Regulation of mouse mammary-gland gamma-glutamyltranspeptidase mRNA during pregnancy, lactation and weaning.

The level of gamma-glutamyltranspeptidase (GGT) activity and of its mRNA were determined in the mouse mammary gland during pregnancy, lactation and weaning. The GGT activity, which is very low in the virgin-mouse mammary gland (5 munits/mg of protein), increases progressively during pregnancy (3-fold), reaches its maximum at the onset of lactation (8-fold) and returns rapidly to basal level at weaning. Although no GGT-specific mRNA is detected in the virgin-mouse mammary gland, a single faint band of 2.2 kb in size is found during pregnancy. During lactation, an additional mRNA of 2.4 kb in size appears, and the level of both mRNAs is higher. This high level of mRNA persists during weaning as well. Southern-blot analysis of mouse mammary-gland DNA provides convincing evidence that there is only one gene which codes for the two mRNAs. The present study provides the first evidence for a physiological regulation of the two GGT mRNAs in the same tissue.     

Identification and characterization of 10 polymorphic microsatellite loci in the German cockroach,Blattella germanica

Primer sequences and initial characterization are presented for 10 microsatellite loci isolated from the German cockroach, Blattella germanica. In a sample of 30 individuals from a single population sample, all loci were polymorphic with two to 12 alleles segregating per locus and levels of observed heterozygosity ranging from 0.27 to 0.92. One locus showed a deficit of heterozygotes. Experimental conditions are described for polymerase chain reaction multiplexing, which enables the genotyping of eight loci in three electrophoretic runs consisting of one set of three and two sets of two markers. Seven primer sets cross‐amplify in the related Blattella asahinai.     

Rubella virus E2 signal peptide is required for perinuclear localization of capsid protein and virus assembly

The rubella virus (RV) structural proteins capsid, E2, and E1 are synthesized as a polyprotein precursor. The signal peptide that initiates translocation of E2 into the lumen of the endoplasmic reticulum remains attached to the carboxy terminus of the capsid protein after cleavage by signal peptidase. Among togaviruses, this feature is unique to RV. The E2 signal peptide has previously been shown to function as a membrane anchor for the capsid protein. In the present study, we demonstrate that this domain is required for RV glycoprotein-dependent localization of the capsid protein to the juxtanuclear region and subsequent virus assembly at the Golgi complex.     

Synthesis and construction of a novel multiple peptide conjugate system: strategy for a subunit vaccine design

We describe the design and synthesis of a novel well characterized multi-peptide conjugate (MPC) system containing antigens from human malaria parasite and the Tat protein of HIV type-1 (HIV-1-Tat). Construction of the MPC utilizes Fmoc solid-phase peptide synthesis coupled with solution chemistry. In the first phase, a core template that serves as primary anchor for the synthesis and attachment of multiple antigens is synthesized. Serine(trityl) and multiple lysine branches with epsilon groups blocked during chain assembly are incorporated forming a tetrameric core. Cysteine whose side chain thiol serves to couple haloacetyl or S-protected haloacetyl peptides is added to complete assembly of the core template. Modification to the coupling solvent, addition of key amino acid derivatives (N-[1-hydroxy-4-methoxybenzyl]) in the peptide sequence allows the synthesis of base peptides on the core template with molecular mass greater than 7500 kDa. Base peptides are then reacted with high performance liquid chromatography purified haloacetyl peptides to generate multiple peptide conjugates with molecular masses of 10 to 13 kDa. MPC constructs thus formed are further characterized by matrix assisted laser desorption-time of flight mass spectroscopy (MALDI-MS), amino acid analysis, size exclusion chromatography, and SDS-polyacrylamide gel electrophoresis (PAGE). To our knowledge, this is the first report describing a chemically well defined multiple conjugate system with potential for development of synthetic subunit vaccines.     

Characterization of a Leishmania stage‐specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulence

Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein‐encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co‐immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene‐deleted parasites (Ldp27^?/?) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27^?/? parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.     

Centrin1 is required for organelle segregation and cytokinesis in Trypanosoma brucei

Centrin is a calcium-binding centrosome/basal body-associated protein involved in duplication and segregation of these organelles in eukaryotes. We had shown that disruption of one of the centrin genes (centrin1) in Leishmania amastigotes resulted in failure of both basal body duplication and cytokinesis. Here, we undertook to define the role of centrin1 (TbCen1) in the duplication and segregation of basal body and its associated organelles kinetoplast and Golgi, as well as its role in cytokinesis of the procyclic form of Trypanosoma brucei by depleting its protein using RNA inhibition methodology. TbCen1-depleted cells showed significant reduction in growth compared with control cells. Morphological analysis of these cells showed they were large and pleomorphic with multiple detached flagella. Both immunofluorescence assays using organelle-specific antibodies and electron microscopic analysis showed that TbCen1-deficient cells contained multiple basal bodies, kinetoplasts, Golgi, and nuclei. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrin. This failure in organelle segregation may be the likely cause of inhibition of cytokinesis, suggesting for the first time a new and unique role for centrin in the segregation of organelles without affecting their multiplication in the procyclic form of T. brucei.     

Protein disulfide isomerase assisted protein folding in malaria parasites

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.