Effect of purified soluble polysaccharides extracted from gray oyster mushroom [Pleurotus sajor-caju (Fr.) Sing.] on 3t3-L1 adipocytes

Functional mushroom polysaccharides have been known to the Asian traditional medicine for many years. The anti-obesity activity of mushroom polysaccharides has been illustrated both in vivo and in vitro. However, the effect of pure polysaccharides on isolated fat cells has not been demonstrated. In this study, we used hot water and ammonium oxalate to extract polysaccharides from gray oyster mushroom [Pleurotus sajor-caju (Fr.) Sing.] and subsequently purified with enzymatic digestion, solvent extraction and ion-exchange chromatography. One of the fractions, 7S1-1, has the highest yield and purity and contains polysaccharides with the molecular weight around 500 kDa. In addition, Fourier transformed infrared spectroscopy (FTIR) and monosaccharide composition analyses showed that this 7S1-1 sample contains mostly β-glucan and some mannan. We find that the 7S1-1 fraction does not affect the total amount of triglycerides in the 3T3-L1 adipocytes but promotes a release of glycerol of the fat cells, suggesting that the fraction does not inhibit adipogenesis but stimulates lipolysis. Laminarin, a small β-glucan, confers similar results to the 7S1-1 sample, but it could not stimulate lipolysis as well as the 7S1-1 sample. Therefore, these results suggest a potential anti-obesity activity of the mushroom polysaccharides

Human Plasmodium knowlesi infection in Ranong province, southwestern border of Thailand

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium knowlesi</it>, a simian malaria parasite, has been reported in humans in many Southeast Asian countries. In Thailand, most of the limited numbers of cases reported so far were from areas near neighbouring countries, including Myanmar.</p> <p>Methods</p> <p>Blood samples collected from 171 Thai and 248 Myanmese patients attending a malaria clinic in Ranong province, Thailand, located near the Myanmar border were investigated for <it>P. knowlesi </it>using nested PCR assays. Positive samples were also investigated by PCR for <it>Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae </it>and <it>Plasmodium ovale</it>, and were confirmed by sequencing the gene encoding the circumsporozoite protein (<it>csp</it>).</p> <p>Results</p> <p>Two samples, one obtained from a Thai and the other a Myanmese, were positive for <it>P. knowlesi </it>only. Nucleotide sequences of the <it>csp </it>gene derived from these two patients were identical and phylogenetically indistinguishable from other <it>P. knowlesi </it>sequences derived from monkeys and humans. Both patients worked in Koh Song, located in the Kawthoung district of Myanmar, which borders Thailand.</p> <p>Conclusion</p> <p>This study indicates that transmission of <it>P. knowlesi </it>is occurring in the Ranong province of Thailand or the Kawthoung district of Myanmar. Further studies are required to assess the incidence of knowlesi malaria and whether macaques in these areas are the source of the infections.</p

Characterization of Transcription from TATA-Less Promoters: Identification of a New Core Promoter Element XCPE2 and Analysis of Factor Requirements

More than 80% of mammalian protein-coding genes are driven by TATA-less promoters which often show multiple transcriptional start sites (TSSs). However, little is known about the core promoter DNA sequences or mechanisms of transcriptional initiation for this class of promoters.Here we identify a new core promoter element XCPE2 (X core promoter element 2) (consensus sequence: A/C/G-C-C/T-C-G/A-T-T-G/A-C-C/A(+1)-C/T) that can direct specific transcription from the second TSS of hepatitis B virus X gene mRNA. XCPE2 sequences can also be found in human promoter regions and typically appear to drive one of the start sites within multiple TSS-containing TATA-less promoters. To gain insight into mechanisms of transcriptional initiation from this class of promoters, we examined requirements of several general transcription factors by in vitro transcription experiments using immunodepleted nuclear extracts and purified factors. Our results show that XCPE2-driven transcription uses at least TFIIB, either TFIID or free TBP, RNA polymerase II (RNA pol II) and the MED26-containing mediator complex but not Gcn5. Therefore, XCPE2-driven transcription can be carried out by a mechanism which differs from previously described TAF-dependent mechanisms for initiator (Inr)- or downstream promoter element (DPE)-containing promoters, the TBP- and SAGA (Spt-Ada-Gcn5-acetyltransferase)-dependent mechanism for yeast TATA-containing promoters, or the TFTC (TBP-free-TAF-containing complex)-dependent mechanism for certain Inr-containing TATA-less promoters. EMSA assays using XCPE2 promoter and purified factors further suggest that XCPE2 promoter recognition requires a set of factors different from those for TATA box, Inr, or DPE promoter recognition.We identified a new core promoter element XCPE2 that are found in multiple TSS-containing TATA-less promoters. Mechanisms of promoter recognition and transcriptional initiation for XCPE2-driven promoters appear different from previously shown mechanisms for classical promoters that show single "focused" TSSs. Our studies provide insight into novel mechanisms of RNA Pol II transcription from multiple TSS-containing TATA-less promoters

The NSL Complex Regulates Housekeeping Genes in Drosophila

MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP–seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP–seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication–related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in Aspergillus nidulans

Extent: 6p.A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB) module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.Paraskevi Georgakopoulos, Robin A. Lockington, Joan M. Kell