Roles of disulfide linkage and calcium ion-mediated interactions in assembly and disassembly of virus-like particles composed of simian virus 40 VP1 capsid protein

The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.     

Cloning and characterization of a candidate nutritive glycoprotein from the albumen gland of the freshwater snail, Helisoma duryi (Mollusca: Pulmonata)

Abstract. The albumen gland is a female accessory sex gland that synthesizes and secretes perivitelline fluid around pulmonate eggs. The perivitelline fluid is composed of mainly galactogen and proteins, and is thought to provide nourishment to the embryos during development. We have previously identified the major secretory protein of the albumen gland of the freshwater snail Helisoma duryi as a native glycoprotein of ∼288 kDa, consisting of four 66-kDa subunits. In this study, the major albumen gland protein in H. duryi was purified, cloned, and the full-length cDNA sequence determined. Nucleotide sequence analysis revealed that the albumen gland protein (HdAGP) shared 83% identity with a partial cDNA sequence from a developmentally regulated albumen gland protein in Biomphalaria glabrata . The HdAGP mRNA was detected by RT-PCR in the albumen gland, ovotestis, mantle and digestive gland. SDS-PAGE analysis of the albumen gland protein in egg masses at different stages of development showed that the amount of HdAGP steadily decreased during embryogenesis, suggesting its possible catabolism by the developing embryos. Protein domain searches suggested that the HdAGP shared limited sequence identity, and adopted a similar three-dimensional conformation to the bactericidal, permeability increasing, protein family, raising the possibility of a potential bactericidal function for this important reproductive/developmental protein.     

Evaluation of methods to assess adequacy of potential soil S supply to crops

Eight methods were evaluated for assessment of the likely soil S-supply to a crop and in particular to identify likely deficiencies in this supply. Of the methods, only sulphate production from incubated soil amended both with a S-containing amino acid and elemental S correlated with field response of grass yield to S-fertilizer applications. Tissue S-concentrations in plants grown for a short-term bioassay also correlated with field S-fertilizer response. Neither sulphate extracted by 3 different salt extractions, nor sulphate production from unamended incubated soil, nor soil arylsulphatase activity, correlated with field fertilizer response.     

Comparison of dynamic responses of cellular metabolites in <Emphasis Type="Italic">Escherichia coli</Emphasis> to pulse addition of substrates

We conducted an integrated study of cell growth parameters, product formation, and the dynamics of intracellular metabolite concentrations using Escherichia coli with genes knocked out in the glycolytic and oxidative pentose phosphate pathway (PPP) for glucose catabolism. We investigated the same characteristics in the wild-type strain, using acetate or pyruvate as the sole carbon source. Dramatic effects on growth parameters and extracellular and intracellular metabolite concentrations were observed after blocking either glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (disruption of pgi gene) or pentose phosphate breakdown of glucose by inactivation of glucose-6-phosphate dehydrogenase (disruption of zwf gene). Reducing power (NADPH) was mainly produced through PPP when the pgi gene was knocked out, while NADPH was produced through the tricarboxylic acid (TCA) cycle by isocitrate dehydrogenase or NADP-linked malic enzyme when the zwf gene was knocked out. As expected, when the pgi gene was knocked out, intracellular concentrations of PPP metabolites were high and glycolytic and concentrations of TCA cycle pathway metabolites were low. In the zwf gene knockout, concentrations of PPP metabolites were low and concentrations of intracellular glycolytic and TCA cycle metabolites were high.     

Potential of Transgenic Crops in Bangladesh: Findings from a Consultation of Bangladeshi Scientific Experts

This note summarizes the results of a consultation of scientific and regulatory experts in July 2005 on the potential of transgenic crops in Bangladesh. We find that Bangladeshi experts are optimistic on the potential of agricultural biotechnology to respond to biotic and abiotic stresses in their country in the future. Public research is constrained by human capacities, infrastructure and capital investment, and transgenic crop development will require the active involvement of outside partners, such as international organizations or collaboration with private companies. We also find that social acceptance of genetic engineering is not considered a major issue, but could become one, and prompted experts to call for a wider awareness campaign on the technology.This research project was conducted as part of the South Asia Biosafety Program (SABP), a project funded by the US Agency for International Development (USAID) and jointly managed by the International Food Policy Research Institute and AGBIOS Canada. The authors would like to thank the Bangladesh Agricultural Research Council and all the participants to the meetings in Dhaka and Mymensingh for their help.