Practical application of the protein C activator Protac from Agkistrodon contortrix venom

The protein C activator Protac from A. contortrix venom is being investigated as a potential antithrombotic agent and as a tool for the preparation of activated protein C. Its established major application is the zymogen activation in functional protein C determinations based on either a clotting assay or a chromogenic substrate technique. The sensitivity of the activated partial thromboplastin time as an indicator reaction for Protac activated protein C depends on the contact activator component of the reagent. Protein C dose-response increased in the following order: kaolin greater than ellagic acid greater than sulfatide. This phenomenon is due to a competition of molecular affinities between Protac, plasma components and the different activating surfaces.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

Characterization of the major protein-tyrosine-phosphatases of human placenta

In the preceding article (Tonks, N. K., Diltz, C. D., and Fischer, E. H. (1988) J. Biol. Chem. 263, 6722-6730), the purification of the major protein-tyrosine-phosphatases from human placenta, some to apparent homogeneity, was described. This report compares the characteristics of these enzymes and clearly identifies at least two distinct protein-tyrosine-phosphatase catalytic subunits. All were absolutely specific for phosphotyrosyl residues and showed no activity on any of the phosphoseryl/phosphothreonyl-containing proteins tested; they exhibited a high affinity for substrate with Km values in the submicromolar range. All were absolutely dependent on sulfhydryl compounds and appeared to contain at least one highly reactive cysteinyl residue essential for activity. Subtypes 1A and 1B could be distinguished by their response to polyanionic and polycationic compounds. The 1B enzymes were activated by EDTA, spermine, spermidine, and myelin basic protein to a greater extent than the 1A subtypes. Furthermore, they were inhibited by approximately 2 orders of magnitude lower concentrations of heparin (IC50 approximately 20 nM) and 1:1 or 4:1 poly (glutamate/tyrosine) (IC50 approximately 50 nM) than the 1A subtypes. Surprisingly, inhibition by the glutamate/tyrosine copolymers was strictly noncompetitive. Peptide mapping following digestion with Achromobacter protease I or Staphylococcus aureus V8 protease supported the view that, whereas protein-tyrosine-phosphatase subtypes 1A and 1B are different, their soluble and particulate counterparts are closely related structurally and are distinct from serine/threonine phosphatases 1 and 2A.     

Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid

In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.     

Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function

The effects of granulocyte-macrophage (GM)-CSF on the synthesis of MHC class II molecules and on the Ag presentation capacity by bone marrow derived macrophages (BMM phi) was investigated. BMM phi obtained by in vitro culture in the presence of macrophage-CSF were negative for synthesis of I-A molecules and induced the Ag-mediated proliferation of insulin-specific T clone cells with lower efficiency than splenic accessory cells. After pulse treatment with GM-CSF for 24 to 48 h, day 12 BMM phi exhibited highly efficient Ag presentation function which was superior to that induced by IFN-gamma. Expression of membrane-bound IL-1 was augmented significantly by GM-CSF, but not by IFN-gamma. However, the T cell clone used to probe for accessory cell function of BMM phi was not dependent on IL-1 for optimal proliferation. Concomitantly, GM-CSF induced the de novo synthesis of I-A molecules, although to a lesser extent than optimal doses of IFN-gamma. Thus GM-CSF appears to elicit properties in addition to Ia molecule synthesis and membrane IL-1 expression in BMM phi being essential for efficient accessory cell function to the T clone cells. The activation of BMM phi by GM-CSF was reversible and could be repeated. These data show that GM-CSF exerts a modulatory influence on preformed BMM phi, reversibly activating cells to Ia biosynthetic potential and pronounced accessory cell capacity, thus rendering the explanation unlikely that differentiation of precursor cells into a constitutively functional state had occurred.     

Role of the LFA3-CD2 interaction in human specific B cell differentiation

We examined the role of the lymphocyte function-associated (LFA)3 molecule in human B cell response. A mAb to this molecule did not influence B cell proliferation induced by anti-mu antibody and IL. In contrast, the same mAb inhibited the specific T-dependent B cell response induced by a particulate Ag. In the same line, two anti-CD2 mAb (directed toward the T11-1 and T11-2 epitopes) inhibited this response, whether used alone or in association. These inhibitions took place at an early stage of the response, and anti-LFA3 and anti-CD2 mAb acted on B cells and T cells, respectively. In contrast, when T cell help was provided by exogenous IL-2, the B cell response was resistant to the inhibitory effect of anti-LFA3 mAb. Taken together, these results indicate that the LFA3-CD2 pair play a major role in the direct T-B interaction required for T cell help.     

Reduction of tumor growth following treatment with a glutamine antimetabolite

Assessment of arterial-venous differences across transplanted methylcholanthrene-induced sarcomas in rats revealed significant decreases in plasma concentrations of glutamine, serine and glucose. Treatment with the glutamine antimetabolite, acivicin, significantly reduced tumor weights by 65% at the conclusion of the experiment 34 days after tumor induction. These results suggest that glutamine is an essential metabolic substrate for tumor growth and that blockade of glutamine utilization can inhibit the growth of these transplantable sarcomas.     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.     

Fast one-step procedure for the detection of nucleic acids in situ by primer-induced sequence-specific labeling with fluorescein-12-dUTP.

We provide fast, simple, one-step procedures for sequence-specific detection of nucleic acids in situ. Tandem repeat sequences in DNA are stained within 30 min, and mRNA is stained within 2 h. The procedures are based on the incorporation of the newly available fluorescein-labeled dUTP into DNA synthesized in situ by primed in situ labeling, with denatured fragments of cloned DNA or oligonucleotides as primers. The extreme speed and simplicity of the reaction make it attractive for automatization in routine laboratory procedures and opens up new diagnostic possibilities.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity