Regulation of immunity by a novel population of Qa-1-restricted CD8alphaalpha+TCRalphabeta+ T cells

Regulatory mechanisms involving CD8+ T cells (CD8 regulatory T cells (Tregs)) are important in the maintenance of immune homeostasis. However, the inability to generate functional CD8 Treg clones with defined Ag specificity has precluded a direct demonstration of CD8 Treg-mediated regulation. In the present study, we describe the isolation of functional lines and clones representing a novel population of TCRalphabeta+ Tregs that control activated Vbeta8.2+ CD4 T cells mediating experimental autoimmune encephalomyelitis. They express exclusively the CD8alphaalpha homodimer and recognize a peptide from a conserved region of the TCR Vbeta8.2 chain in the context of the Qa-1a (CD8alphaalpha Tregs). They secrete type 1 cytokines but not IL-2. CD8alphaalpha Tregs kill activated Vbeta8.2+ but not Vbeta8.2- or naive T cells. The CD8alphaalpha Tregs prevent autoimmunity upon adoptive transfer or following in vivo activation. These findings reveal an important negative feedback regulatory mechanism targeting activated T cells and have implications in the development of therapeutic strategies for autoimmune diseases and transplantation.     

Molecular Dynamic Simulation Reveals Damaging Impact of RAC1 F28L Mutation in the Switch I Region

Ras-related C3 botulinum toxin substrate 1 (RAC1) is a plasma membrane-associated small GTPase which cycles between the active GTP-bound and inactive GDP-bound states. There is wide range of evidences indicating its active participation in inducing cancer-associated phenotypes. RAC1 F28L mutation (RAC^F28L) is a fast recycling mutation which has been implicated in several cancer associated cases. In this work we have performed molecular docking and molecular dynamics simulation (~0.3 μs) to investigate the conformational changes occurring in the mutant protein. The RMSD, RMSF and NHbonds results strongly suggested that the loss of native conformation in the Switch I region in RAC1 mutant protein could be the reason behind its oncogenic transformation. The overall results suggested that the mutant protein attained compact conformation as compared to the native. The major impact of mutation was observed in the Switch I region which might be the crucial reason behind the loss of interaction between the guanine ring and F28 residue.     

A novel approach for extraction of PCR-compatible DNA from activated sludge samples collected from different biological effluent treatment plants

This paper describes a method that facilitates the extraction of PCR-compatible DNA from different activated sludge samples. The approach involves a novel preprocessing step in DNA extraction, which removes potential PCR inhibitors. The sludge was washed with different ratios of acetone and petroleum ether after pretreatment with 0.01% Tween-20 at 50 degrees C. It was observed that an initial washing step with 50 mM Tris-HCl, pH 9.0, before the detergent-solvent step, improved the quality of the extracted DNA. The extraction protocol resulted in amplifiable amounts of DNA when 10 mg of a sludge sample was used, even in the presence of phenol as a sludge contaminant. The usefulness of the extracted template was demonstrated by carrying out different PCR reactions. The random amplified polymorphic DNA (RAPD) patterns demonstrated the diversity of sludge samples.     

Variation in pituitary responsiveness to synthetic gonadotropin releasing hormone (GnRH) during different phases of the menstrual cycle (author's transl)

Synthetic GnRH, at a dose of 100 mcg, was injected intravenously into 12 healthy, single, regulary menstruating women in order to test the capacity of the pituitary to release LH and FSH in response to the administration of the decapeptide. A total of 12 tests was performed during different stages of the menstrual cycle, i.e., on D 3-4, D 13-16 and D 21-29 of the cycle. Following GnRH administration, there was a rapid increase in serum levels of LH. Although there was a pronounced variation of responses in the course of the menstrual cycle, the maximum response was observed 30 to 40 min., after injection. The mean net increases of LH (M +/- SE mIU/ml) were in the following order: 118 +/- 22 in the preovulatory phase, 63 +/- 12 in the midluteal phase, and 35 +/- 7 in the early follicular phase. A concomitant but much smaller rise in serum levels of FSH was observed. These data indicate that the sensitivity of pituitary gonadotrophs to GnRH is preferentially increased during the preovulatory phase of the cycle, thus lending further support to already published data which demonstrated increased pituitary sensitivity to GnRH toward midcycle.     

Genomic annotation and validation of bacterial consortium NDMC-1 for enhanced degradation of sugarcane bagasse

This study aims at designing a consortium using rumen bacterial isolates for enhancing the hydrolysis of sugarcane bagasse (SB) for efficient biofuel formation. The microbial population was screened through biochemical and molecular tools along with enzymatic activity to obtain potential isolates for diverse cellulolytic and hemicellulolytic carbohydrate active enzyme (CAZyme). Five strains (Paenibacillus, Bacillus, Enterobacter, and Microbacterium) were selected for designing the consortium NDMC-1. The hydrolytic efficiency of NDMC-1 was determined based on cellulase production with simultaneous rise in monosaccharides, oligosaccharides, and soluble chemical oxygen demand (sCOD) concentration. Cellulolytic machinery of these isolates was further explored using genome sequencing. The isolates selected for consortia NDMC-1 interacted synergistically leading to enhanced cellulase production. Maximal endoglucanase (1.67 μmol ml−1 min−1), exoglucanase (0.69 μmol ml−1 min−1), and β-glucosidase (2.03 μmol ml−1 min−1) activity were achieved with SB as a sole carbon source after 48 h of incubation. Enhancement in SB hydrolysis employing NDMC-1 was evident by the increase in sCOD from 609 to 2589 mg/l and release of 1295 mg/l reducing sugar, comprising 59.8%, 8.23%, and 6.16% of glucose, cellobiose, and cellotriose, respectively, which resulted in 5.5-fold rise in biogas production. On genome annotation, total 472 contigs from glycoside hydrolase family: 84 from Microbacterium arborescens ND21, 72 from Enterobacter cloacae ND22, 61 from Bacillus subtilis ND23, 116 from Paenibacillus polymyxa ND24, and 140 from Paenibacillus polymyxa ND25 were identified. On further analysis, total 33 cellulases, 59 hemicellulases, and 48 esterases were annotated in the reported genomes. This work proposes the application of consortia-based bioprocessing systems over the conventionally favorable single organism approach for efficient hydrolysis of cellulosic substrates to fermentable sugars.     

Regulation of aromatase activity by cytokines, PGE2 and 2-methoxyoestrone-3-O-sulphamate in fibroblasts derived from normal and malignant breast tissues

Synthesis of oestrone from androstenedione within tumours, by the aromatase enzyme complex, is an important source of oestrogen that is available to support the growth of hormone-dependent breast tumours. In view of the central role that the aromatase enzyme has in oestrogen synthesis there has been considerable interest in understanding its regulation and developing inhibitors to block its action. In the present study we have derived fibroblasts from breast tumours (TFs), tissue proximal to tumours (PFs) and reduction mammoplasty tissue (RMFs) and used them to investigate the regulation of aromatase activity by PGE(2), IL-6 plus its soluble receptor (SR) or TNFalpha. In addition we have examined the ability of 2-methoxyoestrone sulphamate (2-MeOEMATE), a compound which alters microtubule stability, to block the stimulation of aromatase activity by these factors. Basal aromatase activity in PFs was significantly higher (p<0.001) than in TFs or RMFs. The combination of IL-6 plus SR or TNFalpha produced the greatest stimulation of aromatase activity in TFs (up to 61-fold) while having a much lower stimulatory effects on aromatase activity in PFs (up to 60% increase) or RMFs (up to 192% increase). 2-MeOEMATE reduced basal aromatase activity in TFs by 87% and completely abrogated the ability of PGE(2), IL-6 plus SR or TNFalpha to stimulate aromatase activity in these fibroblasts. Results from these studies indicate that while PFs have the highest level of non-stimulated aromatase activity, aromatase activity in TFs shows the greatest response to cytokines. These findings suggest that intrinsic difference may exist for the different types of fibroblasts in the way in which they respond to regulatory factors. The ability of 2-MeOEMATE to block cytokine stimulated aromatase activity suggests that, in addition to its other anti-cancer properties, this compound may also act to inhibit cytokine-stimulated aromatase activity in breast tumours.     

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH

The role of external Ca²⁺ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca²⁺ in the medium. The observed Ca²⁺-mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca²⁺ in the medium. Thus, the ability of Ca²⁺ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca²⁺ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca²⁺ channel blockers tested, such as verapamil and LaCl₃, inhibited the observed Ca²⁺-mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m -chlorophenyl hydrazone, inhibited this Ca²⁺-mediated response, whereas monensin, an inhibitor of the Na⁺/H⁺ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca²⁺ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.     

Antiviral activity and synthesis of quaternized promazine derivatives against HSV-1

N-(4-chlorobenzyl)triflupromazinium chloride, a known antitubercular agent, has been found to also be active against HSV-1. A preliminary structure-activity relation has been explored to determine which groups are crucial to viral inhibition. Antiviral assessments such as GFP reduction, plaque reduction, treatment timing and wash-out studies have also been probed to determine a mode of action for QPD-1. Based on this preliminary data, it appears that QPD-1 is a reversible inhibitor, suspected to inhibit early stages of viral replication of HSV-1 at 50μM, equipotent to acyclovir.     

Isolation and Characterization of <Emphasis Type="Italic">Citrobacter</Emphasis> Strain HPC255 for Broad-Range Substrate Specificity for Chlorophenols

This study aims at development of an approach for selection of strain, which has capability for oxidation of broad-range of chloro-substitute phenols. A multiplex PCR was optimized targeting loci involved in phenol and chlorophenol degradation, which was used to select activated sludge samples and also to assess the degradative genotype of isolates. The isolated strains were screened on the basis of RAPD analysis. In parallel, physiological experiments were carried out with activated sludge samples and isolated bacteria by respirometric analysis. Based on cluster analysis of RAPD pattern and respirometric data, the isolate G20 was selected and identified by using 16S rDNA sequence analysis as Citrobacter freundii strain HPC255. The strain could oxidize different substituted chlorophenol molecules. Such strains could provide the pool of intermediates, which can further be degraded by the associated population, thus helping in maintaining the synergistic association of catabolic activity in activated sludge.     

Discrimination models using variance-stabilizing transformation of metabolomic NMR data

After the extensive work that is being done in the areas of genomics, proteomics, and metabolomics, the study of metabolites has come of interest in its own right. Metabolites in biological systems give an understanding of the state of the system and provide a powerful tool for the study of disease and other maladies. Several analytical techniques such as mass spectrometry and high-resolution NMR spectroscopy have been used to study metabolites. The data, however, from these techniques remains quite complex. Traditionally, multivariate analyses have been used for such data. These methods however have an underlying assumption that the data is multivariate normal with a constant variance. This is not necessarily the case. It has been shown that a generalized log transformation renders the variance of the data constant effectively making the data more suitable for multivariate analysis. We demonstrate the effectiveness of these transformations on NMR data taken on a set of 18 abalone that were categorized as either being healthy, stunted, or diseased. We show how the transformation makes multivariate classification of the abalone into the healthy, stunted and diseased categories much more effective and gives a tool for identifying potential metabolic biomarkers for disease.