Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII

Steady-state kinetic parameters were compared for the action of alpha- and gamma-thrombin on the physiologically important thrombin substrates fibrinogen and factor XIII at 37 degrees C, pH 7.4, and 0.14 M NaCl. gamma-Thrombin, an alpha-thrombin derivative proteolytically cleaved at R-B73 and K-B154, was observed to catalyze the release of fibrinopeptide A (FPA) from fibrinogen with a specificity constant (kcat/Km) of 5 X 10(3) M-1 s-1. This value was approximately 2400-fold lower than the specificity constant for the corresponding alpha-thrombin-catalyzed reaction. The low specificity constant was attributed to an increase in Km and a decrease in kcat for gamma-thrombin-catalyzed release of FPA from fibrinogen. Conversion of alpha-thrombin to gamma-thrombin also resulted in an approximately 800-fold reduction in the specificity constant for thrombin-catalyzed release of fibrinopeptide B (FPB) from fibrin I, as well as a loss in discriminatory power. Whereas alpha-thrombin preferentially released FPA from intact fibrinogen, gamma-thrombin released FPA and FPB from intact fibrinogen at similar rates. In contrast to the large difference in specificity constants observed for alpha- and gamma-thrombin catalysis with fibrin(ogen) as substrate, the specificity constant (2.6 X 10(4) M-1 s-1) observed for gamma-thrombin-catalyzed release of activation peptide from factor XIII was only 5-fold lower than the corresponding value for the alpha-thrombin-catalyzed reaction. Additionally, the promotion of factor XIII activation by fibrin characteristic of the alpha-thrombin-catalyzed reaction did not occur in the gamma-thrombin-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

The contribution of prehatch and posthatch development to protandry in the chrysomelid Diabrotica virgifera virgifera

Laboratory tests with eggs of Diabrotica virgifera virgifera LeConte showed that during a 10-day hatching period, hatch of male eggs predominated on the first and second days, eggs of mixed sex, with ca. 1:1 ratio, hatched on the third and fourth days, and eggs hatching from the fifth to the tenth days were nearly all female. Overall, female eggs hatched a mean of 2.9 days later than male eggs. Not only did female eggs hatch later, but the time for posthatch development to the adult stage was 1.8 days longer for females. The later egg hatch and longer posthatch development for females resulted in female adults emerging a mean of 4.7 days later than male adults. Total adult emergence lasted 14 days; of this, males predominated during the first 5 days, and females predominated during the last 9 days. Males of D. v. virgifera appear to have evolved protandry (the tendency for males to emerge before females) by developing both a postdiapause embryonic stage and a combined larval and pupal stage of shorter duration.

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Résumé L'observation, au laboratoire, pendant une période d'éclosion de dix jours, des oeufs de D. v. virgifera LeConte, a montré que les oeufs mâles prédominent les deux premiers jours d'éclosion, que les oeufs des deux sexes, avec des fréquences 0,5/0,5, ont éclos les troisième et quatrième jours, et que les oeufs éclos du cinquième au dixième jour étaient presque tous femelles. Globalement, les oeufs femelles ont éclos en moyenne 2,9 jours plus tard que les oeufs mâles. De plus, la durée du développement post-embryonnaire des femelles a demandé 1,8 jour en plus. Une éclosion plus tardive et un développement post-embryonnaire plus long ont entrainé une émergence des femelles en moyenne 4,7 jours après les mâles. La période d'émergence des adultes s'est étalée sur 14 jours; les mâles ayant dominé pendant les 5 premiers jours et les femelles pendant les 9 derniers. Les mâles de D. v. virgifera semblent avoir évolué vers la protandrie en acquerant, tant une diapause post-embryonnaire que des stades de développements larvaire et nymphal plus brefs.

     

Variation in length and age at sexual maturity of Atlantic groundfish: a reply

Synopsis The assertion has been made by Halliday (1987) that trends in size and age at maturity of Atlantic groundfish published by Beacham (1983a, b, c, d, e, f) are artifacts induced by errors in determining the sex of an individual, distinguishing between immature and mature fish, sampling fish outside of the regular spawning season, and by nonrandom sampling of the population. In particular, Halliday asserts that for the Atlantic argentine,Argentina silus analysis, the conclusions of Beacham (1983a) that: (1) median length at sexual maturity declined over time; and (2) males matured at older ages than did females are invalid owing to biases in both sampling and analysis. In fact, if some of the biases indicated by Halliday were significant, then the decline in median length at sexual maturity is enhanced and the conclusions of Beacham (1983a) reinforced. Size and age at sexual maturity are dynamic characters in many vertebrate populations, and the fact that they should change for Atlantic groundfish should not be surprising given variable exploitation patterns in the fisheries since 1960.     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

The effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold

The purpose of this investigation was to determine the effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold (PWCFT). Ten adult males (mean age 23 years, SD 3) volunteered as subjects for this study. During the first laboratory visit the subjects performed a maximal bicycle ergometer test for the determination of maximum oxygen consumption (VO2max). Between 48 and 72 h later, the subjects pedaled to exhaustion at a power output which corresponded to a mean of 76% of VO2max (range, 72-80%) for the purpose of glycogen depletion. For the next 3 days, the subjects were fed a 10.5 MJ.day-1 low carbohydrate diet which consisted of 7.5% carbohydrates, 22.0% protein and 70.5% fat. The subjects then performed an incremental cycle ergometer test to the onset of fatigue or PWCFT, which was estimated from integrated electromyographic voltages of the vastus lateralis muscle. For the next 3 days the subjects were fed a 10.5 MJ high carbohydrate diet which consisted of 72.2% carbohydrates, 12.4% protein and 15.4% fats for the purpose of glycogen supercompensation. The subjects then performed a second PWCFT test. A paired t-test indicated that there was no significant (p greater than 0.05) difference between the means of the PWCFT values (depletion 246 W, SD 30; supercompensation 265 W, SD 28) and they were highly correlated at r = 0.884. The results of this investigation suggested that the methods commonly used to affect glycogen depletion or supercompensation had no effect on PWCFT.     

Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates.

The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.     

Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a M_r 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a M_r 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.