An endophytic <Emphasis Type="Italic">Neurospora</Emphasis> sp. from <Emphasis Type="Italic">Nothapodytes foetida</Emphasis> producing camptothecin

The medicinal plant, Nothapodytes foetida, contains a number of important alkaloids like camptothecin (an anticancer drug molecule), but its concentration is less to meet the existing demand of this important molecule, in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as a potential source of anticancer drug lead compound, i.e., camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of an anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of a precursor anticancer drug molecule in the future at a large scale. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 225–231. The text was submitted by the authors in English.     

Molecular cloning and characterization of amylase from soil metagenomic library derived from Northwestern Himalayas

The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase, protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as substrates. The maximal activity (2.46 U/mg) was observed at 40 °C under nearly neutral pH conditions with amylose; whereas it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division.     

Development and validation of molecular markers linked to an Aegilops umbellulata-derived leaf-rust-resistance gene, Lr9, for marker-assisted selection in bread wheat.

An Aegilops umbellulata-derived leaf-rust-resistance gene, Lr9, was tagged with 3 random amplified polymorphic DNA (RAPD) markers, which mapped within 1.8 cM of gene Lr9 located on chromosome 6BL of wheat. The markers were identified in an F2 population segregating for leaf-rust resistance, which was generated from a cross between 2 near-isogenic lines that differed in the alien gene Lr9 in a widely adopted agronomic background of cultivar 'HD 2329'. Disease phenotyping was done in controlled environmental conditions by inoculating the population with the most virulent pathotype, 121 R63-1 of Puccinia triticina. One RAPD marker, S5550, located at a distance of 0.8+/-0.008 cM from the Lr9 locus, was converted to sequence-characterized amplified region (SCAR) marker SCS5550. The SCAR marker was validated for its specificity to gene Lr9 against 44 of the 50 known Lr genes and 10 wheat cultivars possessing the gene Lr9. Marker SCS5550 was used with another SCAR marker, SCS73719, previously identified as being linked to gene Lr24 on a segregating F2 population to select for genes Lr9 and Lr24, respectively, demonstrating the utility of the 2 markers in marker-assisted gene pyramiding for leaf-rust resistance in wheat.     

Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression

Background

Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/Principal Findings

A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/Significance

Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.     

Characterization, Genetic Diversity, and Evolutionary Link of Cucumber mosaic virus Strain New Delhi from India

The genome of Cucumber mosaic virus New Delhi strain (CMV-ND) from India, obtained from tomato, was completely sequenced and compared with full genome sequences of 14 known CMV strains from subgroups I and II, for their genetic diversity. Sequence analysis suggests CMV-ND shares maximum sequence identity at the nucleotide level with a CMV strain from Taiwan. Among all 15 strains of CMV, the encoded protein 2b is least conserved, whereas the coat protein (CP) is most conserved. Sequence identity values and phylogram results indicate that CMV-ND belongs to subgroup I. Based on the recombination detection program result, it appears that CMV is prone to recombination, and different RNA components of CMV-ND have evolved differently. Recombinational analysis of all 15 CMV strains detected maximum recombination breakpoints in RNA2; CP showed the least recombination sites.     

Brassinosteroid-mediated evaluation of antioxidant system and nitrogen metabolism in two contrasting cultivars of Vigna radiata under different levels of nickel

The role of 28-homobrassinolide (HBL) in countering nickel-induced oxidative damage through overexpression of antioxidant enzymes and proline in Vigna radiata has been investigated. Two varieties of V. radiata, one sensitive to Ni (PDM-139) and the other tolerant to Ni (T-44), were sown in the soil fed with different levels (0, 50, 100 or 150 mg kg⁻¹) of Ni, and at 29-day stage, foliage of plants was applied with deionized water (control), 10⁻⁸ or 10⁻⁶ M of HBL. The plants were sampled at 45-day stage of growth to assess various physiological as well as biochemical characteristics. The remaining plants were allowed to grow up to maturity to study the yield characteristics. The growth traits, leghemoglobin, nitrogen and carbohydrate content in the nodules, leaf chlorophyll content, photosynthesis efficiency, leaf water potential, activities of nitrate reductase, carbonic anhydrase and nitrogenase decreased proportionately with the increasing concentrations of nickel, whereas electrolyte leakage, various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and accumulation of proline increased at 45-day stage. However, the exogenously applied HBL to the nickel-stressed or non-stressed plants improved growth, nodulation and photosynthesis and further enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and accumulation of proline. The deleterious impact of Ni on the plants was concentration dependent where HBL applied to the foliage induced overexpression of antioxidant enzyme and accumulation of proline (osmolyte) which could have conferred tolerance to Ni up to 100 mg kg⁻¹, resulting in improved growth, nodulation, photosynthesis and yield attributes.     

Molecular Docking of Known Carcinogen 4- (Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with Cyclin Dependent Kinases towards Its Potential Role in Cell Cycle Perturbation

Cell cycle is maintained almost all the times and is controlled by various regulatory proteins and their complexes (Cdk+Cyclin) in different phases of interphase (G₁, S and G₂) and mitosis of cell cycle. A number of mechanisms have been proposed for the initiation and progression of carcinogenesis by abruption in cell cycle process. One of the important features of cancer/carcinogenesis is functional loss of these cell cycle regulatory proteins particularly in CDKs and cyclins. We hypothesize that there is a direct involvement of these cell cycle regulatory proteins not only at the genetic level but also proteins level, during the initiation of carcinogenesis. Therefore, it becomes significant to determine inconsistency in the functioning of regulatory proteins due to interaction with carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Hence, we investigated the interaction efficiency of NNK, against cell cycle regulatory proteins. We found a different value of ΔG (free energy of binding) among the studied proteins ranging between -3.29 to -7.25 kcal/mol was observed. To validate the results, we considered Human Oxy-Hemoglobin at 1.25 Å Resolution, [PDB_ID:1HHO] as a +ve control, (binding energy -6.06 kcal/mol). Finally, the CDK8 (PDB_ID:3RGF) and CDK2 (PDB_ID:3DDP) regulatory proteins showing significantly strong molecular interaction with NNK -7.25 kcal/mol, -6.19 kcal/mol respectively were analyzed in details. In this study we predicted that CDK8 protein fails to form functional complex with its complementary partner cyclin C in presence of NNK. Consequently, inconsistency of functioning in regulatory proteins might lead to the abruption in cell cycle progression; contribute to the loss of cell cycle control and subsequently increasing the possibility of carcinogenesis.