BASP1 promotes high glucose-induced endothelial apoptosis in diabetes via activation of EGFR signaling.

AIMS: Diabetes mellitus is a common chronic disease of glucose metabolism. Endothelial dysfunction is an early event in diabetes complicated by cardiovascular disease. This study aimed to reveal the expression of BASP1 and its biological roles in endothelial cell dysfunction in diabetes complicated by cardiovascular disease. MATERIALS AND METHODS: By analyzing the databases related to diabetes complicated with coronary heart disease, BASP1 was screened out as an upregulated gene. Human umbilical vein endothelial cells (HUVECs) and primary mouse aortic endothelial cells were treated with high glucose to establish cell models of diabetes-related endothelial dysfunction, and the expression changes of BASP1 were verified by RT-qPCR, western blot, and immunofluorescence. BASP1 was silenced or overexpressed by siRNA or overexpression plasmid, and its effects on cell migration, apoptosis, tube formation, inflammatory response, and ROS were detected. The possible signaling pathway of BASP1 was found and the mechanism of BASP1 on promoting the progression of endothelial dysfunction was explored using the EGFR inhibitor, gefitinib. RESULTS: Bioinformatics analysis indicated that the expression of BASP1 in patients with diabetes mellitus and concomitant coronary heart disease was increased. High glucose induced the upregulation of BASP1 expression in endothelial cells, and showed a time-dependent relationship. Silencing of BASP1 alleviated the damage of high glucose to endothelial cells. BASP1 regulated EGFR positively. The promoting effect of BASP1 on endothelial cell apoptosis may be achieved by regulating the EGFR pathway. CONCLUSION: BASP1 promotes endothelial cell injury induced by high glucose in patients with diabetes, which may be activated by activating the EGFR pathway.

Alternative method for site-directed mutagenesis of complex polyketide synthase in Streptomyces albus JA3453.

Sequence analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces albus JA3453 revealed a gene, ozmH, encoding a hybrid polyketide and non-ribosomal peptide enzyme. Tandem ketosynthase (KS) domains (KS 10-1 and KS 10-2) were characterized and they show significant homology with known KSs. Using an alternative method that involves RecA-mediated homologous recombination, the negative selection marker sacB gene, and temperature-sensitive replications, site-directed mutagenesis of the catalytic triad amino acid cysteines were carried out in each of the tandem KS domains to test the function they play in OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant strains showed that KS 10-2 is essential for OZM biosynthesis but KS 10-1 is not indispensable and might serve as a "redundant" domain. These results confirmed the existence of an "extra domain" in complex polyketide synthase.

Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning the oxazolomycin biosynthetic gene cluster from Streptomyces albus JA3453.

Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.

[Effect of apoptosis in human breast cancer cells and its probable mechanisms by genistein].

OBJECTIVE: To investigate the effects of genistein on human breast cancer cell MCF-7 apoptosis and its probable mechanisms. METHODS: In this study, the methods of MTT, cell apoptosis detecting in fluorescent and electronic microscope and flow cytometry, and expression of Bax and erbB-2 protein were employed. RESULTS: The results showed that genistein could significantly inhibit the growth and induce the apoptosis of MCF-7 cells. Apoptotic cells of morphology from MCF-7 cells treated by different concentrations of genistein were observed by fluorescent and electronic microscope and the frequency of apoptosis in MCF-7 cells by flow cytometry showed increasingly with concentrations of genistein increased. The expression of Bax protein in MCF-7 cells was increased and the expression of erbB-2 protein was decreased with the doses of genistein. CONCLUSION: Genistein can induce MCF-7 cells apoptosis and it may be one of the mechanisms for the inhibitory effect of genistein in human cancer cells.

Blocking effects of genistein on cell proliferation and possible mechanism in human gastric carcinoma.

AIM: To study the blocking effects of genistein on cell proliferation cycle in human gastric carcinoma cells (SGC-7901) and the possible mechanism. METHODS: MTT assay was applied in the detection of the inhibitory effects of genistein on cell proliferation. Flow cytometry was used to analyze the cell cycle distribution. Immunocytochemical technique and Western blotting were performed to detect the protein expression of cyclin D1, cyclin B1 and p21(waf1/cip1). RESULTS: Genistein significantly inhibited the growth and proliferation of human gastric carcinoma cells (SGC-7901). Seven days after treatment with different concentrations of genistein (2.5, 5.0, 10.0, 20.0 microg/mL), the growth inhibitory rates were 11.2%, 28.8%, 55.3%, 84.7% respectively and cell cycles were arrested at the G(2)/ M phase. Genistein decreased cyclin D1 protein expression and enhanced cyclin B1 and p21(waf/cip1) protein expression in a concentration-dependent manner. CONCLUSION: The growth and proliferation of SGC-7901 cells can be inhibited by genistein via blocking the cell cycle, with reduced expression of cyclin D1 and enhanced expression of cyclin B1 and p21(waf/cip1) protein in the concentration range of 0-20 microg/mL.

[Study on mechanisms of human gastric carcinoma cells apoptosis induced by genistein].

In order to study the apoptosic effect of genistein on human gastric carcinoma SGC-7901 cells, DAPI dyeing and DNA electrophoresis were used. Western Blot was used to detect the changes of the expression of p53 protein, Caspase-3 after SGC-7901 cells were exposed to genistein. The results showed that genistein induced cell apoptosis. Genistein also decreased the expression of mutant p53, and increased the protein expression of Caspase-3. These indicated that genistein could induce the apoptosis of gastric carcinoma cells. The inhibition of the expression of mutant p53 and enhance of the protein expression of Caspase-3 would be possible mechanism.

[Inhibitory effects of genistein on the synthesis of DNA and the protein expression of cyclin D1 in human gastric carcinoma cell-line].

In order to study the inhibitory effects of genistein on the growth of human gastric carcinoma cells and to explore its possible mechanism, cell proliferation and DNA incorporation tests are applied. Immunocytochemistry is also applied in this study to detect the protein expression of cyclin D1 after the cell-line is exposed to genistein. The results showed that the growth of the cells and the synthesis of DNA is decreased in a marked dose-dependent manner after the treatment with genistein. Genistein also decreased the protein expression of cyclin D1. The above mentioned results indicate that genistein inhibits the growth of gastric carcinoma cells and the possible mechanism of this inhibition might be resulted from inhibiting the synthesis of DNA and the protein expression of cyclin D1.

[Effect of isoflavone on human breast cancer cell line MCF-7 and its probable mechanism].

The effects of isoflavone on human breast cancer cell line MCF-7 growth and apoptosis and its probable mechanisms were studied in vitro. The results showed that isoflavone could significantly inhibit the growth and induce the apoptosis of MCF-7 cells. Western Blotting showed that isoflavone increased iNOS protein expression. These results suggested that isoflavone significantly inhibited the MCF-7 growth and induced cell apoptosis by regulating iNOS gene expression.