Efficient fertile plant regeneration from protoplasts of the Indica rice breeding line IR72 (Oryza sativa L.)

Protoplasts were isolated from embryogenic cell suspensions obtained from mature seed derived embryogenic callus of the advanced Indica type rice breeding line IR72 available from IRRI (International Rice Research Institute), Manila. Culture of protoplasts with the agarose bead type method without nurse culture led to sustained proliferation of protoplast derived clones. A simple culture protocol was developed which stimulated embryogenic development. Germination of somatic embryos has so far produced 277 green plants from 6 independent experiments. 117 plants have been transferred to soil and are growing in the greenhouse. A few of them have already flowered and set seeds.Abbreviations 2,4-D, 2,4 Dichlorophenoxyacetic acid - BAP Benzylaminopurine - CH Casein hydrolysate - ECS Embryogenie cell suspension - Kn Kinetin - NAA Napthaleneacetic acid; Media: - AA Muller and Grafe 1978 - MS Murashige and Skoog 1962 - N6 Chu et al. 1975 - R2 Ohira et al. 1973     

Gene transfer by electroporation into intact scutellum cells of wheat embryos

Summary Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-F capacitor and with 100 g DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MES 2-N-morpholinoethane sulfonic acid     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hg^r and 170 PPT^r IR72 plants from protoplasts through selection. 31 Hg^r and 70 PPT^r plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.     

Regulation of autophagy as a therapeutic option in glioblastoma

Apoptosis - Around three out of one hundred thousand people are diagnosed with glioblastoma multiforme, simply called glioblastoma, which is the most common primary brain tumor in adults. With a...     

Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.     

Disentangling the role of soil in structuring tropical tree communities at Tarap Hill Reserve of Bangladesh

Tarap Hill Reserve, the largest upland reserve of Bangladesh, is situated along the Indo-Burma Biodiversity Hotspot. It encompasses the last remaining patches of natural vegetation in the Northeastern Tarap Mountain System and harbors 87 % of the nationally declared red-listed vascular plant species. Despite requiring high conservation priority, this is one of the least studied reserves in the tropics. In this study, we collected vegetation and soil (eight variables) data from 68 sample plots. We identified the tree communities by cluster analysis and verified them using the multi-response permutation procedure and detrended correspondence analysis. Species richness, diversity, and compositional similarity between the communities were also estimated. In total, 116 tree species representing 69 genera were recorded within the four identified tree community types. Finally, canonical correspondence analysis with associated Monte Carlo permutation tests (499 permutations) was performed to explore the patterns of variation in tree species distribution explained by the soil variables. Soil phosphorus, organic matter content, and pH were most closely correlated with tree compositional variation. Thus, conservation strategies that take into account variations in these influential soil factors may aid in the conservation of trees in the reserve.     

Natural and Regenerated Saltmarshes Exhibit Similar Soil and Belowground Organic Carbon Stocks,Root Production and Soil Respiration

Ecosystems - Saltmarshes provide many valuable ecosystem services including storage of a large amount of ‘blue carbon’ within their soils. To date, up to 50% of the world’s...     

Addressing the problems of base pairing and strand cyclization in template-directed synthesis: a case for the utility and necessity of 'molecular midwives' and reversible backbone linkages for the origin of proto-RNA

Nucleic acid synthesis is precisely controlled in living organisms by highly evolved protein enzymes. The remarkable fidelity of information transfer realized between template and product strands is the result of both the spatial selectivity of the polymerase active site for Watson-Crick base pairs at the point of nucleotide coupling and subsequent proof-reading mechanisms. In the absence of naturally derived polymerases, in vitro template-directed synthesis by means of chemically activated mononucleotides has proven remarkably inefficient and error-prone. Nevertheless, the spontaneous emergence of RNA polymers and their protein-free replication is frequently taken as a prerequisite for the hypothetical 'RNA world'. We present two specific difficulties that face the de novo synthesis of RNA-like polymers in a prebiotic (enzyme-free) environment: nucleoside base selection and intramolecular strand cyclization. These two problems are inherent to the assumption that RNA formed de novo from pre-existing, chemically-activated mononucleotides in solution. As a possible resolution to these problems, we present arguments and experimental support for our hypothesis that small molecules (referred to as 'molecular midwives') and alternative backbone linkages (under equilibrium control) facilitated the emergence of the first RNA-like polymers of life.