Effects of dietary sugars and saliva and serum on Candida bioflim formation on acrylic surfaces

The effect of two dietary sugars, glucose and galactose, on biofilm formation of the oral fungal pathogen Candida on denture acrylic strips coated with saliva and serum pellicles was examined in vitro using Candida albicans (3 isolates), C. glabrata (2 isolates) and C. tropicalis (2 isolates). The degree of biofilm activity was affected by both the dietary sugar and the nature of the pellicle (ANOVA, p < 0.01). With most isolates the glucose grown yeasts demonstrated significantly more bioflim activity than the galactose grown fungi, in the presence of pellicles (ANOVA, p < 0.01 or P < 0.01). In contrast, one isolate of galactose-grown yeast elicited significantly higher biofilm activity than glucose-grown yeasts on the control strips (ANOVA, p < 0.01). Taken together, these results imply that a saliva or a serum pellicle, and the carbon source in the environment, act a complex manner modulating Candida bioflim formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sequence Variation and Linkage Disequilibrium in the Human T-Cell Receptor β (TCRB) Locus

The T-cell receptor (TCR) plays a central role in the immune system, and > 90% of human T cells present a receptor that consists of the alpha TCR subunit (TCRA) and the beta subunit (TCRB). Here we report an analysis of 63 variable genes (BV), spanning 553 kb of TCRB that yielded 279 single-nucleotide polymorphisms (SNPs). Samples were drawn from 10 individuals and represent four populations-African American, Chinese, Mexican, and Northern European. We found nine variants that produce nonfunctional BV segments, removing those genes from the TCRB genomic repertoire. There was significant heterogeneity among population samples in SNP frequency (including the BV-inactivating sites), indicating the need for multiple-population samples for adequate variant discovery. In addition, we observed considerable linkage disequilibrium (LD) (r(2) > 0.1) over distances of approximately 30 kb in TCRB, and, in general, the distribution of r(2) as a function of physical distance was in close agreement with neutral coalescent simulations. LD in TCRB showed considerable spatial variation across the locus, being concentrated in "blocks" of LD; however, coalescent simulations of the locus illustrated that the heterogeneity of LD we observed in TCRB did not differ markedly from that expected from neutral processes. Finally, examination of the extended genotypes for each subject demonstrated homozygous stretches of >100 kb in the locus of several individuals. These results provide the basis for optimization of locuswide SNP typing in TCRB for studies of genotype-phenotype association.     

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.     

Alterations in inotropy,nitric oxide and cyclic GMP synthesis,protein phosphorylation and ADP-ribosylation in the endotoxin-treated rat myocardium and cardiomyocytes

To evaluate the effects of the in vivo endotoxin treatment of the rat on (1) the contractile responses in the subsequently isolated papillary muscle to adrenergic and cholinergic agonists and (2) the biochemical parameters (cyclic GMP, nitric oxide synthesis, protein phosphorylation and ADP-ribosyslation) in the subsequently isolated cardiomyocytes. Following the in vivo endotoxin treatment (4 mg/kg i.p., 18 h), contractile responses to increasing amounts of isoprenaline or to increasing amounts of oxotremorine in the presence of a fixed amount of isoprenaline were determined in isolated papillary strips. Activities of nitric oxide synthase, guanylyl cyclase, as well as phosphorylation of phospholamban and troponin-inhibitory subunit, and pertussis toxin-catalyzed and endogenous ADP-ribosylations were determined in the intact cardiomyocytes and subcellular fractions. The increase in the force of contraction by isoprenaline was reduced, while its inhibition by oxotremorine was greater in the endotoxin-treated papillary strips. The activities of both nitric oxide synthase, primarily of the inducible form of the enzyme, and cytosolic guanylyl cyclase were higher while the phosphorylations of both phospholamban and troponin-inhibitory subunit were of lesser magnitude in the cardiomyocytes following the in vivo endotoxin treatment. Pertussis toxin-catalyzed ADP-ribosylation of the 41 kDa polypeptide, which is the alpha subunit of Gi, was also decreased. The results of the present study support the postulate that alterations in both the cyclic AMP and cyclic GMP signalling cascade contribute to the myocardial dysfunction caused by endotoxin and cytokines.     

Characterization of the redox components of transplasma membrane electron transport system from Leishmania donovani promastigotes

An investigation has been made of the points of coupling of four nonpermeable electron acceptors e.g., alpha-lipoic acid (ALA), 5,5'-dithiobis (2-nitroaniline-N-sulphonic acid) (DTNS), 1,2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide which are mainly reduced via an interaction with the redox sites present in the plasma membrane of Leishmania donovani promastigotes. ALA, DTNS, NQSA and ferricyanide reduction and part of O2 reduction is shown to take place on the exoplasmic face of the cell, for it is affected by external pH and agents that react with the external surface. Redox enzymes of the transplasma membrane electron transport system orderly transfer electron from one redox carrier to the next with the molecular oxygen as the final electron acceptor. The redox carriers mediate the transfer of electrons from metabolically generated reductant to nonpermeable electron acceptors and oxygen. At a pH of 6.4, respiration of Leishmania cells on glucose substrate shut down almost completely upon addition of an uncoupler FCCP and K+-ionophore valinomycin. The most pronounced effects on O2 uptake were obtained by treatment with antimycin A, 2-heptadecyl-4-hydroxyquinone-N-oxide, paracholoromercuribenzene sulphonic acid and trifluoperazine. Relatively smaller effects were obtained by treatment with potassium cyanide. Inhibition observed with respect to the reduction of the electron acceptors ALA, DTNS, NQSA and ferricyanide was not similar in most cases. The redox chain appears to be branched at several points and it is suggested that this redox chain incorporate iron-sulphur center, b-cytochromes, cyanide insensitive oxygen redox site, Na+ and K+ channel, capsaicin inhibited energy coupling site and trifluoperazine inhibited energy linked P-type ATPase. We analyzed the influence of ionic composition of the medium on reduction of electron acceptors in Leishmania donovani promastigotes. Our data suggest that K+ have some role for ALA reduction and Na+ for ferricyanide reduction. No significant effects were found with DTNS and NQSA reduction when Na+ or K+ was omitted from the medium. Stimulation of ALA, DTNS, NQSA and ferricyanide reduction was obtained by omitting Cl- from the medium. We propose that this redox system may be an energy source for control of membrane function in Leishmania cells.     

TGF-alpha as a candidate tumor antigen for renal cell carcinomas

Objectives  Patients with renal cell carcinomas (RCC) have few treatment options, underscoring the importance of developing new approaches such as immunotherapy. However, few tumor associated antigens (TAA), which can be targeted by immunotherapy, have been identified for this type of cancer. von Hippel-Lindau clear cell RCC (VHL^−/−RCC) are characterized by mutations in the VHL tumor suppressor gene. Loss of VHL function causes the overexpression of transforming growth factor (TGF)-α, leading us to hypothesize that TGF-α could be a potential TAA for immunotherapy of kidney cancer, which was evaluated in this study. Methods and results  We first confirmed the absent or weak expression of TGF-α in important normal tissues as well as its overexpression in 61% of renal tumors in comparison to autologous normal kidney tissues. In addition, we demonstrated the immunogenicity of TGF-α, by expanding many T cell lines specific for certain TGF-α peptides or the mature TGF-α protein, when presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. Interestingly, some of these TGF-α-specific T cells were polyfunctionals and secreted IFN-γ, TNF-α and IL-2. Conclusion  We have shown that TGF-α is a valid candidate TAA, which should allow the development of a targeted immunotherapy.     

Altering conserved lipid binding sites in cytochrome c oxidase of Rhodobacter sphaeroides perturbs the interaction between subunits I and III and promotes suicide inactivation of the enzyme

Subunit III of the three-subunit catalytic core of cytochrome c oxidase (CcO) contains no metal centers, but it does bind two lipids, within a deep cleft, in binding sites conserved from bacteria to humans. Subunit III binds to subunit I, where it prevents the spontaneous suicide inactivation of CcO by decreasing the probability of side reactions at the heme-Cu O2 reduction site in subunit I. Subunit III prevents suicide inactivation by (1) maintaining adequate rates of proton delivery to the heme-Cu active site and (2) stabilizing the structure of the active site during turnover [Mills and Hosler (2005) Biochemistry 44, 4656]. Here, we first show that mutating several individual residues of the conserved lipid binding sites in subunit III disturbs the subunit I-III interface. Then, two lipid binding site mutants were constructed with an affinity tag on subunit III such that the mutant CcOs could be isolated with 100% subunit III. R226A eliminates an ion pair to the phosphate of the outermost lipid of the cleft, while W59A-F86A disrupts interactions with the fatty acid tails of both lipids. Once these mutant CcOs are placed into soybean phospholipid vesicles, where extensive exchange of bacterial for soybean lipids takes place, it is shown that altering the lipid binding sites mimics a major loss of subunit III, even though subunit III is completely retained, in that suicide inactivation becomes much more probable. The rate of proton delivery to the active site remains rapid, ruling out slow proton uptake as the primary reason for increased suicide inactivation upon alteration of the lipid binding sites. We conclude that altering the lipid binding sites of subunit III may promote side reactions leading to suicide inactivation by allowing greater movement to occur in and around the O2 reduction site of subunit I during the catalytic cycle.