Prostaglandin E2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts

The effects of prostaglandin E₂ (PGE₂) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of ³⁵S-methione labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE₂ treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE₂ was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE₂ to culture dishes presumably constitutes an environmental change to promote the functional seen in the blood erythroid bursts in terms of Hb synthesis and switching.     

HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.     

Niche-Specific Requirement for Hyphal Wall protein 1 in Virulence of Candida albicans

Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.     

Hfr formation by I pilus-determining plasmids in Escherichia coli K-12.

R483, an atypical, I pilus-determining plasmid, and also R144, a typical one, were shown to suppress the DnaA phenotype by integration into the Escherichia coli chromosome.     

Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.