Genomic insights into secondary metabolites of pharmaceutical utility for Hyphococcus flavus MCCC 1K03223T, isolated from bathypelagic seawater.

During an attempt to screen secondary metabolites of pharmaceutical utility, we sequenced the complete genome of type strain of a novel marine bacterial genus, named genus Hyphococcus. The type strain, Hyphococcus flavus MCCC 1K03223T, was isolated from bathypelagic seawater of South China Sea at a depth of 2500 m. The complete genome of strain MCCC 1K03223T is composed of a circular chromosome of 3,472,649 bp with a mean G + C content of 54.8%. Functional genomic analysis showed that this genome encodes five biosynthetic gene clusters, which were annotated to synthesize medicinally important secondary metabolites. Secondary metabolites annotated include ectoine which acts cytoprotection, ravidomycin which is an antitumor antibiotic and three other different metabolites of terpene type. The secondary metabolic potentials of H. flavus revealed in this study provide more evidences on mining bioactive substances from marine bathypelagic microorganisms.

Schisandra polysaccharide inhibits hepatic lipid accumulation by downregulating expression of SREBPs in NAFLD mice.

BACKGROUND: Hepatoprotective effects of Chinese herbal medicine Schisandra Chinensis (Schisandra) have been widely investigated. However, most studies were focused on its lignan extracts. We investigated the effects of Schisandra polysaccharide (SCP) in a mouse model of non-alcoholic fatty liver disease (NAFLD), and studied its effect on sterol regulatory element binding proteins (SREBPs) and the related genes. METHODS: The mouse model of NAFLD was established by feeding mice with a high-fat diet for 16 weeks. Effect of SCP-treatment (100 mg/kg, once daily for 12 weeks) on biochemical parameters and liver histopathology was assessed. Relative levels of sterol regulatory element-binding proteins (SREBPs) and their gene expressions were determined by quantitative real-time polymerase chain reaction and Western Blot. RESULTS: SCP significantly reduced the liver index by 12.0%. Serum levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol, alanine aminotransferase and aspartate aminotransferase were decreased by 31.3, 28.3, 42.8, 20.1 and 15.5%, respectively. Serum high-density lipoprotein cholesterol was increased by 26.9%. Further, SCP lowered hepatic TC and TG content by 27.0% and 28.3%, respectively, and alleviated fatty degeneration and necrosis of liver cells. A significant downregulation of mRNA and protein expressions of hepatic lipogenesis genes, SREBP-1c, fatty acid synthase and acetyl-CoA carboxylase, and the mRNA expression of liver X receptor α (LXRα) was observed in NAFLD mice treated with SCP. SCP also significantly reduced the hepatic expression of SREBP-2 and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). CONCLUSION: These findings demonstrate the hepatoprotective effects of SCP in a mouse model of NAFLD; the effects may be mediated via downregulation of LXRα/SREBP-1c/FAS/ACC and SREBP-2/HMGCR signaling pathways in the liver.

Cloning and primary immunological study of TGF-ß1 and its receptors TßR I /TßR II in tilapia(Oreochromis niloticus).

The transforming growth factor ß (TGF-ß) superfamily plays critical roles in tumor suppression, cell proliferation and differentiation, tissue morphogenesis, lineage determination, cell migration and apoptosis. Recently, TGF-ß1, one important member of TGF-ß superfamily, is suggested as an immune regulator in the teleost. In this study, we cloned the cDNAs of TGF-ß1 and its receptors, TßR I and TßR II (including three isoforms) from tilapia (Genbank accession numbers: KP754231- KP754235). A tissue distribution profile analysis indicated that TGF-ß1 was highly expressed in the head kidney, gill, spleen, kidney and PBLs (peripheral blood leukocytes); TßR I only showed considerable expression in the liver; and TßR II-2 was highly expressed in the kidney, gill, liver, head kidney and heart. We determined that the mRNA expressions of TGF-ß and TßR I /TßR II-2 were significantly increased in tilapia head kidney and spleen leukocytes by the stimulation of Lipopolysaccharide (LPS) or Poly I: C. We also examined their expressions in the spleen and head kidney of tilapia after IP injection of streptococcus agalactiae. The results showed that the mRNA expressions of these three genes all increased in the head kidney as early as 6 h post infection, and in the spleen 3 d post infection. In addition, the protein level of TGF-ß1 was also up-regulated in the head kidney and the spleen after infection. Taken together, our data indicate that the TGF-ß1-TßR I /TßR II-2 system functions potentially in tilapia immune system.

Molecular cloning, functional identification and expressional analyses of FasL in Tilapia, Oreochromis niloticus.

FasL is the most extensively studied apoptosis ligand. In 2000, tilapia FasL was identified using anti-human FasL monoclonal antibody by Evans's research group. Recently, a tilapia FasL-like protein of smaller molecule weight was predicted in Genbank (XM_003445156.2). Based on several clues drawn from previous studies, we cast doubt on the authenticity of the formerly identified tilapia FasL. Conversely, using reverse transcription polymerase chain reaction (RT-PCR), the existence of the predicted FasL-like was verified at the mRNA level (The Genbank accession number of the FasL mRNA sequence we cloned is KM008610). Through multiple alignments, this FasL-like protein was found to be highly similar to the FasL of the Japanese flounder. Moreover, we artificially expressed the functional region of the predicted protein and later confirmed its apoptosis-inducing activity using a methyl thiazolyl tetrazolium (MTT) assay, Annexin-V/Propidium iodide (PI) double staining, and DNA fragment detection. Supported by these evidences, we suggest that the predicted protein is the authentic tilapia FasL. To advance this research further, tilapia FasL mRNA and its protein across different tissues were quantified. High expression levels were identified in the tilapia immune system and sites where active cell turnover conservatively occurs. In this regard, FasL may assume an active role in the immune system and cell homeostasis maintenance in tilapia, similar to that shown in other species. In addition, because the distribution pattern of FasL mRNA did not synchronize with that of the protein, post-transcriptional expression regulation is suggested. Such regulation may be dominated by potential adenylate- and uridylate-rich elements (AREs) featuring AUUUA repeats found in the 3' untranslated region (UTR) of tilapia FasL mRNA.

Molecular cloning and expression of orange-spotted grouper (Epinephelus coioides) CD8α and CD8ß genes.

T-cell surface glycoprotein CD8 consists of two distinguished chains, termed α and ß chains, and functions as a co-receptor for the T-cell receptor by binding to MHC class I proteins. In this study we report the cloning and identification of both CD8α and CD8ß genes from orange-spotted grouper (Epinephelus coioides). The predicted grouper CD8α and CD8ß proteins were structurally similar to other fish especially to those of Pleuronectiformes. Real-time RT-PCR revealed that the CD8 mRNA was much higher in the thymus than in other immune organs, and the expression level were very low in stomach, liver, and brain. During embryonic development of the grouper, the highest CD8 transcripts were detected in the multi-cell stage, followed by muscle burl stage, which suggested that the multi-cell stage may be critical in CD8 transcript synthesis. Moreover, CD8 mRNA levels were examined in lymphocytes at different time treated with lipopolysaccharide (LPS), polyriboinosinic polyribocytidylic acid (PolyI:C), phytohemagglutinin (PHA), and concanavalin A (ConA). The result showed that the CD8 mRNA levels were significantly affected in time-dependent manner by PolyI:C, PHA, and ConA, but not by LPS.

5-(2-Fur-yl)-3-methyl-1-(3-nitro-phen-yl)-4,5-dihydro-1H-pyrazole.

In the title compound, C(14)H(13)N(3)O(3), the pyrazoline ring assumes an envelope conformation with the furanyl-bearing C atom at the flap position. The dihedral angle between the furan and nitrobenzene rings is 84.40 (9)°. Weak inter-molecular C-H⋯O hydrogen bonding is present in the crystal structure.

3-Methyl-1-(3-nitro-phen-yl)-5-phenyl-4,5-dihydro-1H-pyrazole.

In the title compound, C(16)H(15)N(3)O(2), the planar [maximum deviation 0.156 (2) Å] pyrazoline ring is nearly coplanar with the 3-nitro-phenyl group and is approximately perpendicular to the phenyl ring, making dihedral angles of 3.80 (8) and 80.58 (10)°, respectively. Weak inter-molecular C-H⋯O hydrogen bonding is present in the crystal structure.

[Effect of Xuesaitong drop pills on experimerntal thrombosis and thrombolysis in rats].

OBJECTIVE: To study the effect and the mechanism of Xuesaitong drop pills (total saponins in Radix Notoginseng; XDP) on experimental thrombosis, thrombolysis and blood theology. METHOD: First, the rats were randomly divided into five groups: control, XDP (90, 30, 10 mg x kg(-1)), Xuesaitong tablet (XP) 30 mg x kg(-1). Then the effect of the drugs on thrombus and thrombosis was studied after the ratsthrombosis was induced by the arteriovenous shunt. Second, the rats were randomly divided into seven groups: model, XDP (90, 30, 10 mg x kg(-1)), XT (90, 30 mg x kg(-1)), lumbrukinase capsule. Then the effect of the drugs on thrombus and thrombosis was studied after the rats'thrombosis was induced by the electrical stimulation of common carotid artery. Third, the rats were randomly divided into six groups: control, model, XDP (80, 40 mg x kg(-1)), XT (40, 20 mg x kg(-1)). Then the effect of the drugs on blood circulation promoting was observed after the rats'acute blood stasis induced by adrenalin and icy water. RESULT: XDP 90, 30 mg x kg(-1) could notably lighten the wet-weight and dry-weight of thrombus in the arteriovenous shunt model in rats in a dose-dependent manner (P < 0.01). XDP 90 mg x kg(-1) with intragastric administration for 3 days had the satisfactory effect on thrombolysis after the rat's thrombosis was induced by the electrical stimulation of common carotid artery (P < 0.01). XDP 80, 40 , 20 mg x kg(-1) reduced significantly erythrocyte aggregation (P < 0.01) and decreased the whole blood viscosity at low shear rate (P < 0.05). XDP 80, 40 mg x kg(-1) reduced the whole blood viscosity at high shear rate and plasma viscosity (P < 0.05). XDP 80 mg x kg(-1) decreased the whole blood viscosity at high shear rate (P < 0.05). CONCLUSION: XDP can significantly inhibit the thrombosis and has the satisfactory effect on thrombolysis. One kind of the mechanism is related to the effect on blood rheology.

Regulation of the stability of P-glycoprotein by ubiquitination.

Ubiquitination plays a crucial role in regulating protein turnover. Here we show that ubiquitination regulates the stability of the MDR1 gene product, P-glycoprotein, thereby affecting the functions of this membrane transporter that mediates multidrug resistance. We found that P-glycoprotein was constitutively ubiquitinated in drug-resistant cancer cells. Transfection of multidrug-resistant cells with wild-type ubiquitin or treatment with an N-glycosylation inhibitor increased the ubiquitination of P-glycoprotein and increased P-glycoprotein degradation. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), a proteasome inhibitor, induced accumulation of ubiquitinated P-glycoprotein, suggesting the involvement of the proteasome in the turnover of the transporter. Treatment of multidrug-resistant cells with 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester that increases the phosphorylation of P-glycoprotein through activation of protein kinase C, or substituting phosphorylation sites of P-glycoprotein by nonphosphorylatable residues did not affect the ubiquitination of the transporter. Enhanced ubiquitination of P-glycoprotein resulted in a decrease of the function of the transporter, as demonstrated by increased intracellular drug accumulation and increased cellular sensitivity to drugs transported by P-glycoprotein. Our results indicate that the stability and function of P-glycoprotein can be regulated by the ubiquitin-proteasome pathway and suggest that modulating the ubiquitination of P-glycoprotein might be a novel approach to the reversal of drug resistance.