Q2N and E64G double mutation of ubiquitin confers a stress sensitive phenotype on Saccharomyces cerevisiae.

The eukaryotic protein, ubiquitin harbours a parallel β-bulge in its structure which is formed by residues Glu64(1), Ser65(2) and Gln2(X). Despite their low % frequency of occurrence in parallel β-bulges, the residues Gln2 and Glu64 have been totally conserved in ubiquitin. In a previous study, two single mutants UbQ2N and UbE64G were constructed by replacing the residues Gln2 and Glu64 with Asn and Gly, respectively to understand their importance. The choice of the residues for substitution was made on the basis of their high preference for existence in parallel β-bulge, so that the structure of mutants remains unaltered and any functional differences observed would highlight the importance of Gln2 and Glu64 in ubiquitin biology. The results from this study established that yeast cells expressing either UbQ2N or UbE64G, displayed functional differences with respect to survival upon exposure to cycloheximide and degradation of substrates by ubiquitin fusion degradation (UFD) pathway. It describes construction of the double mutant UbQ2N-E64G and its characterization. Our results showed expression of UbQ2N-E64G in stress hypersensitive SUB60 cells led to significant decrease in growth rate and prolonged half-life of substrates of UFD pathway, besides failure of complementation under heat and antibiotic stresses, providing the reason for conservation of Gln2 and Glu64 in ubiquitin sequence.

Construction and functional characterization of double and triple mutants of parallel -bulge of ubiquitin.

Ubiquitin, a small eukaryotic protein serving as a post-translational modification on many important proteins, plays central role in cellular homeostasis and cell cycle regulation. Ubiquitin features two -bulges, the second -bulge, located at the C-terminal region of the protein along with type II turn, holds 3 residues Glu64(1), Ser65(2) and Gln2(X). Percent frequency of occurrence of such a sequence in parallel -bulge is very low. However, the sequence and structure have been conserved in ubiquitin through out the evolution. Present study involves replacement of residues in unusual -bulge of ubiquitin by introducing mutations in combination through site directed mutagenesis, generating double and triple mutants and their functional characterization. Mutant ubiquitins cloned in yeast expression vector YEp96 tested for growth profile, viability assay and heat stress complementation study have revealed significant decrease in growth rate, loss of viability and non-complementation of heat sensitive phenotype with UbE64G-S65D and UbQ2N-E64G-S65D mutations. However, UbQ2N-S65D did not show any negative effects in the above assays. Present results show that, replacement of residues in -bulge of ubiquitin exerts severe effects on growth and viability in Saccharomyces cerevisiae due to functional failure of the mutant ubiquitins UbE64G-S65D and UbQ2N-E64G-S65D.

Leptin response in patients with tuberculous pleuritis.

BACKGROUND & OBJECTIVES: Tuberculous pleuritis is used as a model to understand the protective immune response in tuberculosis. It is predominated by Th1 response at the site of infection, where a possible role for the leptin, a known enhancer of Th1 response, could be speculated. Hence, we investigated leptin levels in pleural effusions in patients with both tuberculous (TP) and non-tuberculous (NTP) pleural effusion. METHODS: Leptin and cytokine levels were assessed in serum and pleural fluid of TP and NTP patients (N = 20 each) by ELISA. Multivariate regression analysis were performed to find the possible determinants of leptin taking leptin as the dependent and body mass index (BMI), gender, source of leptin [i.e., serum or pleural fluid (PF)], age and disease status as independent variables. RESULTS: PF leptin levels were significantly higher than serum leptin levels in both the groups however the PF leptin levels were significantly lower in TP subjects compared to NTP. The results showed that the leptin was found to be dependent on BMI but not on the other parameters. However, regression analysis based on the source of leptin showed males to be a better predictor of leptin. No correlation was observed between leptin and measured immune parameters. INTERPRETATION & CONCLUSION: Our findings demonstrated that the decreased leptin levels were associated with reduction in BMI but not with the disease status in tuberculous pleuritis.

Differential T helper cell response in tuberculous pleuritis.

PURPOSE: This study was conducted to understand the in vivo and in vitro immune responses and to find whether there exists any difference in the systemic and localized immune responses in tuberculous pleuritis. METHODS: The in vivo levels of IFN-gamma and IL-4 were compared in plasma (BL) and pleural fluid (PF) of 47 tuberculous (TB) and 31 nontuberculous pleuritis (Non-TB) patients. In vitro cytokine response to various mycobacterial antigens was studied in 19 TB patients by ELISA. Both ex vivo and in vitro cytokine responses were further ascertained by intracellular cytokine staining on purified CD4+ T cells from pleural fluid mononuclear cells (PFMC) of 10 TB patients. RESULTS: The ex vivo results showed a significant increase in IFN-gamma levels and higher IFN-gamma + T cells in PF. On the other hand, in vitro results showed simultaneous increase in both IFN-gamma and IL-4 levels in the supernatants of antigen stimulated PFMC. Similarly antigen specific increase was observed in both IFN-gamma + and IL-4+ T cells in all cultured conditions. However, the percentile increase was more in IL-4 secreting T cells, significant for PPD stimulation (P < 0.05), indicating that in vitro cellular response was dominated by Th2 type. CONCLUSIONS: These results showed a differential T-helper response in TB pleuritis suggestive of predominant Th1 in vivo and mixed response (Th1 and Th2) under in vitro conditions.

Juvenile hyaline fibromatosis--a rare case report.

Fibromatosis is a group of disorders characterized by infiltrating fibroblastic proliferation. Among them Juvenile hyaline fibromatosis (J.H.F) is a rare, progressive, crippling autosomal recessive disorder diagnosed based on the characteristic clinicopathological findings of generalized cutaneous nodular lesions, gingival hypertrophy, flexion contractures of large joints with osteolytic lesions, and proliferating fibroblasts set within a hyalinized stroma. This disorder commonly manifests in childhood with family history of consanguinity and in siblings. A case of seven year old boy born to consanguinous parents is reported who presented with multiple subcutaneous nodules and gingival hypertrophy. Histopathological examination revealed proliferating fibroblasts embedded in an abundant homogeneous eosinophilic hyalinized matrix. The matrix showed PAS stain positivity, supporting the pathogenesis of this disorder as an inborn error of glycosaminoglycans metabolism. The differential diagnosis is discussed and the literature reviewed.

Isolated pure lipoma of the uterus--a case report.

Lipoma primary to uterus is a rare entity among uterine fatty tumors. These create preoperative diagnostic confusion and their histogenesis is still unclear. Only a few cases of pure lipoma of uterus have been reported in the last few decades. A case of isolated pure lipoma primary to uterus diagnosed postoperatively by pathological examination is presented.

Conformational features of reduced and disulfide intact forms of hen egg white lysozyme in aqueous solution in presence of 3-chloro-1, 2-propanediol and dioxane: implications for protein folding intermediates.

Conformational features of reduced and disulfide intact hen egg white lysozyme in aqueous 1,4-dioxane and 3-chloro-1, 2-propanediol solutions have been examined using circular dichroism and fluorescence spectroscopy. We find that in presence of 1, 4-dioxane, reduced lysozyme assumes a relatively compact conformational form with secondary structure closer to native state and no tertiary structure as judged by peptide and aromatic CD spectra and ANS binding studies monitored by fluorescence. Further, in presence of 40% (v/v) 3-chloro-1, 2-propanediol, disulfide intact lysozyme (DI-lysozyme) assumes a conformational form with native like secondary structure and no tertiary structure akin to a molten globule state. We correlate our results to kinetic hydrogen- deuterium exchange NMR results of the refolding of lysozyme available in literature and suggest that the conformational forms observed in our study could be models for kinetic intermediates in the refolding of lysozyme.