Aims:  Isolation of bacterial antagonist for use in the biological control of phytopathogenic fungi like rice blast fungus, Magnaporthe grisea, and to further purify and characterize the antifungal molecule produced by the antagonist. Methods and Results:  Bacterial antagonist exhibiting highest antifungal activity against the rice blast fungus M. grisea was isolated from soil and identified as Bacillus licheniformis BC98. Besides M. grisea, the isolate also inhibited the growth of other phytopathogens such as Curvularia lunata and Rhizoctonia bataticola. Biologically active fractions were isolated from the culture filtrate and further fractionated by reverse‐phase high‐performance liquid chromatography (HPLC) enabling detailed structural characterization of a component of molecular mass 1035 Da. The active peptide was identified as surfactin after 500 MHz 1H NMR analysis. Microscopic analysis of the effect of the antagonist on M. grisea revealed bulbous hyphae showing patchy and vacuolated cytoplasm when observed under the electron microscope. Conclusions:  The antagonistic lipopeptide secreted by B. licheniformis BC98 and identified as surfactin, induced morphological changes in M. grisea, inhibiting its further growth, and thus exhibiting fungicidal activity. Significance and Impact of the Study:  The antagonist inhibits germination of M. grisea, a potent rice phytopathogen, and therefore appears to be a potential candidate for control of rice blast disease.

DNA methylation is a major determinant of epigenetic inheritance. DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division, and deregulated expression of DNMT1 leads to cellular transformation. We show herein that AU-rich element/poly(U)-binding/ degradation factor 1 (AUF1)/heterogenous nuclear ribonucleoprotein D interacts with an AU-rich conserved element in the 3 untranslated region of the DNMT1 mRNA and targets it for destabilization by the exosome. AUF1 protein levels are regulated by the cell cycle by the proteasome, resulting in cell cycle-specific destabilization of DNMT1 mRNA. AUF1 knock down leads to increased DNMT1 expression and modifications of cell cycle kinetics, increased DNA methyltransferase activity, and genome hypermethylation. Concurrent AUF1 and DNMT1 knock down abolishes this effect, suggesting that the effects of AUF1 knock down on the cell cycle are mediated at least in part by DNMT1. In this study, we demonstrate a link between AUF1, the RNA degradation machinery, and maintenance of the epigenetic integrity of the cell.DNA methylation patterns are a critical component of the epigenome, controlling gene expression in vertebrates (37,38). The enzyme DNA methyltransferase 1 (DNMT1) is responsible for maintenance and propagation of DNA methylation patterns. These patterns are altered in tumorigenesis (2, 14). The overexpression of DNMT1 in NIH 3T3 mouse fibroblasts causes cell transformation (55), while DNMT1 overexpression in human fibroblasts results in aberrant methylation of endogenous CpG islands (51). In parallel, the down regulation of DNMT1 inhibits cancer growth in animal models (20,28,35). On the basis of these reports, DNMT1 was therefore proposed as a target for anticancer therapy (46, 47).As was expected from its critical role in maintaining epigenomic integrity, DNMT1 expression was shown to be controlled by cell growth (39,48,49). Multiple mechanisms, such as transcriptional (3,17,27,29,40), posttranscriptional (11), and posttranslational (1, 12) mechanisms, ensure a tight regulation of its expression during the cell cycle. It was suggested that deregulated expression of DNMT1 during the cell cycle might be critical for cell growth control (39, 50) and DNA replication (18, 31). Deregulated cell cycle control of DNMT1 was observed in breast cancer and colorectal cancers (10, 33).Our previous study showed that DNMT1 3Ј untranslated region (3Ј-UTR) contains a highly conserved noncanonical AU-rich region, which is responsible for regulating its expression level during the cell cycle (11). Deletion of this conserved region resulted in cellular transformation. We also observed binding of a protein with an apparent size of ϳ40 kDa on this region, which triggered the destabilization of DNMT1 transcript in G 0 /G 1 phase.Using affinity capture with the 3ЈUTR of DNMT1 mRNA and matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS-MS) analysis, we identified AU-rich element ...

The beta-amyloid peptide (Ab) is considered responsible for the pathogenesis of Alzheimer's disease. Despite the magnitude of reports describing a neurotoxic role of extracellular Ab, the role for intracellular Ab (iAb) has not been elucidated. We previously demonstrated that in rat pheochromocytoma cells expression of moderate levels of Ab results in the up-regulation of phospho-extracellular signal-regulated kinases (ERK1)/2 along with an elevation of cyclic AMP-response element (CRE)-regulated gene expression; however, the effect of high intracellular levels of Ab were not examined. Towards this goal we generated constructs that endogenously produce different expression levels of iAb in a human cell line. We show a bimodal response to Ab in a neural human cell line. A moderate increase of endogenous Ab up-regulates certain cyclic AMP-response element-binding protein (CREB) responsive genes such as presenilin 1, presenilin 2, brain-derived neurotrophic factor, and mRNA and protein levels by CREB activation and Synapsin 1 nuclear translocation. On the other hand, high-loads of iAb resulted in sustained hyper-phosphorylation of CREB that did not translocate to the nucleus and did not stimulate activation of CRE-regulated gene expression. Our study suggests that variations in levels of iAb could influence signaling mechanisms that lead to phosphorylation of CREB, its nuclear translocation and CRE-regulated genes involved in production of Ab and synaptic plasticity in opposite directions.

Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s. The quantity of DNA was good enough for PCR analysis and Dot blot hybridization. This technique can be used for various studies, such as DNA fingerprinting to study the population structure of the phytopathogen in different regions, and for a quick screening of M. grisea transformants.

The optimization of process parameters for the production of an antifungal molecule produced by Bacillus licheniformis BC98 was carried out using novel statistical tools. The parameters studied were pH, temperature and agitation rate. Fed batch cultivations were carried out since the maximum production of the molecule was observed in the late log phase. The statistical design used allows the evaluation of the effects of several different process variables in a single batch. Data from several batches indicated that while the effects of two of the variable factors, viz., temperature and agitation rate, were significant at 95% confidence intervals, the agitation rate was most critical for the production of the molecule, and pH had no significant effect. The cultivation of the bacterium under optimized conditions (fed batch, 150 rpm, 32 degrees C, pH 5.8) resulted in a 30-fold increase compared with that under unoptimized conditions (shake flask, 100 rpm, 29 degrees C, pH 5.8) in the production of the antifungal molecule.