Influence of broth dilution on the disruption of Escherichia coli

Summary The effect of cell concentration (5 to 150 g/L wet wt after broth dilution) on homogenizer disruption efficiency and homogenate viscosity is reported for E. coli. Broth dilution increases homogenizer efficiency and decreases feed and homogenate viscosity. However, this increase in disruption efficiency is not sufficient to warrant dilution of the broth prior to homogenization. The optimal feed concentration is the maximum possible that does not lead to practical handling difficulties due to high viscosity.     

The effect of acetaldehyde concentrations on the relative rates of formation of acetaldehyde-modified hemoglobins

The effect of various concentrations of acetaldehyde (0, 0.05, 0.1, 0.25, 0.5, 1.0, and 5.0 mM) on the relative rates of formation of hemoglobin acetaldehyde adducts detected in fractions eluted from cation exchange high-pressure liquid chromatography (HPLC) was investigated. When the hemoglobin and acetaldehyde mixtures were incubated at 37 degrees C for various time intervals up to 24 hr, increased amounts of HbA1c could be observed after 2 hr incubation with 1 mM or greater concentrations of acetaldehyde, or after 4 hr incubation with at least 0.5 mM acetaldehyde. An increase in the HbA1a + b fraction was not observed with 4 hr incubation time until the acetaldehyde level reached 1 mM. The HPLC method detected no difference in minor hemoglobins from alcoholic and normal subjects. Incubation of red blood cells at 37 degrees C for 1 hr with six consecutive pulses of 0.05 mM [14C]acetaldehyde showed no differences in the amounts of minor hemoglobins determined chromatographically at various pulse intervals. However, the measure of the 14C-label incorporation into hemoglobin showed that adducts eluting in the HbA1a+b fraction were formed at a faster rate than those eluting in the HbA1c or HbA0 fraction, respectively. The specific activities of the HbA1a+b fractions at 2, 4, and 6 pulses were 34, 128, and 949 cpm/mg hemoglobin; those of the HbA1c fraction were 15, 58, and 174 cpm/mg hemoglobin. This evidence of modification of hemoglobin by physiological levels of acetaldehyde from 14C-label incorporation suggests that an assay more sensitive than chromatographic separation of adducts might be clinically useful in detecting alcoholism or monitoring alcohol detoxification programs.     

Prostaglandin modulation of N-formylmethionylleucylphenylalanine-induced transmembrane potential changes in rat neutrophils

Prostaglandins of the E-series (PGEs) and PGI2 will inhibit formylmethionylleucylphenylalanine-(f-Met-Leu-Phe) induced lysosomal enzyme release and superoxide-anion (O2-) production by neutrophils. The inhibitory effects of PGEs and PGI2 on neutrophil functional responses have been correlated with their ability to increase intracellular cAMP. In this study we have examined the effects of PGEs and PGI2 on f-Met-Leu-Phe- and phorbol-myristate-acetate-induced rat neutrophil membrane potential changes using an optical probe of membrane potential 3,3-dipropylthiodicarbocyanine iodide. 15-(S)-15-methyl-PGE1 (15-methyl-PGE1), a stable analogue of PGE1 and PGI2 inhibited f-Met-Leu-Phe-induced transmembrane potential changes in a dose-dependent manner. This inhibition was correlated with the ability of these agents to increase intracellular cAMP levels and inhibit O2- production and degranulation. In contrast, 15-methyl-PGE1 and PGI2, did not inhibit phorbol-myristate-acetate-induced transmembrane potential changes and O2- production. These results suggest independent mechanisms of activation of neutrophils by phorbol myristate acetate and f-Met-Leu-Phe, and they also suggest that the inhibitory effects of prostaglandins and cAMP on f-Met-Leu-Phe-stimulated cells is at a step or steps prior to activation of those processes involved in effecting changes in transmembrane potential, which are common to both stimuli.     

Human genes for glutathione S-transferases

The tissue distribution of different glutathione S-transferases (GST) is analysed by electrophoresis. The existence of GST"e" (erythrocyte), GST3, GST1, and GST2 is confirmed. GST"e" the fastest and most thermolabile of different GST analysed is observed only in erythrocyte cells. GST3 which migrates more slowly than GST"e" is present in all tissues and cells analysed, excepted for erythrocyte cells in which only GST"e" is observed. GST1 presents a polymorphism with four phenotypes, 1, 1/2, 2, and 0 controlled by three alleles 1, 2, and 0 (null). With the sample of 56 livers analysed the different frequencies obtained are 9%, 5%, 43%, 43% for the phenotypes 1, 1/2, 2, and 0 respectively and 0.074 (p), 0.279 (q), 0.647 (r) for the alleles, 1, 2, and 0 (null). GST2 presents variant patterns due probably, in the majority of cases, to post-synthetic modifications rather than allelic variation. Two new GST are described, GST4 and GST5. GST4 abundant in muscle tissue is a dimeric protein. GST4 forms with GST1 a heterodimeric band. GST5 is observed in brain homogenates. For the chromosome localization the results obtained by man (leucocytes)-mouse somatic cell hybrid analysis indicate that the gene for leucocytes GST is on chromosome 11. This gene is the structural GST3 gene.     

Activation of oxidative metabolism of granulocytes and monocytes by IGG-coated platelets from patients with thrombocytopenia

We have previously shown that monoclonal anti-T cell antibodies bound to their specific targets can trigger the activation of monocyte/macrophage oxidative metabolism through an Fc receptor-mediated interaction. The present study demonstrates that IgG coated platelets from patients with thrombocytopenia-associated diseases can induce a similar respiratory burst activation in polymorphonuclear and mononuclear phagocytes from normal individuals. The intensity of the oxidative reaction as measured by luminol-dependent chemiluminescence is in close correlation with the level of surface-bound IgG molecules as determined by a radioactive anti-immunoglobulin assay. This new methodology to evaluating IgG fixed on human platelets by their capacity to trigger the generation of highly reactive oxygen species by granulocytes and monocytes has also suggested a new mechanism in the genesis of thrombocytopenia associated with autoimmune diseases.     

A convenient apparatus and tracking dye for anaerobic analytical polyacrylamide-gel electrophoresis

An anaerobic modification of conventional polyscrylamide-gel electrophoretic equipment is described. The modified apparatus has been applied to the separation of Azotobacter vinelandii nitrogenase components and should prove useful in the analysis of other O₂-sensitive proteins. Electrophoresis in reducing gels can be followed with a dithionite-resistant tracking dye, potassium gualazulene-1-sulfonate.     

Localisation régionale des gènes humains IDHS,MDHS, PGK, α-GAL,G6PD par l'hybridation cellulaire interspécifique

Summary 22 independent man-hamster (HGPRT^–) hybrids using male human cells with balanced reciprocal translocation t(X;2)(p22;q32) were analysed for human genes localized on chromosome 2 (IDH_S, MDH_S), on chromosome X (PGK, GAL, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.2, chr.2q^–, chr.Xp⁺).The following results were obtained:The chromosomes 2 and 2q^– are absent in the 22 hybrids.In 9 hybrids, the absence of MDH_S in spite of the presence of the chromosome Xp⁺ indicates that the gene for MDH_S is not localized on this chromosome (or that the gene for MDH_S is not on the segment 2q32-2qter translocated on X).In 14 hybrids, the three markers of X (PGK, GAL, G6PD) and IDH_S are expressed in the presence of the chromosome Xp⁺. This result indicates that the genes for these markers are on Xp⁺ or that the genes PGK, GAL, G6PD are on X without the Xp22-Xter segment, translocated on the chr.2, and that the gene for IDH_S is on the 2q32-2qter segment translocated on X.In 8 hybrids, in the absence of the intact chromosome Xp⁺, the higher percentage of the presence of G6PD (7 hybrids) and the lower percentage of the presence of IDH_S (3 hybrids) are explained by the fact that these hybrids selected in HAT medium had to retain a segment of Xp⁺ bearing the human gene HGPRT. G6PD appeared very close to HGPRT and IDH_S very distant from HGPRT.The study of the different correlations between the presence and the absence of these four markers on Xp⁺ in the different hybrids indicates the following order on the chromosome Xp⁺ from p to q: IDH_s — PGK — GAL — G6PD.

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Groupe INSERM: Directeur J. Frézal

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Groupe CNRS, ER, 149: Directeur J. de Grouchy     

Evaluation of the role of conserved His and Met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase

The 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form His-(X)4-His-(X)4-His-(X)17-His-(X)8-His which represents a potential binding site for non heme iron to the protein. The importance of selected amino acids within this His cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems. After single mutation of each of the 5 His residues at positions 363, 368, 373, 391, and 400 by Ser, Cys, or Lys, measurable levels of 5-lipoxygenase activity could be recovered in Escherichia coli only for the Ser363 and Cys363 mutants, with most amino acid substitutions causing a decrease in the levels of expression of the soluble protein. In contrast, 25-80% of soluble 5-lipoxygenase activity was recovered after the replacement of several of the hydrophobic amino acids in this region: Tyr384 by Ser or Phe; Phe394 by Trp and Val375 by Ala. Met436 could be replaced by Leu with little effect on 5-lipoxygenase activity or turnover inactivation half-time. High levels of mutant 5-lipoxygenases containing a Ser residue instead of His at each of the five positions were also expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus. The specific activity (58-75% of control) and the reaction time course of the Ser363, Ser391, and Ser400 mutants were comparable with that of native 5-lipoxygenase whereas inactive proteins were obtained for the Ser368 and Ser373 mutants. These results show that His368 and His373 residues are important for 5-lipoxygenase activity and that the other conserved His363, His391, His400, and Met436 residues are not crucial for the catalytic cycle or for the mechanism of self-inactivation of 5-lipoxygenase.     

Retention of enzyme by electropolymerized film: a new approach in developing a hypoxanthine biosensor

An amperometric biosensor for hypoxanthine was constructed by forming a layer of crosslinked xanthine oxidase on a platinum electrode, followed by electropolymerization of a submonolayer film of resorcinol and para-diaminobenzene. The fabricated electrodes were evaluated for speed of response, sensitivity, and reusability. Optimal performance was obtained with enzyme-based electrodes sparsely covered with film which was formed by electropolymerization in less than 6 min. The resulting electrodes exhibited linear response to hypoxanthine in the. range 5-300 muM with a response time of 2 min. Application of the biosensor in monitoring hypoxanthine content of fish extracts yielded results which agreed well with spectrophotometric assays using soluble xanthine oxidase. The biosensor was stable for 60 days when stored at 4 degrees C in phosphate buffer and it could be used continuously for 6 h with over 50 assays.     

Inhibitors of Urokinase and Thrombin in Cultured Neural Cells

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with 125I-urokinase or 125I-thrombin. Rinsed glioblastoma possessed two components that complexed 125I-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the 125I-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with 125I-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I-urokinase; one was PAI-1, and the other was PN-1. The secreted PN-1 also formed complexes with 125I-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I-urokinase or 125I-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with 125I-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)