[Exploring the active ingredient of Chinese yellow wine which could inhibit the Hcy induced proliferation and migration of vascular smooth muscle cells].

OBJECTIVE: To explore the active ingredients in the Chinese yellow wine could inhibit the proliferation and migration of rat vascular smooth muscle cells induced by homocysteine (Hcy). METHODS: The primary culture and identification of rat vascular smooth muscle cells (VSMCs) was conducted, and the VSMCs in passage 4-7 were used in the following experiments. The VSMCs were divided into 7 groups: control, Hcy (1 mmol/L), Hcy + oligosaccharide, Hcy + polypeptides, Hcy + polyphenols, Hcy + alcohol, Hcy + Chinese yellow wine and were given the corresponding treatment. The proliferation of VSMCs was determined by MTT. Transwell chambers and would healing were employed to test the migratory ability of VSMCs. Wester blot and gelatin zymography were used to investigate the expressions and activities of metal matrix proteinase 2/9 (MMP-2/9) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in VSMCs of each group. RESULTS: Compared with control group, the proliferation, migration and the expression and activity of MMP-2/9 of VSMCs were significantly increased in the VSMCs of Hcy group (P < 0.01). Compared with Hcy group, the proliferation, migration and the expression and activity of MMP-2/9 of VSMCs were significantly decreases in the VSMCs of polypeptides group, polyphenols group and Chinese yellow wine group. However, the expression of TIMP-2 among each group had no significant difference. CONCLUSION: Polypeptides and polyphenols in the Chinese yellow wine could inhibit the proliferation and migration of VSMCs induced by Hcy.

Characterization of a novel serine protease inhibitor gene from a marine metagenome.

A novel serine protease inhibitor (serpin) gene designated as Spi1C was cloned via the sequenced-based screening of a metagenomic library from uncultured marine microorganisms. The gene had an open reading frame of 642 base pairs, and encoded a 214-amino acid polypeptide with a predicted molecular mass of about 28.7 kDa. The deduced amino acid sequence comparison and phylogenetic analysis indicated that Spi1C and some partial proteinase inhibitor I4 serpins were closely related. Functional characterization demonstrated that the recombinant Spi1C protein could inhibit a series of serine proteases. The Spi1C protein exhibited inhibitory activity against α-chymotrypsin and trypsin with K(i) values of around 1.79 × 10(-8) and 1.52 × 10(-8) M, respectively. No inhibition activity was exhibited against elastase. Using H-d-Phe-Pip-Arg-pNA as the chromogenic substrate, the optimum pH and temperature of the inhibition activity against trypsin were 7.0-8.0 and 25 °C, respectively. The identification of a novel serpin gene underscores the potential of marine metagenome screening for novel biomolecules.

A novel ß-glucosidase with lipolytic activity from a soil metagenome.

Moonlighting proteins have two different functions within a single polypeptide chain. Exploring moonlighting enzymes from the environment using the metagenomic approach is interesting. In the present study, a novel ß-glucosidase gene, designated as bgl1D, with lipolytic activity (renamed Lip1C) was cloned through function-based screening of a metagenomic library from uncultured soil microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis also indicated that Lip1C and other putative lipases are closely related. Biochemical characterization demonstrated that the maximum activity of the recombinant Lip1C protein occurs at pH 8.0 and 30°C using 4-nitrophenyl butyrate as substrate. The putative lipase had an apparent K(m) value of 0.88 mmol/L, a k(cat) value of 212/min, and a k(cat)/K(m) value of 241 L/mmol/min. Lip1C exhibited habitat-specific characteristics with 5 mmol/L AlCl(3), CuCl(2), and LiCl. The characterization of the biochemical properties of Lip1C enhances our understanding of this novel moonlighting enzyme isolated from a soil metagenome.

[Cloning and diversity analysis of microorganism genes from alkalescence soil].

The metagenomic DNAs were extracted and purified from alkalescence environmental samples directly. On the basis of the metagenomic DNA, the alkaline soil 16S rDNA library composed of 5,562 positive clones was constructed. The phylogenic tree indicated that the bacteria from the alkaline soils were bio-diversity. The metagenomic DNA library named AL01 was constructed by inserting restriction fragments of the purified DNAs into plasmids pGEM-3Zf(+) vector. This library contained 23,650 positive clones and the average foreign DNA fragments were about 3.2 kb. The length of the library covered 75.68 Mb. The efficiency of the metagenomic library was approximately 6,000 clones from 1g dry soil samples. After screening AL01 DNA library with the screening tactics of enzymes, we confirmed that a positive clone, designated pGXAA2011, contained an alkaline protease gene AP01. Enzymatic analysis proved that its reaction optimum pH was 9.5 and the optimum temperature was 40 degrees C. Furthermore, a clone, designated pGXAG142 was screened from metagenomic DNA library, which expresses beta-glucosidase. DNA sequence indicated that the potential ORF of pGXAG142, which was named unglu01, there was no DNA or amino acids identity with the known beta-glucosidase genes in the Genbank. The integrated ORF was cloned into pETBlue-2 vector and was then transformed into Tuner(DE3)pLacI. The recombinant expression clone could express beta-glucosidase on the screening plate clearly and the analysis of SDS-PAGE indicated that the target protein was about 29 kDa.