Heterologous expression and characterization of glycoside hydrolase with its potential applications in hyperthermic environment

With the progressive focus on renewable energy via biofuels production from lignocellulosic biomass, cellulases are the key enzymes that play a fundamental role in this regard. This study aims to unravel the characteristics of Thermotoga maritima MSB8 (Tma) (a hyperthermophile from hot springs) thermostable glycoside hydrolase enzyme. Here, a glycoside hydrolase gene of Thermotoga maritima (Tma) was heterologously expressed and characterized. The gene was placed in the pQE-30 expression vector under the T5 promotor, and the construct pQE-30-Gh was then successfully integrated into Escherichia coli BL21 (DH5α) genome by transformation. Sequence of the glycoside hydrolase contained an open reading frame of 2.124 kbp, encoded a polypeptide of 721 amino acid residues. The molecular weight of the recombinant protein estimated was 79 kDa. The glycoside hydrolase was purified by Ni⁺²-NTA affinity chromatography and its enzymatic activity was investigated. The recombinant enzyme is highly stable within an extreme pH range (2.0–7.0) and highly thermostable at 80 °C for 72 h indicating its viability in hyperthermic environment and acidic nature. Moreover, the Ca²⁺ and Mn²⁺ introduction stimulated the residual activity of recombinant enzyme. Conclusively, the thermostable glycoside hydrolase possesses potential to be exploited for industrial applications at hyperthermic environment.     

3D QSAR pharmacophore-based virtual screening for the identification of potential inhibitors of tyrosinase

Abstract

Tyrosinase plays an important role in melanin biosynthesis and protects skin against ultraviolet radiations. Functional deficiency of tyrosinase results in serious dermatological diseases. Tyrosinase also participates in neuromelanin formation in the human brain, which leads to neurodegeneration resulting in Parkinson’s disease. In fruits and vegetables, tyrosinase plays a critical role in senescence, causing undesired browning that results in faster deterioration and shorter shelf lines. The only commercially available tyrosinase is mushroom tyrosinase and it shows the highest homology to the mammalian tyrosinase. Although kojic acid is currently used as a tyrosinase inhibitor, they have serious side effects such as dermatitis, carcinogenesis and hepatotoxicity. Therefore, in order to develop a more active and safer tyrosinase inhibitor, 3D QSAR pharmacophore models were generated based on experimentally known inhibitors. The pharmacophore model, Hypo1, was developed with a large cost difference, high correlation coefficient and low RMS deviation. Hypo1 showed a good spatial arrangement; consisting of five-point features including two hydrogen bond acceptor, one hydrogen bond donor and two hydrophobic features. Hypo1 was further validated by cost analysis, test set and Fisher’s randomisation method. Hypo1 was used as a 3D query for screening the in-house drug-like databases, and the hits were further selected by applying ADMET, Lipinski’s rule of five and fit value criteria. To identify binding conformations, the obtained hits were subjected to molecular docking. Finally, molecular dynamics simulations revealed the appropriate binding modes of hit compounds. To conclude, we propose the final three hit compounds with new structural scaffolds as a virtual candidate as tyrosinase inhibitors.

Communicated by Ramaswamy H. Sarma     

Interaction of antiprogestins with progesterone receptors in rat uterus

Cytosolic and nuclear progesterone receptors (PRc and PRn) under antiprogestin treatment were measured in rat deciduoma and compared with values for contralateral (nondeciduomatous) rat uterine tissue. Uterine PRc and PRn of the progesterone treated group were 101 +/- 8.7 and 4770 +/- 590 fmol/mg DNA respectively. After treatment with antiprogestins STS-557, 5 alpha-DNE, (5 alpha-dihydronorethisterone), 5 alpha-DNG (5 alpha-dihydronorgestrel), RU-22092 and RU-16556, PRc in the nondeciduomatous control horn ranged from 127 to 377 fmol/mg DNA and PRn from 2785 to 17925 fmol/mg DNA. In the decidual tissue, PRc decreased significantly (4.6 +/- 0.8 fmol/mg DNA) on 5 alpha-DNG treatment as compared with the progesterone alone treatment group (147 +/- 3.8). PRn in decidual tissue also decreased maximally on 5 alpha-DNG treatment. These results suggest that the interaction of antiprogestins may not be identical in control uterine tissue and in deciduoma.     

Salt tolerance potential of wild resources of the tribe Triticeae

The salt tolerance potential of variousAegilops species of different genome combinationsviz., Aegilops squarrosa, Ae. cylindrica, Ae. ovata. Ae. triuncialis, Ae. variabilis, Ae. bicornis, Ae. longissima, Ae. umbellulata, andAe. sharonensis was tested to identify the high salt-tolerant genotype(s). Screening was done in cement tanks filled with gravel and Hoagland nutrient solution. Salinity was created by mixing Na₂SO₄, CaCl₂, MgCl₂ and NaCl in the ratio of 10∶5∶1∶4 and induced by a stepwise increase in electrical conductivity number of tillers and number of leaves. Inter-and intragenomic variations for cation uptake were also significant. Species with DD and CD genome were found to be highly tolerant. Possible factors responsible for these observations have been discussed.     

Production and cytogenetics of Triticum aestivum L. hybrids with some rhizomatous Agropyron species

Summary Intergeneric hybrids between Triticum aestivum L. and conventional rhizomatous Agropyron species were produced in variable frequencies. They were recovered in high percentage frequencies for T. aestivum cultivars with A. acutum (14.6%), A. intermedium (48.0%), A. pulcherrimum (53.3%), and A. trichophorum (46.6%). The crossability percentages with the highly crossable cultivar Chinese Spring for these Agropyron species accessions were 33.12%, 65.0%, 53.3%, and 65.4%, respectively. Autosyndetic associations of two of their three genomes gave mean meiotic chromosome association data of 17.0 I (univalents) +1.53 II (ring bivalents) + 7.04 II (rod bivalents) +1.43 III (trivalents) +0.05 IV (quadrivalents) +0.01 IV (pentavalents) for A. acutum and of 21.8 I + 1.56 II (rings) +7.22 II (rods) +0.84 III + 0.04 IV for A. intermedium. Chromosome pairing at metaphase I was comparatively lower for A. pulcherrimum (34.4 I + 0.2 II (rings) +3.4 II (rods) +0.14 III) and A. trichophorum (36.7 I + 0.35 II (rings) +2.26 II (rods) + 0.04 III) hybrids with T. aestivum. Hybrids of wheat with A. campestre and A. repens were obtained in low frequency. Direct crossing did not permit T. aestivum/ A. desertorum hybridization. However, by utilizing the 2n=10x=70 A. repens/A. desertorum amphiploid as the pollen source, hybridization with T. aestivum did indeed occur. Aneuploidy was prevalent in this hybrid combination while all other hybrid combinations were apparently normal.     

Bacterial secondary production in freshwater measured by3H-thymidine incorporation method

Bacterial secondary production in lake water was measured by³H-thymidine incorporation into DNA. The application of the method to freshwater systems studied required (1) thymidine concentration > 10 nM (10–25 nM) evaluated from isotope dilution by varying the specific activity of labeled thymidine, (2) short incubation periods less than 1 hour, and (3) partial purification of the DNA fraction for measuring³H-thymidine incorporation. During 2 diel studies, bacterial productivity was compared to phytoplankton primary production and extracellular release of organic carbon. Diel changes in bacterial growth suggested substantial activity during the morning and evening. Possible mechanisms of control of bacterial growth, such as extracellular release of organic carbon, are discussed.     

The Bulk Isolation of Oligodendroglia from Whole Rat Forebrain: A New Procedure Using Physiologic Media

Abstract: A method for the isolation of oligodendroglia from undissected rat forebrain is described. The method has been applied to brains from 10-, 30- and 60-day-old rats. The procedure uses a balanced salt solution at pH 7.2 throughout. Tissue is briefly exposed to trypsin and DNase and dissociated and the cells are purified on a discontinuous sucrose gradient. The isolates were composed of 90% phase-bright rounded cells having diameters after fixation of 7-12 μm. The contamination was primarily by red blood cells and phase-dark nuclei. Neurons and astroglia were lysed by the procedure. The method is reproducible and should be applicable to other ages of rat or to other species. The cells have been examined by light and electron microscopy and analyzed for protein and nucleic acids. None of the cell parameters measured, including total protein (58 pg/cell), varied significantly with age. With this new method it should be possible to carry out studies on the development and metabolism of oligodendroglia in small laboratory animals.     

Severe autosomal recessive retinitis pigmentosa maps to chromosome 1p13.3–p21.2 between D1S2896 and D1S457 but outside ABCA4

A severe form of autosomal recessive retinitis pigmentosa (arRP) was identified in a large Pakistani family ascertained in the Punjab province of Pakistan. All affected individuals in the family had night blindness in early childhood, early complete loss of useful vision, and typical RP fundus changes plus macular degeneration. After exclusion of known arRP loci, a genome-wide scan was performed using microsatellite markers at about 10 cM intervals and calculating two-point lod scores. PCR cycle dideoxynucleotide sequencing was used to sequence candidate genes inside the linked region for mutations. RP in this family shows linkage to markers in a 10.5 cM (8.9 Mbp) region of chromosome 1p13.3-p21.2 between D1S2896 and D1S457. D1S485 yields the highest lod score of 6.54 at theta=0. Sequencing the exons and intron-exon boundaries of five candidate genes and six ESTs in this region, OLFM3, GNAI3, LOC126987, FLJ25070, DKFZp586G0123, AV729694, BU662869, BU656110, BU171991, BQ953690, and CA397743, did not identify any causative mutations. This novel locus lies approximately 4.9 cM (7.1 Mbp) from ABCA4, which is excluded from the linked region. Identification and study of this gene may help to elucidate the phenotypic diversity of arRP mapping to this region.