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.     

Salt Tolerance ofEchinochloa crusgalli

Salt tolerance ofEchinochloa crusgalli was studied using gravel culture with root medium electrical conductivity between 3 to 25 dS m^-1. Salinity depressed germination and shoot yield. A 50 % reduction in shoot yield occurred at 15.9 dS m^-1. The plant was able to maintain its tissue water content and K concentration in the tissue water while Na, Ca and Cl increased and Mg decreased with increasing root zone salinity.     

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     

Growth and ionic relations of six triticale cultivars as affected by salinity

Growth and ionic relations were studied in six triticale cultivars of different geographical origins grown in a greenhouse in nutrient solution with or without the addition of 100 mM NaCl. In 21 d old plants of all the six cultivars growth was little affected in the salt treatment, whereas in the subsequent three harvests during vegetative phase (after 31, 38 and 45 d), growth reduction effects of salinity were progressively pronounced. Generally, shoots of all the six cultivars accumulated relatively more K+ as compared to Na+ or Cl-. Differential accumulation of K+, Na+ and Cl- by various cultivars was coupled with variable rates of Na+ and Cl- transport from root to shoot which were — to some extent- related to cultivar differences in growth in saline root media. Chloride content of shoots of the six cultivars was negatively correlated with the relative growth reduction due to salinity at the four harvests.     

In vitro determination of phagocytic indices of Candida berkhout species by rat peritoneal macrophages

The aims of this investigation were to study and describe the behaviour of 13 different species of Candida, as compared with C. albicans, by means of phagocytosis assays in vitro.Tests were carried out with rat peritoneal macrophages in contact with quantified suspensions of live yeasts. Phagocytic indices, candidacidal activity and filamentation rat were tested microscopically after 3 h incubation at 37 ° C.The phagocytic indices obtained allowed us to separate the fungi into four groups. Candida albicans and tropicalis belong to Group I; diddensii and shehatae, among others, belong to Group II; sake, krusei, viswanathii, etc., Group III; and C. glaebosa and haploid strains of Pichia ohmeri (C. guilliermondii var. membranaefaciens), Group IV. These data would suggest a possible correlation between pathogenesis and phagocytic indices.There were no evidences of any phagocytes ability to kill yeasts. Candidacidal activity was absent in the species assayed. Yeast lysis may have been observed if our assays would have taken longer than 3 h.     

Bombesin-induced behavioural changes: Antagonism by neuroleptics

Adult male Sprague-Dawley rats were administered bombesin (BN) intracerebroventricularly (ICV), at a dose of 0.001, 0.01, 0.1, or 1.0 μg, and the behavioural effects monitored longitudinally across time for up to 24 hr. Administration of BN significantly increased the locomotor, rearing and grooming activity at all doses. The time-course of behavioural activation was dose-related (lasting up to 2.5 hr). There was no significant difference in the behavioural response of rats receiving the BN doses in an ascending or descending order. To test the effects of dopamine receptor blockade on the BN-induced behavioural changes, groups of animals were treated with fluphenazine or haloperidol (0.1 to 2.5 mg/kg, IP) 30 min prior to BN (1 μg, ICV) administration. The results revealed that the neuroleptics could effectively antagonize the BN-induced activation of locomotor, rearing and grooming activity. These data are concordant with the view that centrally administered BN stimulates spontaneous exploratory and grooming behaviours in rats, in a time- and dose-related manner. Furthermore, since neuroleptics block these effects, it remains possible that the BN-induced behavioural changes may be mediated, at least in part, through the dopaminergic system(s).     

Nucleotide sequence encoding the 5'' end of Xenopus laevis 18S rRNA.

We have sequenced a region of cloned Xenopus laevis ribosomal DNA encompassing the last 24 nucleotides of the external transcribed spacer and the first 275 nucleotides of the 18S gene. The start of the 18S gene was identified by correlating the results obtained from RNA hybridization and fingerprinting with the DNA sequence. This 5' region of 18S rRNA contains five 2'-O-methyl groups and at least six pseudouridine residues. Several of these modified nucleotides are clustered into a relatively short region from nucleotides 99-124. Nucleotides 227-250 constitute a distinctive sequence of 24 consecutive G and C residues. Comparison with the first 160 nucleotides of a yeast 18S gene (25) reveals three blocks of high sequence homology separated by two short tracts where homology is low or absent. The external transcribed spacer sequences diverge widely from within a few nucleotides of the start of the 18S gene.     

Umbilical cord-derived mesenchymal stem cells preconditioned with isorhamnetin: potential therapy for burn wounds

BACKGROUNDImpaired wound healing can be associated with different pathological states. Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide. Mesenchymal stem cells (MSCs) possess the ability to regenerate tissues by secreting factors involved in promoting cell migration, proliferation and differentiation, while suppressing immune reactions. Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.AIMTo enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.METHODSHuman umbilical cord MSCs (hU-MSCs) were isolated and characterized by surface markers, CD105, vimentin and CD90. For preconditioning, hU-MSCs were treated with isorhamnetin after selection of the optimized concentration (5 µmol/L) by cytotoxicity analysis. The migration potential of these MSCs was analyzed by the in vitro scratch assay. The healing potential of normal, and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound. Normal, and preconditioned MSCs (IH + MSCs) were transplanted after 72 h of burn injury and observed for 2 wk. Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.RESULTSThe scratch assay analysis showed a significant reduction in the scratch area in the case of IH + MSCs compared to the normal untreated MSCs at 24 h, while complete closure of the scratch area was observed at 48 h. Histological analysis showed reduced inflammation, completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH + MSCs. Gene expression analysis was time dependent and more pronounced in the case of IH + MSCs. Interleukin (IL)-1β, IL-6 and Bcl-2 associated X genes showed significant downregulation, while transforming growth factor β, vascular endothelial growth factor, Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound, showing increased angiogenesis and reduced inflammation and apoptosis.CONCLUSIONPreconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation, and improving tissue architecture and wound healing. The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.