The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Patterns of Reproductive Isolation in Toads

Understanding the general features of speciation is an important goal in evolutionary biology, and despite significant progress, several unresolved questions remain. We analyzed an extensive comparative dataset consisting of more than 1900 crosses between 92 species of toads to infer patterns of reproductive isolation. This unique dataset provides an opportunity to examine the strength of reproductive isolation, the development and sex ratios of hybrid offspring, patterns of fertility and infertility, and polyploidization in hybrids all in the context of genetic divergence between parental species. We found that the strength of intrinsic postzygotic isolation increases with genetic divergence, but relatively high levels of divergence are necessary before reproductive isolation is complete in toads. Fertilization rates were not correlated to genetic divergence, but hatching success, the number of larvae produced, and the percentage of tadpoles reaching metamorphosis were all inversely related with genetic divergence. Hybrids between species with lower levels of divergence developed to metamorphosis, while hybrids with higher levels of divergence stopped developing in gastrula and larval stages. Sex ratios of hybrid offspring were biased towards males in 70% of crosses and biased towards females in 30% of crosses. Hybrid females from crosses between closely related species were completely fertile, while approximately half (53%) of hybrid males were sterile, with sterility predicted by genetic divergence. The degree of abnormal ploidy in hybrids was positively related to genetic divergence between parental species, but surprisingly, polyploidization had no effect on patterns of asymmetrical inviability. We discuss explanations for these patterns, including the role of Haldane''s rule in toads and anurans in general, and suggest mechanisms generating patterns of reproductive isolation in anurans.     

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.     

Human cytomegalovirus immediate-early two protein region involved in negative regulation of the major immediate-early promoter.

The immediate-early two (IE2) gene products of human cytomegalovirus negatively regulate gene expression from the major immediate-early promoter in permissive human fibroblasts. A mutational analysis of the IE2 proteins indicated that the carboxyl-terminal region is required for negative regulation. The IE2 proteins that lack amino acid residues 365 to 519, or the carboxyl-terminal amino acids failed to negatively regulate. Most of the amino-terminal portion of the IE2 protein was not required for negative regulation. A possible explanation of the negative effect on downstream expression by the IE2 proteins is discussed.     

Effect of chronic cocaine administration and cocaine withdrawal on coronary flow rate and heart rate responses to epinephrine and cocaine in isolated perfused rat hearts

The acute dose-dependent effects of epinephrine and cocaine on heart rate and coronary flow rate (CFR) were examined in isolated, perfused (Langendorff) rat hearts from animals: i) pretreated with daily cocaine injections (20 mg/kg/day) for 8 weeks; ii) after 2-day withdrawal from 8-week cocaine pretreatment; iii) vehicle-treated controls. Chronic cocaine (CC) hearts were significantly less sensitive to the chronotropic effects of epinephrine than control (C) or withdrawal (CW) hearts. CW hearts exhibited significantly higher heart rates in response to epinephrine than C and CC hearts. Epinephrine alone (2.5 x 10(-7) M) decreased CFR 11% (C), 9%(CC), 14%(CW) from respective baseline levels. Cocaine alone had no significant effect on CFR in C hearts but produced slight dose-dependent decrements in CFR in CC and particularly CW hearts at higher doses. Cocaine plus epinephrine markedly decreased CFR in all groups, particularly in CW hearts. The results indicate that chronic daily cocaine administration produces a functional tolerance of the heart to the chronotropic actions of epinephrine but a 2-day withdrawal from chronic cocaine results in a rebound supersensitivity to adrenergic stimulation and cocaine's sympathomimetic effects. In addition, cocaine produces only minor decrements in coronary flow in the rat heart, while cocaine acts synergisticallly with epinephrine to produce a marked decrease in CFR.     

Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.