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.     

Molecular genetics of met 17 and met 25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene

Summary A method for transposon mutagenesis in Azospirillum lipoferum 29708 is reported with transposon Tn5. The suicide plasmid pSUP2021 was used to deliver Tn5 in A. lipoferum using Escherichia coli SM10 as the donor. Neomycin-resistant transconjugants were detected at a frequency of 6x10^-6 per recipient. Different types of mutants were isolated, e.g. auxotrophic, coloured, IAA-negative, and IAA-overproducers. Among the auxotrophic mutants, cysteine and methionine requirers prevailed. Random Tn5-insertion with only one copy per mutant was demonstrated by Southern blotting and hybridization. Tn5-induced mutants are relatively stable, with reversion rates of 2–20×10^-8. A gene which is a part of the carotenoid pathway is closely linked to the histidine genes. The existence of two pathways for IAA production in A. lipoferum is discussed.     

Plant protoplasts and genetic engineering II

Book Review

Plant protoplasts and genetic engineering IIY.P.S. Bajaj (Ed.), (Biotechnology in Agriculture and Forestry, Vol. 9). Berlin: Springer-Verlag, 1989. 510 pages. DM398.00. ISBN 3-540-50789-2     

Competition between siratro and kleingrass for15N labelled mineralized nitrogen

Summary Isotope dilution provides a method for measuring plant competition for mineral N and transfer of biologically fixed N from a legume to a grass. A plant growth medium was enriched with¹⁵N, and used to grow Siratro (Macropitilium atropurpureum D.C. Urb.) and Kleingrass 75 (Panicum coloratum L.) in 20 liter pots for 98 days in a glasshouse. The plants were grown in pure stand and in mixtures. When grown in 50∶50 mixture the grass obtained 59% of the labelled N and the legume obtained 41%. The grass produced nearly as much root mass as the legume even though biomass of the shoots were less than half that of the legume. Reducing the proportion of either plant species in the mixture reduced the proportion of the mineralized N absorbed by that species. The shoots of the grass were significantly more enriched (1.166 atom%¹⁵N excess) than the roots (1.036). The grass received 12% of its N as biologically fixed N from the legume.     

Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function

Summary The mechanism of the inhibition and of the recovery of DNA synthesis in E. coli following UV-irradiation was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response. Several lines of evidence indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement.Recovery of DNA synthesis after UV was largely unaffected by mutations in the uvrA, recB or umuC genes. Resumption of DNA synthesis does however require protein synthesis and the regulatory action of recA. Experiments with a recA constitutive mutant and recA 200 (temperature sensitive RecA) demonstrated that RecA protein itself is directly required but is not sufficient for recovery of DNA synthesis. We therefore propose that recovery of DNA synthesis depends upon the concerted activity of RecA and the synthesis of an inducible Irr (induced replisome reactivation) factor under RecA control. We suggest that the mechanism of recovery involves the action of Irr and RecA to promote movement of replisomes past non-instructive lesions, uncoupled from polymerisation and/or that Irr and RecA are required to promote re-initiation of a stalled replication complex downstream of a UV-lesion subsequent to such an uncoupling step.     

Quadriacanthus aegypticus n. sp., a monogenean gill parasite from the Egyptian teleost Clarias lazera

Summary Quadriacanthus aegypticus n. sp., a monogenean from the gills of Clarias lazera inhabiting Nile delta waters in Egypt, is described. The genus Quadriacanthus Paperna, 1961 is reported for the first time in Egypt. Particular attention has been paid to the reproductive system, the digestive system, the anterior adhesive glands, the posterior body glands and haptoral sclerites. Possible functions of the different internal organs are discussed. The diagnosis of the genus Quadriacanthus is emended. ac]19840926