Association of Xmn1 −158 γG variant with severity and HbF levels in β-thalassemia major and sickle cell anaemia

Haemoglobinopathies including β-thalassemia and sickle cell anaemia (SCA) are considered to be classical monogenic diseases. There is considerable clinical variability between patients inheriting identical β-globin mutations. The reasons for this variability are not well understood. Previous studies have suggested that a variety of genetic determents influence different clinical phenotypes. The genetic variants that modulate HbF levels have a very strong impact on ameliorating the clinical phenotype. In the present study 6,500 blood samples from suspected cases were analysed using HPLC, ARMS-PCR, RDB techniques. Patients with β-thalassemia and SCA were classified into mild, moderate, severe according to the severity score based on Hb levels, age of onset, age at which patients received their first blood transfusion, the degree of growth retardation and splenectomy. Patients with β-thalassemia and SCA were analysed for Xmn1 polymorphism and association between this polymorphism and severity of β-thalassemia and SCA was evaluated. We found a significant difference in genotypic and allelic frequencies of Xmn1 polymorphism between mild and moderate and mild and severe cases. There was a significant difference in high and low percentage of HbF in CC, CT and TT bearing individuals. The TT bearing individuals were found to have a high percentage of HbF in β-thalassemia as well as SCA. This study confirms that increased γ^G-globin expression associated with Xmn1 polymorphism ameliorates the clinical severity in β-thalassemia as well as SCA in the study population.     

Cross Talk between NDR Kinase Pathways Coordinates Cytokinesis with Cell Separation in Schizosaccharomyces pombe

NDR (nuclear Dbf-2-related) kinases constitute key regulatory nodes in signaling networks that control multiple biological processes such as growth, proliferation, mitotic exit, morphogenesis, and apoptosis. Two NDR pathways called the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR) exist in the fission yeast Schizosaccharomyces pombe. The SIN promotes cytokinesis, and the MOR drives cell separation at the end of cytokinesis and polarized growth during interphase. We showed previously that cross talk exists between these two pathways, with the SIN inhibiting the MOR during cytokinesis through phosphorylation of the MOR component Nak1 by the SIN Sid2 kinase. The reason for this inhibition remained uncertain. We show here that failure to inhibit MOR signaling during cytokinesis results in cell lysis at the site of septum formation. Time-lapse analysis revealed that MOR signaling during cytokinesis causes cells to prematurely initiate septum degradation/cell separation. The cell lysis phenotype is due to premature initiation of cell separation because it can be rescued by mutations in genes required for cell separation/septum degradation. We also shed further light on how the SIN inhibits the MOR. Sid2 phosphorylation of the MOR proteins Sog2 and Nak1 is required to prevent cell lysis during cytokinesis. Together, these results show that SIN inhibition of the MOR enforces proper temporal ordering of cytokinetic events.     

Rationally selected single-site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates

Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. Using a rational mutagenesis approach based on structural bioinformatics and evolutionary analysis, two positions (F16S and Y95F) were identified as priority sites for mutagenesis. The mutant and parent enzymes were expressed and secreted from Pichia pastoris and the single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in k(cat) and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent enzyme, the two variants were active between pH 4.0 and 8.0 and showed optimal activity at temperature 70°C at pH 5.0. The purified enzymes were active at 50°C for over 12 h and retained at least 80% of initial activity for 2 h at 70°C. In contrast to the improved hydrolysis seen with the single mutation enzymes, no improvement was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.     

Phytochemical Profiling,Antimicrobial, Antiproliferative and Apoptotic Effects of Stemodia viscosa Roxb. of Western Ghats Region,India

The present study shows the chemical profile, antimicrobial, antiproliferative, and apoptotic effects of Stemodia viscosa extracts. Thirteen bioactive compounds were identified in the 80 % ethanolic extract by GC/MS analysis. The acetone extract exhibited a higher content of flavonoids and phenols of 805.10 μg QE/mg DW and 89.31 μg GAE/mg DW extracts, respectively. Furthermore, the acetone extract possessed the highest antioxidant activity (IC₅₀=9.96 μg/mL). The 80 % ethanolic extract exhibited significant antimicrobial activity; the highest activity was observed against Staphylococcus aureus with a zone of inhibition of 25±0.51 mm, MIC value of 4 mg/mL, and MBC value of 8 mg/mL. The antiproliferative results revealed the presence of anticancer activity with an IC₅₀=91.562 and 74.362 μg/mL against the B16F10 skin and COLO205 colon cancer cells, respectively. The flow cytometric analysis shows that the plant extracts cause cancer cell death through the induction of apoptosis. Our findings confirmed that Stemodia viscosa is a potential source of biologically active compounds.     

QSAR studies on benzene sulfonamide carbonic anhydrase inhibitors: need of hydrophobic parameter for topological modeling of binding constants of sulfonamides to human CA-II

The binding constants (logK) of benzene sulfonamides to human CA-II have been modeled using a large series of distance-based topological indices. The need or otherwise of the hydrophobic parameter (logP) for such topological modeling of logK has been examined critically. In both the cases excellent results have been obtained. In multiparametric models involving indicator parameters we observed that introduction of hydrophobic parameter (logP) yields much improved statistics. The results are discussed on the basis of statistical parameters and also by using cross-validation method.