Genetic control of experience-dependent plasticity in the visual cortex.

Depriving one eye of visual experience during a sensitive period of development results in a shift in ocular dominance (OD) in the primary visual cortex (V1). To assess the heritability of this form of cortical plasticity and identify the responsible gene loci, we studied the influence of monocular deprivation on OD in a large number of recombinant inbred mouse strains derived from mixed C57BL/6J and DBA/2J backgrounds (BXD). The strength of imaged intrinsic signal responses in V1 to visual stimuli was strongly heritable as were various elements of OD plasticity. This has important implications for the use of mice of mixed genetic backgrounds for studying OD plasticity. C57BL/6J showed the most significant shift in OD, while some BXD strains did not show any shift at all. Interestingly, the increase in undeprived ipsilateral eye responses was not correlated to the decrease in deprived contralateral eye responses, suggesting that the size of these components of OD plasticity are not genetically controlled by only a single mechanism. We identified a quantitative trait locus regulating the change in response to the deprived eye. The locus encompasses 13 genes, two of which--Stch and Nrip1--contain missense polymorphisms. The expression levels of Stch and to a lesser extent Nrip1 in whole brain correlate with the trait identifying them as novel candidate plasticity genes.

The effects of inulin supplementation of diets with or without hydrolysed protein sources on digestibility, faecal characteristics, haematology and immunoglobulins in dogs.

Dogs with food allergy are often treated by giving a diet with hydrolysed protein sources. Prebiotics might also be successful in prevention and treatment of allergic disease through their effect on the colonic microflora, analogous to studies on probiotics in allergic children. The present study was set up to investigate the effect of supplementing inulin (IN) to commercial hypoallergenic dog diets on apparent nutrient digestibility, faecal characteristics, haematology and Ig in dogs. Supplementation of 3 % IN did not affect faecal pH, food and water intake and urine production. Compared with the intact protein diet with a limited number of ingredients (L), the diet with a hydrolysed protein source (H) resulted in an increased water intake (P<0.001), which could be due to the osmotic effect of free amino acids. Faeces production was increased by IN due to increased faecal moisture content. Increased faeces production on the H diet was mainly due to a higher DM excretion. Subsequently, the apparent digestibility coefficient (ADC) of DM was lower in the H diet group. A similar result was noted for ADC of diethyl ether extract and crude ash. The ADC of crude protein was higher in the H diet group, whereas IN decreased the ADC of crude protein. Differences in the ADC of crude protein among the different diets disappeared after correction for a higher faecal biomass, except for the dogs fed the L+IN diet. Total faecal IgA concentrations were lower in the H group (P<0.05) because of lower antigenic stimulation of hydrolysed protein, which implies that hydrolysed protein is really hypoallergenic. The present study indicates that the use of hydrolysed protein diets for canine food allergy treatment can affect digestibility and that combination with IN affected apparent protein digestibility but not IgA response.

Association of stress during delivery with increased numbers of nucleated cells and hematopoietic progenitor cells in umbilical cord blood.

OBJECTIVE: Umbilical cord blood can be used as a source of bone marrow repopulating cells for allogeneic stem cell transplantation. Large variations in the frequencies of white blood cells and hematopoietic progenitor cells have been found for umbilical cord blood. These variations may be due in part to specific circumstances during labor and delivery. STUDY DESIGN: In this study we analyzed the relationship between stress factors occurring during parturition and the frequencies of nucleated cells, leukocyte subsets, CD34(+) cells, and hematopoietic progenitor cells, as determined in semisolid medium cultures of umbilical cord blood. RESULTS: We observed that a prolonged first stage of labor resulted in increases in the numbers of nucleated cells, granulocytes, CD34(+) cells, and hematopoietic progenitor cells in umbilical cord blood. Evaluation of parameters that indicate stress of the infant during delivery demonstrated higher numbers of nucleated cells, granulocytes, CD34(+) cells, and hematopoietic progenitor cells in umbilical cord blood from children with lower venous pH. CONCLUSION: Longer duration stress during delivery increased the numbers of nucleated cells, granulocytes, CD34(+) cells, and hematopoietic progenitor cells, possibly by causing mobilization of various cell populations by endogenous cytokines. As long as umbilical cord blood harvesting does not interfere with the delivery, umbilical cord blood collected after stressful deliveries may provide optimal units for hematopoietic stem cell transplantation.

Isolation and characterization of a trypsin from the slipper lobster, Thenus orientalis (Lund).

Trypsin has been isolated and purified from the digestive glands of the slipper lobster, Thenus orientalis. It is a glycoprotein with a molecular mass of approximately 35 kDa as judged by both SDS-PAGE and gel filtration. The N-terminal amino acid sequence has strong homology to crustacean trypsins. This is confirmed by the cross-reaction of crustacean trypsins with antibodies to the T. orientalis enzyme. Despite a 40% identity with the bovine trypsin N-terminal sequence, there was no cross-reaction with the mammalian serine proteases. The optimum kcat and kcat/Km values for N-alpha-benzoylarginine-p-nitroanalide were 0.91 s-1 and 9.7 x 10(3) M-1 s-1, respectively, with this specificity constant being lower than those reported for other crustacean trypsins. Inhibition studies indicated the presence of serine and histidine at the active site and pKa of the catalytic histidine residue was found to be 5.7 in the free enzyme and 4.7 in the Michaelis complex.

Inhibitory mechanism of serpins. Interaction of thrombin with antithrombin and protease nexin 1.

The mechanism for the inhibition of thrombin by the serpins antithrombin and protease nexin 1 has been investigated using several kinetic techniques at pH 7.9 and 37 degrees C with an ionic strength of 0.3 M. Rapid kinetic studies demonstrated that a two-step mechanism for the formation of the stable thrombin-serpin complex applied to both serpins. The inhibition constant for the initial thrombin-antithrombin complex was 265 microM, and the rate constant for the conversion of this complex to the final one was 3.9 s-1; the corresponding values for PN1 were 3.4 microM and 6.0 s-1. By using slow-binding kinetics, it was possible to obtain estimates of the second-order rate constants for the formation of the stable thrombin-serpin complexes (1.2 x 10(4) and 1.5 x 10(6) M-1 s-1 for antithrombin and protease nexin 1, respectively) and the dissociation constants for these complexes (< 1 nM for both serpins). The influence of viscosity on the reactions indicated that the rate of interaction of both serpins with thrombin was diffusion-controlled. Moreover, the results indicated that the initial complex reacted more rapidly to form the stable complex than it dissociated to free enzyme and inhibitor; i.e., the behavior of the serpins was analogous to that of "sticky" substrates. By using the results from slow-binding, viscosity, and rapid kinetic studies, it was possible to set values for all of the rate constants for the interactions of antithrombin and protease nexin 1 with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of acrosin by serpins. A suicide substrate mechanism.

The serpins antithrombin, protease nexin 1, and alpha 1-antitrypsin with a reactive-center arginine (Arg-alpha 1-antitrypsin) were found to inhibit the sperm protease acrosin with varying efficiency. The serpins were titrated against acrosin to determine their specific activity with respect to this enzyme. While antithrombin was fully active against acrosin, more than one molecule of Arg-alpha 1-antitrypsin and protease nexin 1 was required to inhibit one molecule of acrosin. In particular, only 2.7% of protease nexin 1 molecules interacting with acrosin formed stable complexes with the enzyme at 37 degrees C and this value decreased to 0.03% at 12 degrees C. N-terminal sequence analysis indicated that acrosin had cleaved protease nexin 1 at its reactive-center Arg-Ser bond. The results could be interpreted in terms of protease nexin 1 acting as a suicide substrate for acrosin; after the formation of an initial complex, the serpin partitioned between pathways yielding either inactivated (cleaved) serpin or a stable serpin-enzyme complex. The association rate constant (k(ass)) and inhibition constant (Ki) for the stable complexes were determined for each of the serpins by using slow-binding kinetics. The values of k(ass) were 2 x 10(5), 4 x 10(4), and 5 x 10(3) M-1 s-1 for Arg-alpha 1-antitrypsin, antithrombin, and protease nexin 1, respectively. The Ki values for the serpins were 1 nM or less. Heparin markedly accelerated the inhibition of acrosin by antithrombin and protease nexin 1; at the optimal concentration, the degree of heparin acceleration of the inhibition rate was 250- and 500-fold for antithrombin and protease nexin 1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid inhibition of the sperm protease acrosin by protein C inhibitor.

Heparin was found to be an allosteric modulator of the amidolytic activity of the protease acrosin. In the presence of saturating concentrations of heparin, there was a 4.9-fold decrease in the value of the Michaelis constant for the substrate D-Ile-Pro-Arg-p-nitroanilide and the value of kcat was 2.5-fold lower. Analysis of the data yielded a dissociation constant of 0.22 +/- 0.04 microM for the heparin-acrosin complex. The presence of relatively high concentrations of protein C inhibitor in seminal plasma [Laurell, M., Christensson, A., Abrahamson, P., Stenflo, J., & Lilja, H. (1992) J. Clin. Invest. 89, 1094-1101] suggests that this serpin may be involved in the control of the activity of acrosin. Acrosin was found to be rapidly inhibited by protein C inhibitor with the association rate constant (kass) for the formation of the complex being (2.41 +/- 0.03) x 10(5) M-1 s-1. The value of kass showed a bell-shaped dependence on the concentration of heparin; it was maximal at concentrations of heparin between 0.08 and 3 microM and decreased at lower and higher concentrations. At the optimal heparin concentration, the value of kass for the acrosin-protein C inhibitor reaction was 230-fold higher ((5.6 +/- 0.1) x 10(7) M-1 s-1) than in the absence of heparin. The results suggest that protein C inhibitor may be important in the physiological control of acrosin activity, particularly where the presence of heparin-like glycosaminoglycans would stimulate the acrosin-protein C inhibitor reaction.

Interaction of activated protein C with serpins.

The inhibition of activated protein C by six different serine protease inhibitors (serpins) that have arginine residues in the P1 position has been investigated. Micromolar concentrations of C1-inhibitor failed to inhibit the enzyme, and it was inhibited only slowly by antithrombin III with an association rate constant (kass.) of 0.15 M-1.s-1. The kass. values for the other serpins tested (protease nexin I, protein C inhibitor, and mutants of alpha 1-antichymotrypsin and alpha 1-antitrypsin with P1 arginine residues) were at least 1000-fold higher, with P1-Arg-alpha 1-antitrypsin (kass. = 7 x 10(4) M-1.s-1) being the most effective inhibitor. The inhibition with these four serpins appeared to be reversible, with inhibition constants in the nanomolar range. The relatively high value of kass. for protease nexin I (5 x 10(3) M-1.s-1) suggested that it may be involved in the control of activated protein C on the surface of platelets where protein nexin I is present at relatively high concentrations. The value of kass. for protease nexin I, protein C inhibitor and antithrombin III showed a bell-shaped dependence on heparin concentration. At optimal concentrations, heparin accelerated the rate of inhibition by protease nexin I, protein C inhibitor and antithrombin III by 44-, 18- and 13-fold respectively. The kinetic constants for the inhibition of thrombin were also determined, and in all cases the serpins were more effective inhibitors of thrombin. Comparison of the sequences of the active-site regions of activated protein C and thrombin suggested that the more hydrophobic active site of thrombin may be more favourable for interactions with serpins.

Purification and characterization of the NADP-dependent glutamate dehydrogenase from Bacillus fastidiosus.

Ammonia assimilation in Bacillus fastidiosus proceeds via the NADP-dependent glutamate dehydrogenase. The enzyme, purified to homogeneity, is composed of identical subunits with a molecular weight of about 48,000 dalton. Presumably the enzyme is a hexamer. The enzyme is specific for NADP(H). The pH optima for the amination and deamination reactions are 7.7 and 8.6, respectively. The temperature optimum is 60 degrees C. Furthermore, temperature stability and apparent Km values for substrates of both the amination and deamination reactions were determined. Several metabolites were tested for their effect on the enzyme activity. Only malate and fumarate showed some inhibitory effect.

Interconversion of F430 derivatives of methanogenic bacteria.

F430 is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F430 is thermolabile and in the presence of acetonitrile or C10-4 two epimerization products are obtained upon heating; in the absence of these compounds F430 is oxidized to 12,13-didehydro-F430. The latter is stereoselectively reduced under H2 atmosphere to F430 by cell-free extracts of M. barkeri or M. thermoautotrophicum. H2 may be replaced by the reduced methanogenic electron carrier coenzyme F420.

High incidence of sensitivity to yeast killer toxins among Candida and Torulopsis isolates of human origin.

Among yeast strains of human origin belonging to the genera Candida, Cryptococcus, Torulopsis, and Rhodotorula which were examined for killer and sensitive characteristics with killer and sensitive strains of Cryptococcus, Hansenula, Kluyveromyces, Pichia, Saccharomyces, and Torulopsis as screening organisms, a high incidence of sensitivity to killer toxins was observed within the genera Candida and Torulopsis. Of 142 strains tested, 116 strains distributed over all Candida and Torulopsis species examined were sensitive to one or more killers. Several new intergeneric killer-sensitive relationships are described. Furthermore, killing activity was exhibited by six strains of Candida (C. krusei, C. guilliermondii) and three strains of Torulopsis (T. glabrata).

Physiological conditions affecting the sensitivity of Saccharomyces cerevisiae to a Pichia kluyveri killer toxin and energy requirement for toxin action.

The interaction between the killer toxin of Pichia kluyveri 1002 and cells of Saccharomyces cerevisiae SCF 1717 is strongly affected by the physiological state of sensitive cells. The killing effect is maximal for cells in the lag and early exponential phase of growth, whereas stationary cells are completely resistant. Furthermore, sensitivity is markedly enhanced by a rise of the pH (from 3.2 to 6.8) at which cells are cultured. Three successive stages can be distinguished in the killing process: (I) binding of the toxin to the primary binding site; (II) transmission of the toxin to its reactive site in the plasma membrane; (III) occurrence of functional damage (K+-leakage; decrease of intracellular pH). The transition from stage I to II is prevented in the absence of metabolic energy or at low temperature (below 10 degrees C). Sensitive cells in stage I can be rescued from toxin-induced killing by a short incubation at pH 7.0, which treatment is not effective for cells in stage II. Cells in stage II are able to resume growth when plated in a rich medium containing suitable concentrations of potassium and hydrogen ions. Rescue was not observed for cells in stage III of the killing process.

Production, purification and properties of a Pichia kluyveri killer toxin.

Production of the killer toxin of Pichia kluyveri 1002 was stimulated in the presence of yeast extract. In a minimal medium production was optimal at pH 3.8-4.0 and 22--25 degrees C. Addition of gelatin and nonionic detergents, like Brij-58 (polyoxyethylene 20 cetyl ether) and Triton-X-100, to this medium enhanced production significantly. The killer toxin was purified 140-fold by use of a stepwise ethanol precipitation and butyl Sepharose column chromatography. The purified killer toxin, which still contained some carbohydrates, appeared to be glycoprotein with a mol wt of about 19 000 and an isoelectric point of 4.3. It was stable between pH 2.5 and 4.7 and up to 40 degrees C.

Killer-sensitive relationships in yeasts from natural habitats.

Yeast strains (157) belonging to at least 9 genera were isolated from natural habitats and screened for killer-sensitive relationships. Killer and sensitive characteristics were exhibited by 17 and 11% of the isolates, respectively. The strains belong to either one of two mutually exclusive killer-sensitive groups.

Chemotaxis and transport of amino acids in Allomyces arbuscula.

Among a number of amino acids tested, L-lysine and L-arginine are the principal attractants in the chemotaxis of the zygotes of Allomyces arbuscula. The reaction can be stimulated to a greater or lesser extent by a number of compounds chemically related to L-leucine. No relationship between transport of attracting amino acids and their effect on chemotaxis has been found.