Epigallocatechin gallate induced apoptosis in Sarcoma180 cells in vivo: Mediated by p53 pathway and inhibition in U1B, U4-U6 UsnRNAs expression

The aim of this study was to understand the mode of action of tea polyphenol epigallocatechin gallate (EGCG) in vivo. Swiss albino mice were treated i.p. with EGCG at two different doses i.e. 12-mg/kg body weight and 15-mg/kg body weight, for 7 days prior to inoculation of Sarcoma180 (S180) cells and continued for another 7 days. The growth of the S180, harvested 7 days after inoculation, was significantly reduced due to treatment with EGCG. The flowcytometric analysis of S180 cells, showed significant increase in apoptosis and reduction in the number of cells in G2/M phase of cell cycle due to treatment with EGCG. The induction of apoptosis has also been confirmed by the TUNEL and DNA fragmentation assays. Both RT-PCR and Western blot analysis showed significant up-regulation of p53 and bax, and down-regulation of bcl-2 and c-myc due to EGCG treatment. No changes in the expression pattern of p21, p27, bcl-xl, mdm2 and cyclin D1 were seen. Interestingly, there was significant down-regulation of spliceosomal uridylic acid rich small nuclear RNAs (UsnRNAs) U1B and U4-U6 due to EGCG treatment. This indicates that these UsnRNAs may be involved in the apoptosis process. Taken together, our study suggests that in vivo EGCG could induce apoptosis in S180 cells through alteration in G2/M phase of the cell cycle by up-regulation of p53, bax and down-regulation of c-myc, bcl-2 and U1B, U4-U6 UsnRNAs.     

Inhibition of LRRK2 kinase activity stimulates macroautophagy

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific targets for disease modifying therapies in patients.     

First report on molecular detection of a stolbur phytoplasma (Group16SrXII-A) associated with kenaf (Hibiscus cannabinus L.) in India

Infection of stolbur phytoplasma was detected in kenaf (Hibiscus cannabinus) plants at CRIJAF research farm, Barrackpore, India. The infected plants formed profuse short branches at the top with bushy and bunchy top appearance. PCR with universal 16S rDNA phytoplasma primers P1/P7 yielded amplicons of 1.5 kb from all symptomatic leaf samples. Nested PCR with 16S-rDNA-specific nested primer pair R16F2n/R2 generated an amplicon of 1241 bp confirming the presence of a phytoplasma. The nested PCR products were sequenced and BALSTn analysis revealed 100% identity with 16S rRNA gene of phytoplasma. Phylogenetic analysis showed kenaf phytoplasma having 99% identity with both “Bois noir” stolbur phytoplasma 16SrXII group (Accession no: JQ181540). The RFLP data also supported the phylogenetic analysis. Multi-locus sequence characterisation assay was conducted by using different locus-specific primers viz. tuf, rpsC-rplV, rplF-rplR, map-SecY and uvrB-degV. The infected phytoplasma samples amplified only SecY gene and generated 1224 bp product which was deposited at NCBI (accession no: KC508636).     

Translocation of heme oxygenase-1 to mitochondria is a novel cytoprotective mechanism against non-steroidal anti-inflammatory drug-induced mitochondrial oxidative stress, apoptosis, and gastric mucosal injury

The mechanism of action of heme oxygenase-1 (HO-1) in mitochondrial oxidative stress (MOS)-mediated apoptotic tissue injury was investigated. MOS-mediated gastric mucosal apoptosis and injury were introduced in rat by indomethacin, a non-steroidal anti-inflammatory drug. Here, we report that HO-1 was not only induced but also translocated to mitochondria during gastric mucosal injury to favor repair mechanisms. Furthermore, mitochondrial translocation of HO-1 resulted in the prevention of MOS and mitochondrial pathology as evident from the restoration of the complex I-driven mitochondrial respiratory control ratio and transmembrane potential. Mitochondrial translocation of HO-1 also resulted in time-dependent inhibition of apoptosis. We searched for the plausible mechanisms responsible for HO-1 induction and mitochondrial localization. Free heme, the substrate for HO-1, was increased inside mitochondria during gastric injury, and mitochondrial entry of HO-1 decreased intramitochondrial free heme content, suggesting that a purpose of mitochondrial translocation of HO-1 is to detoxify accumulated heme. Heme may activate nuclear translocation of NF-E2-related factor 2 to induce HO-1 through reactive oxygen species generation. Electrophoretic mobility shift assay and chromatin immunoprecipitation studies indicated nuclear translocation of NF-E2-related factor 2 and its binding to HO-1 promoter to induce HO-1 expression during gastric injury. Inhibition of HO-1 by zinc protoporphyrin aggravated the mucosal injury and delayed healing. Zinc protoporphyrin further reduced the respiratory control ratio and transmembrane potential and enhanced MOS and apoptosis. In contrast, induction of HO-1 by cobalt protoporphyrin reduced MOS, corrected mitochondrial dysfunctions, and prevented apoptosis and gastric injury. Thus, induction and mitochondrial localization of HO-1 are a novel cytoprotective mechanism against MOS-mediated apoptotic tissue injury.     

AFLP based assessment of genetic relationships among shiitake (<Emphasis Type="Italic">Lentinula</Emphasis> ssp.) mushrooms

Despite the economical importance of shiitake (Lentinula ssp.) mushrooms, until the present date little information exists on cultivated and wild species in correlation with geographic origin applying molecular techniques. Use of a high resolution molecular tool like AFLP for assessing genetic similarity and geographical diversity would be an important step towards understanding of different Lentinula species. Thirteen wild and 17 cultivated accessions of 3 Lentinula species were analysed with 64 EcoRI–MseI primer combinations and finally 32 reproducible and polymorphic primer combinations were considered for the analysis. A total of 816 informative AFLP markers were generated and scored as binary data. These data were analysed using various method packages for cluster analysis, genetic diversity and genetic differentiation. Percentage polymorphism was high (62.99%) among the species studied. Different clustering analysis segregated the wild and the cultivated species into two major branches, with the wild samples being further grouped according to their geographic location. Overall polymorphisms among cultivated strains in the USA were higher than that of the cultivated strains in Japan (58.9%).     

Targeted spatio-temporal expression based characterization of state of infection and time-point of maximum defense in wheat NILs during leaf rust infection

Leaf rust, caused by the fungus Puccinia triticina, is the most devastating disease of wheat worldwide, which sometimes becomes epidemic. The pathogen evolves into new strains, making its control difficult. Though more than 60 leaf rust resistant genes are now known, only limited insight is available into the molecular mechanism involved in this host pathogen interaction. In the present study, quantitative real-time PCR based differential gene expression profiling was examined for five target genes encoding for chitinase3, β-1,3/1,4 glucanase, thaumatin-like protein, peroxidase2 and mitogen activated protein kinase1 to unravel their coordinated action during compatible and incompatible interaction, to inhibit the pathogen progression and to identify the time-period of maximum defense activity. Spatio-temporal expression profiling suggested that the maximum defense activity occurred at 12-24?hours post inoculation, whereas the state of infection and degree of resistance was predicted using coordinated unique expression signatures of target genes. The significant differences of targeted gene expression between resistant mock inoculated, resistant infected and susceptible infected plants were evaluated using t test at significance level of p?相似文献   

Study of polyacrylamide grafted starch based algal flocculation towards applications in algal biomass harvesting

Microalgae may be the source of high amount of lipid and protein. It has the property for carbon dioxide sequestration, recycling and also can remove pollutants from wastewater. Using traditional methods, collection of algal biomass is either cost effective, time consuming or may be toxic due to use of chemical salts. The aim of this study is to harvest freshwater microalgae (Chlorella sp. CB4) biomass by using polymer. Polyacrylamide grafted starch (St-g-PAM) has been synthesized by microwave assisted method involving a synergism of microwave radiation and ceric ammonium nitrate (CAN) to initiate the grafting reaction. The synthesis was optimized in terms of CAN and monomer (acrylamide) concentration. The algal flocculation efficacy of all the grades of this graft copolymer was studied through standard 'Jar test' procedure. Effects of percentage grafting, pH and zeta potential on percentage recovery of algal biomass were thoroughly investigated.