Structure of pre-pro-von Willebrand factor and its expression in heterologous cells

Von Willebrand factor (vWF), a multifunctional haemostatic glycoprotein derived from endothelial cells and megakaryocytes, mediates platelet adhesion to injured subendothelium and binds coagulation factor VIII in the circulation. Native vWF is a disulphide-bonded homopolymer; the monomeric subunits, of apparent relative molecular mass (Mr) 220,000 (220K) are derived from an intracellular precursor estimated at 260-275K. Multimer assembly is preceded by the formation of dimers, linked near their C-termini, which then assemble into filamentous polymers. The importance of the removal of the large vWF pro-polypeptide during multimer assembly, and whether this or other stages of the complex post-translational processing require components specific to endothelial cells or megakaryocytes, is unknown. Here we report an analysis of the complete sequence of pre-pro-vWF and expression of the molecule in heterologous cells. The vWF precursor is composed of several repeated subdomains. When expressed in COS and CHO cells, it is cleaved and assembled into biologically active high relative molecular mass disulphide bonded multimers. This suggests that the information for assembly of this complex molecule resides largely within its primary structure.     

Genetic dissection of monoamine neurotransmitter synthesis in Drosophila

The biogenic monoamine neurotransmitters octopamine, dopamine and serotonin have been detected in nervous tissue from many insects. We report here that intact Drosophila melanogaster brains, when incubated with the radioactive amino acids tyrosine and tryptophan, synthesized and accumulated labelled monoamines. In two mutant strains monoamine synthesis was abnormal. The per o mutation abolishes the normal circadian rhythm. Brains from per o flies, when incubated in tritiated tyrosine, accumulated one-third as much labelled octopamine as did brains from wild-type flies, but had normal dopamine and serotonin synthesis. In contrast, dopa decarboxylase (Ddc) mutations decreased dopamine and serotonin synthesis but did not affect octopamine synthesis. These results suggest that there are two different aromatic amino acid decarboxylases in Drosophila brains, one that decarboxylates L-dopa and 5-hydroxytryptophan and another that decarboxylates tyrosine. Direct measurement of L-dopa, 5-hydroxytryptophan and tyrosine decarboxylase activities in the different strains confirmed this suggestion.     

Steady-state kinetic studies with the polysulfonate U-9843, an HIV reverse transcriptase inhibitor

The tetramer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT^* and possesses excellent antiviral activity at nontoxic doses in HIV-1 infected lymphocytes grown in tissue culture. Kinetic studies of the HIV-1 RT-catalyzed RNA-directed DNA polymerase activity were carried out in order to determine if the inhibitor interacts with the template: primer or the deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using steady-state Briggs-Haldane kinetics assuming that the template:primer binds to the enzyme first, followed by the binding of the dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived which allows the calculation of all the specific forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The calculated rate constants are in agreement with this model and the results indicated that U-9843 acts as a noncompetitive inhibitor with respect to both the template:primer and dNTP binding sites. Hence, U-9843 exhibits the same binding affinity for the free enzyme as for the enzyme-substrate complexes and must inhibit the RT polymerase by interacting with a site distinct from the substrate binding sites. Thus, U-9843 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either an event leading up to the formation of the phosphoester bond, the formation of the ester bond itself or translocation of the enzyme relative to its template:primer following the formation of the ester bond.     

Age-related lipid peroxidation in the digestive gland of mussels: The role of the antioxidant defence systems

Summary The main cellular defence systems against free radical-mediated oxidative stress are significantly reduced in the dige⁺ive gland of aged (>10 years old) compared to younger (2–4 years old) mussels (Mytilus edulis L.). Moreover, the concentration of lipid peroxidation products (malondialdehyde) is increased in the same age group with respect to younger animals. The obtained data indicate that an impairment of the antioxidant defence systems would render the older animals more susceptible to peroxidative stress, thus supporting the general significance of the free radical theory of aging.     

Microorganism-associated variation in host infestation efficiency in a parasitoid wasp,Trichogramma bourarachae (Hymenoptera: Trichogrammatidae)

InTrichogramma bourarachae (Hymenoptera: Trichogrammatidae), the infestation efficiency presents important variation among strains: 60 eggs/5 days/female in the High (H) strain and 25 eggs/5 days/female in the Low (L) strain. Crosses show that between-strains variation is inherited by the matrocline route. Antibiotic and heat treatments reduce infestation efficiency in the H strain and have no effect on the L strain. The hypothesis of cytoplasmic-symbiotic microorganisms accounting for the higher infestation efficiency in the H strain is supported by microscopic observations. The origin and the significance of this between-strains variation are discussed.     

Evolution and ecological correlates of uniparental reproduction in freshwater snails

We review the spatial and temporal correlates of uniparental reproduction in freshwater snails as they pertain to the ecological hypotheses for the maintenance of biparental sex. The biogeographic evidence from two species (Potamopyrgus antipodarum andBulinus truncatus) presently supports the Red Queen hypothesis that biparental reproduction is selected as a way to reduce the risk to progeny of parasite attack. Uniparental reproduction in these species is associated with low levels of infection by parasites (castrating digenetic trematodes), suggesting that parthenogenesis or self-fertilization can replace cross-fertilization when the risk of infection is low. In addition, inB. truncatus, the opportunity for cross-fertilization coincides with the season in which parasite attack is highest. In a third species (Campeloma decisum), parthenogenetic reproduction is correlated with latitude and the presence of a non-castrating trematode that may prevent cross-fertilization; these patterns suggest that parthenogenesis has been selected as a mechanism to assure reproduction. Finally, we discuss the spotty taxonomic distribution of parthenogenetic species.