The activation of bovine protein C by factor Xa

A complex composed of factor Xa and phospholipid vesicles assembled in the presence of calcium ions catalyzes a discrete cleavage of the heavy chain of bovine protein C that is indistinguishable from that produced by thrombin as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cleavage generates an active site capable of hydrolyzing small substrates and inactivating factor Va function in the prothrombinase complex. Activation of protein C by factor Xa requires both calcium ions and phospholipid vesicles and proceeds at a rate an order of magnitude greater than that observed for alpha-thrombin in solution. gamma-Carboxyglutamic acid-domainless protein C is not activated by factor Xa, consistent with the requirement for phospholipid and distinguishing this reaction from protein C activation by thrombin. Thrombomodulin serves as a cofactor for the factor Xa-catalyzed reaction, forming a 1:1 complex with factor Xa (apparent Kd = 5.7 X 10(-10) M) and stimulating the saturated rate of protein C activation by factor Xa (kcat = 149 min-1) to levels comparable with the thrombin-thrombomodulin complex. Protein C activation by factor Xa is not inhibited by the specific thrombin inhibitor dansyl-N-(3-ethyl-1,5-pentanediyl)amide but is inhibited by antithrombin III, tripeptide-chloromethyl ketones, and the monoclonal antibody alpha-BFX-2b that is highly specific for factor Xa. These data indicate that thrombomodulin is promiscuous in its role as a cofactor and suggest the existence of an alternative pathway for protein C activation in vivo.     

IL-2-activated human killer lymphocytes but not their secreted products mediate increase in albumin flux across cultured endothelial monolayers. Implications for vascular leak syndrome

When cultured with IL-2, human lymphoid cells acquire the ability to lyse various NK-resistant tumor targets. Due to their anti-tumor cytolytic effect, clinical trials with IL-2 alone or IL-2 + IL-2-activated killer (IAK) lymphocytes have been undertaken. However, infusion of therapeutically effective doses of IL-2 is associated with the development of systemic toxicity characterized by exaggerated endothelial permeability, also known as vascular leak syndrome. The present study was designed to examine the effects of IAK cells and their secreted products on vascular endothelial permeability by using an in vitro endothelial permeability model in which the flux of FITC-albumin across endothelial cell (EC) monolayers was measured. When endothelial monolayers were exposed to IAK cells for 2 h, significant increases in the transendothelial permeability to albumin were observed. Exposure of EC to lymphocytes cultured in the absence of IL-2 did not induce significant alteration in the endothelial permeability. In addition, neither culture supernatants of IAK cells nor purified recombinant cytokines, including IL-1 beta, IL-2, IL-3, IL-4, IL-6, TNF-alpha, GM-CSF, M-CSF, and IFN-gamma, had any effect on endothelial permeability in this model. Prior activation of EC with TNF-alpha did not alter the increased permeability induced by IAK cells or lack of it by nonactivated lymphocytes. Dexamethasone treatment of IAK cells abolished their anti-tumor cytolytic effect but only partially inhibited their ability to induce increased endothelial permeability. Pretreatment of IAK cells with mAb directed at the CD11a/CD18 (LFA-1) adhesion complex, and that of EC with mAb directed at the ICAM-1 molecule, inhibited the IAK cell-induced increase in endothelial permeability. These results demonstrate that direct cell-to-cell contact between IAK cells and EC is necessary and sufficient to cause increased endothelial permeability in this model system, and may therefore be an important factor contributing to the development of the vascular leak syndrome observed clinically.     

Influence of mineral supplementation on bovine serum, liver and endometrium at day 1 and day 12 of the estrous cycle

The effects of diet on mineral concentrations in serum, liver and endometrium were determined at points in the reproductive cycle in heifers. Dietary treatments extended 134 d and included feeding a basal hay (negative control), basal hay with concentrate (feed control), feed control with phosphorus and feed control with both phosphorus and trace minerals. Samples of serum and liver were taken at the beginning and end of the trial. Within an estrous cycle during the trial (endometrial biopsy) the cows were sampled either at Day 1 or Day 12 for determining progesterone levels and mineral elements in the blood, liver and endometrial tissue. Trace element concentrations in serum and liver did not differ among collection periods pretrial, endometrial biopsy and post breeding nor among treatment groups. However, endometrial tissue concentrations of copper, manganese and zinc were higher at Day 1 than at Day 12 (P < 0.05) in reverse of serum progesterone, which was higher at Day 12 (P < 0.05). Supplemental trace minerals appeared to increase concentrations of copper (P < 0.20), manganese (P < 0.10) and zinc (P < 0.20) at Day 1 but decrease concentrations of these same elements at Day 12 (P < 0.05, P < 0.10 and P < 0.05, respectively). The large differences in trace element concentrations observed in endometrial tissue at the estral phases and under different diets suggest the possible importance of trace elements and trace element nutrition in fertilization and (or) embryo survival.     

Cooperative oxygen binding, subunit assembly, and sulfhydryl reaction kinetics of the eight cyanomet intermediate ligation states of human hemoglobin.

Correlations between the energetics of cooperativity and quaternary structural probes have recently been made for the intermediate ligation states of Hb [Daugherty et al. (1991) Proc. Natl. Acad. Sci. US 88, 1110-1114]. This has led to a "molecular code" which translates configurations of the 10 ligation states into switch points of quaternary transition according to a "symmetry rule"; T-->R quaternary structure change is governed by the presence of at least one heme-site ligand on each of the alpha beta dimeric half-molecules within the tetramer [see Ackers et al. (1992) Science 255, 54-63, for summary]. In order to further explore this and other features of the cooperative mechanism, we have used oxygen binding to probe the energetics and cooperativities for the vacant sites of the cyanomet ligation species. We have also probed structural aspects of all eight cyanomet ligation intermediates by means of sulfhydryl reaction kinetics. Our oxygen binding results, obtained from a combination of direct and indirect methods, demonstrate the same combinatorial aspect to cooperativity that is predicted by the symmetry rule. Overall oxygen affinities of the two singly-ligated species (alpha +CN beta)(alpha beta) and (alpha beta +CN)(alpha beta) were found to be identical (pmedian = 2.4 Torr). In contrast, the doubly-ligated species exhibited two distinct patterns of oxygen equilibria: the asymmetric species (alpha +CN beta +CN)(alpha beta) showed very high cooperativity (nmax = 1.94) and low affinity (pmedian = 6.0 Torr), while the other three doubly-ligated species showed diminished cooperativity (nmax = 1.23) and considerably higher oxygen affinity (pmedian = 0.4 Torr). Extremely high oxygen affinities were found for the triply-ligated species (alpha +CN beta +CN)(alpha beta +CN) and (alpha +CN beta +CN)(alpha +CN beta) (pmedian = 0.2 Torr). Their oxygen binding free energies are considerably more favorable than those of the alpha and beta subunits within the dissociated alpha beta dimer, demonstrating directly the quaternary enhancement effect, i.e., enhanced oxygen affinity at the last binding step of tetramer relative to the dissociated protomers. Oxygen binding free energies measured for the alpha subunit within the isolated (alpha beta +CN) dimer and for the beta subunit within the isolated (alpha +CN beta) dimer sum to the free energy for binding two oxygens to normal hemoglobin dimers (-16.3 +/- 0.2 versus -16.7 +/- 0.2, respectively), arguing against cooperativity in the isolated dimer. Correlations were established between cooperative free energies of the 10 cyanomet ligation microstates and the kinetics for reacting their free sulfhydryl groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of indolepyruvic acid as an intermediate of rebeccamycin biosynthesis

Summary Experimental evidence is presented to demonstrate that indolepyruvic acid is an intermediate in the rebeccamycin biosynthetic pathway. [3-¹⁴C]Indolepyruvic acid was prepared and efficiently incorporated (8%) into rebeccamycin bySaccharothrix aerocolonigenes.     

A statistical power analysis of the 'internal reference' technique for comparing growth and growth depensation of tilapia strains

Experiments were conducted to compare the growth and growth compensation of three strains of juvenile Oreochromis niloticus . Ten full sib families (10 replicates) per strain were split and grown under crowded and uncrowded conditions for 3 weeks (the treatment). Both treatments were then grown an additional 12 weeks under less crowded conditions (the compensation). Standard length measurements were made at the end of crowding and the end of compensation. Each replicate included a size-matched control population of a fourth (red) reference strain. ANCOVA with the reference strain used as a cofactor revealed significant strain effects on specific growth throughout the experiment. The reference strain removed most of the random among-replicate error variance as shown by an increase of r 2 from 0.06 to 0.91 when it was included in the statistical models. If the reference fish had not been used, approximately 450 replicate families would have been needed to achieve the sensitivity of the present experiment (a difference of 7% among strains significant at P =0.05). We conclude that the CLSU strain grows significantly more slowly than the Israel and NIFI strains under the experimental conditions, that the crowding effect was essentially eliminated after 12 weeks of compensation, and that the reference strain greatly improved the resolution of the strain-testing experiment.     

Multiple glucan-binding proteins of Streptococcus sobrinus.

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.     

Nefiracetam (DM-9384) reverses apomorphine-induced amnesia of a passive avoidance response: Delayed emergence of the memory retention effects

Nefiracetam is a novel pyrrolidone derivative which attenuates scopolamine-induced learning and post-training consolidation deficits. Given that apomorphine inhibits passive avoidance retention when given during training or in a defined 10–12h post-training period, we evaluated the ability of nefiracetam to attenuate amnesia induced by dopaminergic agonism. A step-down passive avoidance paradigm was employed and nefiracetam (3 mg/kg) and apomorphine (0.5 mg/kg) were given alone or in combination during training and at the 10–12h post-training period of consolidation. Co-administration of nefiracetam and apomorphine during training or 10h thereafter produced no significant anti-amnesic effect. However, administration of nefiracetam during training completely reversed the amnesia induced by apomorphine at the 10h post-training time and the converse was also true. These effects were not mediated by a dopaminergic mechanism as nefiracetam, at millimolar concentrations, failed to displace either [³H]SCH 23390 or [³H]spiperone binding from D₁ or D₂ dopamine receptor subtypes, respectively. It is suggested that nefiracetam augments molecular processes in the early stages of events which ultimately lead to consolidation of memory.     

Control of flower development and phyllotaxy by meristem identity genes in antirrhinum.

The flower meristem identity genes floricaula (flo) and squamosa (squa) promote a change in phyllotaxy from spiral to whorled in Antirrhinum. To determine how this might be achieved, we have performed a combination of morphological, genetic, and expression analyses. Comparison of the phenotypes and RNA expression patterns of single and double mutants with the wild type showed that flo and squa act together to promote flower development but that flo is epistatic to squa with respect to early effects on phyllotaxy. We propose that a common process underlies the phyllotaxy of wildtype, flo, and squa meristem development but that the relative timing of primordium initiation or growth is altered. This process depends on two separable events: setting aside zones for potential primordium initiation and partitioning these zones into discrete primordia. Failure of the second event can lead to the formation of continuous double spirals, which are occasionally seen in flo mutants.     

The spectra of base substitutions induced by the impCAB,mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate

We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methane-sulfonate (MMS). We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT → GC transitions. However, we found that the three operons produced different responses to alkylating agents. We found that with MMS the chromosomal umuDC operon produced almost exclusively AT → GC transitions, whilst both mucAB and impCAB produced predominantly transversions. In the case of the impCAB operon the mutation spectrum contained more AT → TA than GC → TA transversions; this balance was reversed with mucAB. The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied. The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed. However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT → TA transversions. It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis. We propose that the type of misincorporation/chain extension which DNA polymerase III is allowed to synthesize on a damaged DNA template is extremely sensitive to both the amount and type of error-prone repair proteins present. The modulation of these events by the different proteins can result in widely different mutagenic changes in the repaired DNA.