The role of the Candida albicans histidine kinase [CHK1) gene in the regulation of cell wall mannan and glucan biosynthesis

The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.     

Groundwater Contaminated with Hexavalent Chromium [Cr (VI)]: A Health Survey and Clinical Examination of Community Inhabitants (Kanpur,India)

Background

We assessed the health effects of hexavalent chromium groundwater contamination (from tanneries and chrome sulfate manufacturing) in Kanpur, India.

Methods

The health status of residents living in areas with high Cr (VI) groundwater contamination (N = 186) were compared to residents with similar social and demographic features living in communities having no elevated Cr (VI) levels (N = 230). Subjects were recruited at health camps in both the areas. Health status was evaluated with health questionnaires, spirometry and blood hematology measures. Cr (VI) was measured in groundwater samples by diphenylcarbazide reagent method.

Results

Residents from communities with known Cr (VI) contamination had more self-reports of digestive and dermatological disorders and hematological abnormalities. GI distress was reported in 39.2% vs. 17.2% males (AOR = 3.1) and 39.3% vs. 21% females (AOR = 2.44); skin abnormalities in 24.5% vs. 9.2% males (AOR = 3.48) and 25% vs. 4.9% females (AOR = 6.57). Residents from affected communities had greater RBCs (among 30.7% males and 46.1% females), lower MCVs (among 62.8% males) and less platelets (among 68% males and 72% females) than matched controls. There were no differences in leucocytes count and spirometry parameters.

Conclusions

Living in communities with Cr (VI) groundwater is associated with gastrointestinal and dermatological complaints and abnormal hematological function. Limitations of this study include small sample size and the lack of long term follow-up.     

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.     

Detection of double stranded RNA in phytopathogenic <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> causing charcoal rot in <Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>

One hundred one isolates of Macrophomina phaseolina from various hosts and eco-geographical locations were employed for elucidating relationships among genetic diversity and virulence. Highly pathogenic, moderately pathogenic, and hypovirulent cluster bean specific isolates were identified. In order to correlate respective phenotypes of plant pathogenic fungus multiple and complex patterns of dsRNA elements were analyzed. Double-stranded ribonucleic acids (dsRNA) are ubiquitous in all major groups and most of them have vast potential as biological control agents for fungi. Rate of virulence and its further association could ascertain by host plant and their fungal genotypes. Variability of the fungal genotypes decides the link between the complexity of dsRNA with different variants and the change in virulence pattern. Double-stranded RNA was identified in approximately 21.7% of M. phaseolina isolates from charcoal rot infected cluster bean varieties. After recurrent laboratory transfer on culture media, the preponderance of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to cluster bean. Macrophomina has successfully showed diversified and reproducible banding profile in dsRNA containing/free isolates. This is the first report of hypovirulence and detection of dsRNA in Macrophomina phaseolina isolates of cluster bean origin.     

Calcium supplementation modulates arsenic-induced alterations and augments arsenic accumulation in callus cultures of Indian mustard (Brassica juncea (L.) Czern.)

In the present study, the effect of arsenate (AsV) exposure either alone or in combination with calcium (Ca) was investigated in callus cultures of Brassica juncea (L.) Czern. cv. Pusa Bold grown for a period up to 24 h. The AsV?(250 μM) + Ca (10 mM) treatment resulted in a significantly higher level of As (464 μg g^?1 dry weight (DW)) than AsV without Ca (167 μg g^?1 DW) treatment at 24 h. Furthermore, AsV + Ca-treated calli had a higher percent of AsIII (24–47%) than calli subjected to AsV treatment (12–14%). Despite this, AsV + Ca-treated calli did not show any signs of hydrogen peroxide (H₂O₂) accumulation or cell death upon in vivo staining, while AsV-exposed calli had increased H₂O₂, shrinkage of cytoplasmic contents, and cell death. Thus, AsV treatment induced oxidative stress, which in turn elicited a response of antioxidant enzymes and metabolites as compared with control and AsV + Ca treatment. The positive effects of Ca supplementation were also correlated to an increase in thiolic constituents', viz., cysteine, reduced glutathione, and glutathione reductase in AsV + Ca than in AsV treatment. An analysis of selected signaling related genes, e.g., mitogen-activated protein kinases (MAPK3 and MAPK6) and jasmonate ZIM-domain (JAZ3) suggested that AsV and AsV + Ca followed variable pathways to sense and signal the As stress. In AsV-alone treatment, jasmonate signaling was seemingly activated, while MAPK3 was not involved. In contrast, AsV + Ca treatment appeared to specifically inhibit jasmonate signaling and activate MAPK3. In conclusion, Ca supplementation may hold promise for achieving increased As accumulation in plants without compromising their tolerance.     

Carboxymethyl tamarind kernel polysaccharide nanoparticles for ophthalmic drug delivery

Tropicamide-loaded carboxymethyl tamarind kernel polysaccharide (CMTKP) nanoparticles were prepared and evaluated for ocular delivery. Preparation of ionotropically gelled CMTKP nanoparticles was optimized employing three-levels, two-factor central composite design. Concentration of polymer and crosslinker had significant synergistic effect on particle size and % encapsulation efficiency. The optimal calculated parameters were concentrations of CMTKP 0.10% (w/v) and calcium chloride 0.11% (w/v). The optimized tropicamide-loaded CMTKP formulation showed ex vivo corneal permeation of tropicamide across isolated goat cornea comparable to its aqueous solution. Further, the mucoadhesive and non-irritant nature of CMTKP nanoparticles indicate their suitability as ocular delivery system.     

Synthesis, characterization and evaluation of thiolated tamarind seed polysaccharide as a mucoadhesive polymer

In the present study, thiol-functionalization of tamarind seed polysaccharide was carried out by esterification with thioglycolic acid. Thiol-functionalization was confirmed by SH stretch in Fourier-transformed infra-red spectra at 2586cm(-1). It was found to possess 104.5mM of thiol groups per gram. The results of differential scanning calorimetry and X-ray diffraction study indicate increase in crystallinity. Polymer compacts of thiolated tamarind seed polysaccharide required 6.85-fold greater force to detach from the mucin coated membrane than that of tamarind seed polysaccharide. Comparative evaluation of Carbopol-based metronidazole gels containing thiolated tamarind seed polysaccharide with gels containing tamarind seed polysaccharide for mucoadhesive strength using chicken ileum by modified balance method revealed higher mucoadhesion of gels containing thiolated tamarind seed polysaccharide. Further, the gels containing tamarind seed polysaccharide and thiolated tamarind seed polysaccharide released the drug by Fickian-diffusion following the first-order and Higuchi's-square root release kinetics, respectively.     

HIV-1 diseases progression associated with loss of Th17 cells in subtype 'C' infection

Th17 cells play a crucial role in host immune response. We examined the role of Th17 cells in HIV-1 'subtype-C' infection and report that HIV-1 specific Th17 cells are induced in early infection and slow progressors but are significantly reduced at late stage of infection. There was a further decline in Th17 cells in late stage subjects with gastrointestinal infections. Additionally, we observed expanded population of IL-21 (needed for Th17 population expansion) producing CD4 T cells in early and slow progressors compared to subjects with late stage infection. A significant positive correlation existed between virus specific IL-17 and IL-21 producing CD4 T cells suggesting that HIV-1 infection induces a demand for Th17 cells. A significant negative correlation between virus specific Th17 cells and HIV-1 plasma viral load (pVL) was also observed, indicating a gradual loss of Th17 cells with HIV-1 disease progression.     

Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females

The single-cell gel electrophoresis or Comet assay measures qualitative and quantitative DNA damage in single cells. Its simplicity and non-invasive nature has made it widely accepted for the monitoring of human genotoxicity, employing peripheral blood lymphocytes. Factors, such as gender, age, and dietary and smoking habits are known to affect the Comet assay responses in lymphocytes. However, there is no information regarding the influence of the menstrual cycle on the results of the assay in lymphocytes of females.A study was therefore undertaken among 18 healthy Indian female volunteers to assess the effect of the menstrual cycle on Comet assay responses. During a complete menstrual cycle, only minor changes were observed in the basal levels of DNA damage in the lymphocytes as evident by Comet parameters, such as tail length (μm), tail DNA (%) and Olive tail moment (arbitrary units).To assess the effect of the estrogen 17β-estradiol (at physiological concentrations of 0.5, 1.0 and 2.0 nM) on the Comet assay responses, an in vitro study was conducted in the human lymphocyte cell line JM-1 and the breast cancer cell line MCF-7. As was evident from the Comet parameters, a significant (p < 0.01) concentration-dependent increase in the level of DNA damage was observed in the MCF-7 cells while no significant change was found in the JM-1 cells.The results indicate that the menstrual cycle does not influence the Comet assay responses in lymphocytes; hence, these can serve as a model for monitoring genotoxicity in females.