Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dichlorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 microM for 2,4-dichlorophenol, and 100 microM for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.     

Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel.

This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction.     

Effect of phytohormones on nuclear RNA synthesis in germinating seeds ofTrigonella foenumgraeceum and its callus

Treatment ofTrigonella foenumgraeceum (fenugreek) seedlings with naphthalene acetic acid plus gibberellic acid enhanced the RNA synthesising capacity of nuclei isolated from the hypocotyl and cotyledonary regions. This increase was more pronounced in the nuclei from the hypocotyl region than from the cotyledonary region.In vitro addition of these phytohormones did not stimulate RNA synthesis by nuclei. The RNA synthesis by mitochondria was not affected by preincubating the seedlings with the hormones. The nuclei isolated from callus cultures of fenugreek hypocotyl treated with the hormone also showed increased RNA synthesis.     

DESI-MSI and METASPACE indicates lipid abnormalities and altered mitochondrial membrane components in diabetic renal proximal tubules

Metabolomics - Food and dietary ingredients have significant effects on metabolism and health. To evaluate whether and how different diets affected the serum lipidomic profile of dogs. Sixteen...     

Assessment of genetic stability on in vitro and ex vitro plants of Ficus carica var. black jack using ISSR and DAMD markers

Molecular Biology Reports - Clonal propagation is one of the attributes of plant tissue culture. Therefore, analysis of genetic stability among the in vitro cultured plants is a crucial step. It...     

Synthesis of Zidovudine Derivatives with Anti-HIV-1 and Antibacterial Activities

Twelve novel zidovudine derivatives were prepared by modifying 5 ′-hydroxyl group of sugar moiety (1–8) and 5-methyl group of thymidine nucleus (9–12) and characterized spectrally. The compounds were evaluated for anti-HIV-1, antitubercular and antibacterial activities. Compound (3-azido-tetrahydro-5- (3,4-dihydro-5-methyl-2,4-dioxopyrimidin- 1 (2H)-yl) furan-2-yl)methyl 7- (4- (2-phenylacetoyloxy) -3,5- dimethylpiperazin-1-yl) -5- (2-phenylacetoyloxyamino) -1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate (5) was found to be the most potent anti-HIV-1 agent with EC₅₀ of 0.0012 μM against HIV-1_IIIB and CC₅₀ of 34.05 μM against MT-4 with selectivity index of 28,375. Compound 5 inhibited Mycobacterium tuberculosis with MIC of 1.72 μM and inhibited four pathogenic bacteria with MIC of less than 1 μM.     

Connectivity of Tiger (Panthera tigris) Populations in the Human-Influenced Forest Mosaic of Central India

Today, most wild tigers live in small, isolated Protected Areas within human dominated landscapes in the Indian subcontinent. Future survival of tigers depends on increasing local population size, as well as maintaining connectivity between populations. While significant conservation effort has been invested in increasing tiger population size, few initiatives have focused on landscape-level connectivity and on understanding the effect different landscape elements have on maintaining connectivity. We combined individual-based genetic and landscape ecology approaches to address this issue in six protected areas with varying tiger densities and separation in the Central Indian tiger landscape. We non-invasively sampled 55 tigers from different protected areas within this landscape. Maximum-likelihood and Bayesian genetic assignment tests indicate long-range tiger dispersal (on the order of 650 km) between protected areas. Further geo-spatial analyses revealed that tiger connectivity was affected by landscape elements such as human settlements, road density and host-population tiger density, but not by distance between populations. Our results elucidate the importance of landscape and habitat viability outside and between protected areas and provide a quantitative approach to test functionality of tiger corridors. We suggest future management strategies aim to minimize urban expansion between protected areas to maximize tiger connectivity. Achieving this goal in the context of ongoing urbanization and need to sustain current economic growth exerts enormous pressure on the remaining tiger habitats and emerges as a big challenge to conserve wild tigers in the Indian subcontinent.     

Emodin Suppresses Migration and Invasion through the Modulation of CXCR4 Expression in an Orthotopic Model of Human Hepatocellular Carcinoma

Accumulating evidence(s) indicate that CXCL12-CXCR4 signaling cascade plays an important role in the process of invasion and metastasis that accounts for more than 80% of deaths in hepatocellular carcinoma (HCC) patients. Thus, identification of novel agents that can downregulate CXCR4 expression and its associated functions have a great potential in the treatment of metastatic HCC. In the present report, we investigated an anthraquinone derivative, emodin for its ability to affect CXCR4 expression as well as function in HCC cells. We observed that emodin downregulated the expression of CXCR4 in a dose-and time-dependent manner in HCC cells. Treatment with pharmacological proteasome and lysosomal inhibitors did not have substantial effect on emodin-induced decrease in CXCR4 expression. When investigated for the molecular mechanism(s), it was observed that the suppression of CXCR4 expression was due to downregulation of mRNA expression, inhibition of NF-κB activation, and abrogation of chromatin immunoprecipitation activity. Inhibition of CXCR4 expression by emodin further correlated with the suppression of CXCL12-induced migration and invasion in HCC cell lines. In addition, emodin treatment significantly suppressed metastasis to the lungs in an orthotopic HCC mice model and CXCR4 expression in tumor tissues. Overall, our results show that emodin exerts its anti-metastatic effect through the downregulation of CXCR4 expression and thus has the potential for the treatment of HCC.