In vitro expression of vital virulent genes of clinical and environmental isolates of Cryptococcus neoformans/gattii in endothelial cells of human blood-brain barrier

BackgroundEvaluation of the pathogenesis of clinical and environmental cryptococcal isolates to the central nervous system is necessary for understanding the risk. This study was designed to determine the in vitro expression of six important virulent genes of Cryptococcus neoformans/gattii in Human Brain Microvascular Endothelial cells (hBMEC).MethodsThe hBMEC were infected with Cryptococcus to determine invasion and survival rate at 3, 12 and 24 hours by subsequent colony count of internalized yeasts. The whole RNA of the intracellular Cryptococcus was extracted to quantify the expression of CAP10, PLB1, ENA1, URE1, LAC1, and MATα genes by real-time quantitative PCR for 3 and 12 hours of infection.ResultsInvasion and survival rates were higher in clinical and standard strains of C. neoformans. A significant difference was observed among the clinical and environmental isolates for the expression of CAP10, ENA1, LAC1, MATα and URE1 at 3 hours, and ENA1, LAC1, MATα, PLB1 and URE1 at 12 hours. Clinical isolates showed significant upregulation of all the genes except PLB1, which was higher in environmental isolates. Relative expressions at the two time-points showed statistically significant (P = 0.043) changes for the clinical isolates and no significance (P = 0.063) for environmental isolates.ConclusionThe C. gattii (VGI) isolates showed significantly lower invasion and survival than C. neoformans (VNI, and VNII) irrespective of their sources. Clinical isolates exhibited higher expression for the majority of the virulent genes until 12 hours of infection, probably due to their better adaptation in the host system and enhanced pathogenicity than the environmental counterparts.     

Sexually dimorphic development and binding characteristics of NMDA receptors in the brain of the platyfish.

This study investigated age- and gender-specific variations in properties of the glutamate N-methyl-d-aspartate receptor (NMDAR) in a freshwater teleost, the platyfish (Xiphophorus maculatus). Prior localization of the immunoreactive (ir)-R1 subunit of the NMDAR protein (R1) in cells of the nucleus olfactoretinalis (NOR), a primary gonadotropin-releasing hormone (GnRH)-containing brain nucleus in the platyfish, suggests that NMDAR, as in mammals, is involved in modulation of the platyfish brain-pituitary-gonad (BPG) axis. The current study shows that the number of cells in the NOR displaying ir-R1 is significantly increased in pubescent and mature female platyfish when compared to immature and senescent animals. In males, there is no significant change in ir-R1 expression in the NOR at any time in their lifespan. The affinity of the noncompetitive antagonist ((3)H)MK-801 for the NMDAR is significantly increased in pubescent females while maximum binding of ((3)H)MK-801 to the receptor reaches a significant maximum in mature females. In males, both MK-801 affinity and maximum binding remain unchanged throughout development. This is the first report of gender differences in the association of NMDA receptors with neuroendocrine brain areas during development. It is also the first report to suggest NMDA receptor involvement in the development of the BPG axis in a nonmammalian vertebrate.     

Reduction of infarct size in the rat heart by LPS preconditioning is associated with expression of angiogenic growth factors and increased capillary density.

Inflammation induces the expression of angiogenic growth factors in tissues, which leads to microvascular growth. Bacterial lipopolysaccharide (LPS) provokes a transient inflammatory response in the heart and induces delayed cardiac resistance to post-ischemic contractile dysfunction. In this study, we examined: 1) the effects of LPS on myocardial expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), 2) whether an increase in the density of myocardial microvessels follows the expression of angiogenic growth factors, and 3) the effect of LPS on myocardial resistance to infarction and its relationship with microvascular growth. Rats were treated with LPS (from Salmonella typhimurium, 0.5 mg/kg i.p.). The expression of bFGF and VEGF in the myocardium was examined at 6 and 12 h after LPS treatment by immunofluorescent staining. Myocardial capillary and arteriole densities were determined 3 days after LPS treatment by morphometry, using immunofluorescent staining of von Willebrand factor (a marker protein of endothelial cells) and alpha-smooth muscle actin (a marker protein of smooth muscle cells). To examine cardiac resistance to infarction, hearts were subjected to 40 min of regional ischemia and 2 h of reperfusion by reversible occlusion of left coronary artery at 3 days after LPS treatment. LPS induced cardiac bFGF and VEGF at 6 and 12 h after treatment. The expression of these growth factors was followed by an increase in myocardial capillary density (2032 +/- 78/mm2 vs. 1617 +/- 47/mm2 in saline control, P < 0.05), but not arteriole density, at 3 days. Meanwhile, infarct size was significantly reduced by LPS preconditioning (infarct/left ventricle 12.3 +/- 1.04% vs. 21.7 +/- 1.65% in saline control, 43% reduction, P < 0.05). These results suggest that LPS preconditioning induces cardiac bFGF and VEGF, and an increase in myocardial capillary density. This increased myocardial capillary density is associated with a reduced infarct size after in vivo regional ischemia-reperfusion.     

Influence of dietary ginger (Zingiber officinales Rosc) on oxidative stress induced by malathion in rats.

Pesticide chemicals may induce oxidative stress leading to generation of free radicals and alterations in antioxidants or oxygen free radical (OFR) scavenging enzymes. Hence, the effect of subchronic malathion (O,O-dimethyl-S-1,2, bis ethoxy carbonyl ethyl phosphorodithioate) exposure was evaluated on lipid peroxidation, glutathione and related enzymes and OFR scavenging enzymes in albino rats. Administration of malathion (20 ppm) for 4 weeks increased the malondialdehyde (MDA) levels in serum, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in erythrocytes and glutathione reductase (GR) and glutathione S-transferase (GST) in serum. However, it decreased the glutathione (GSH) level in whole blood. Concomitant dietary feeding of Zingiber officinales Rosc (ginger 1%, w/w) significantly attenuated malathion induced lipid peroxidation and oxidative stress in these rats. These results indicate the possible involvement of free radicals in organophosphate-induced toxicity and highlight the protective action of ginger, an indigenous medicinal plant product.     

Interaction of the cyanobacterial thiazoline-containing lipid curacin A with bovine brain tubulin

Curacin A is a thiazoline-containing lipid from the marine cyanobacterium Lyngbya majuscula. Despite being a potent inhibitor of microtubule assembly and of colchicine binding to tubulin, curacin A bears little or no structural resemblance to colchicine or to any other tubulin ligand. We investigated the interaction of curacin A with bovine brain tubulin using three different approaches. We first examined its effect on the intra-chain formation of a cross-link in β-tubulin by N,N′- ethylenebis(iodoacetamide). Formation of this cross-link, between cys²³⁹ and cys³⁵⁴, is blocked by colchicine and its A-ring analogues as well as by various other inhibitors of colchicine binding; C-ring analogues do not inhibit its formation. Curacin A strongly inhibited formation of this cross-link. Second, we examined the effect of curacin A on the time-dependent exposure of sulfhydryl groups on tubulin as measured by alkylation with iodo[¹⁴C]acetamide. Curacin A inhibited this very strongly, more so than either colchicine or podophyllotoxin. Last, we investigated the effect of curacin A on the time-dependent exposure of hydrophobic areas on the tubulin molecule. We found that curacin A had only a small effect on this process, comparable in magnitude to that of podophyllotoxin. Curacin A thus appears to have an unusual interaction with tubulin. Its binding site on tubulin is likely to overlap with that of the A-ring of colchicine. Drug Dev. Res. 40:223–229, 1997. © 1997 Wiley-Liss, Inc.