vaginalis, and hemophilus vaginalis, whereas lactobacilli decreased. Duration of pill use also corresponded to increase in vaginal pH. Longer duration of OC use, practice of bad hygiene, and illiteracy were factors associated with an alkaline pH, changed pattern of vaginal flora, and greater susceptibility to infection by staph aureus and E. coli.

Amines and the rat exocrine pancreas: (2) Effects of receptor blockers on turnover of L-5HTP.

The metabolism of L-5HTP by the rat exocrine pancreas, and effects of blockers on the metabolism were studied by fluorescent histochemical and chemical methods. Histochemically, 5-hydroxytryptamine (5-HT) blockers (methyserigide and cyprohepatdine) and dopamine (DA) blockers (haloperidol and sulpiride) produced no apparent changes in fluorescence pictures after injection of L-5HTP. alpha-blockers (phenoxybenzamine and phentolamine) and monoamine oxidase (MAO) inhibitor (iproniazide) produced an increased accumulation of 5-HT fluorescence in the apical regions of acinar cells where the zymogen granules are stored. Chemically, the 5-HT blockers decreased the 5-HT content after injection of L-5HPT. Sulpiride had no effect. Haloperidol decreased the 5-HT content. MAO inhibitor resulted in a vast accumulation of L-dopa: e.g. (1) L-5HTP was more slowly eliminated, and (2) 5-HT blockers produced a decreased content of 5-HT after injection of L-5HTP, in contrast to the finding that DA-blockers produced an incresed content of DA after injection of L-dopa. The mechanism responsible for the differences is discussed in relation to the possible pharmacological effects of L-5HTP and L-dopa on the secretion from the exocrine pancreas of rats.