Technological and Genomic Analysis of Roles of the Cell-Envelope Protease PrtS in Yoghurt Starter Development.

The cell-envelope protease PrtS was proved to be efficient in optimal bacterial growth and fast acidification in pure culture, while its positive effect on the performance of mixed-cultures in milk fermentation was not defined. The aim was to analyze effects of the PrtS on the symbiosis between strains during yoghurt production and cold storage. Two Streptococcus thermophilus strains, KLDS3.1012 and KLDS SM, and two different proteolytic strains of Lactobacillus delbrueckii subsp. Bulgaricus, L7 and L12, were used. Technological properties (viability, acid production, and proteolysis) were determined. Comparative genomics was used to analyze the proteolytic system (cell-envelope protease, transport system, intracellular peptidase) of Streptococcus thermophilus strains. S. thermophilus KLDS SM possesses an intact gene encoding PrtS (A9497_00420), which was not found in the genome of S. thermophilus KLDS3.1012. This gene is the main difference in the proteolytic system between the two genomes. PrtS endowed KLDS SM high levels of viability during fermentation and cold storage. When combined with a weaker lactobacillus strain during fermentation, the acceleration of acid production of mixed-culture by KLDS SM would start at an earlier time. KLDS SM increased the post-acidification of yoghurts during cold storage, but the pH was steadily maintained during 14-28 days. Results suggest that strains of Streptococcus thermophilus with strong proteolytic ability could be used in a wide range of dairy production. The present study provided data for yoghurt starter development from the point of view of proteolysis.

Short communication: Genomic and phenotypic analyses of exopolysaccharides produced by Streptococcus thermophilus KLDS SM.

Streptococcus thermophilus plays important roles in the dairy industry. Streptococcus thermophilus KLDS SM could produce a high amount of exopolysaccharides (EPS). To understand the possible link between the genotype and the phenotype regarding EPS, the complete genome of S. thermophilus KLDS SM was sequenced and investigated in silico for genes related to carbohydrate fermentation, nucleotide sugars synthesis, and EPS gene cluster. We found that S. thermophilus KLDS SM is able to ferment sucrose, mannose, glucose, galactose, and lactose from the genomic research, which was confirmed by API 50 CH (bioMérieux, Marcy l'Etoile, France). The genetic analysis of nucleotide sugars and EPS cluster revealed that the EPS produced by this strain are composed of galactose and glucose, in accordance with the biochemical result. Furthermore, differences in the molecular mass of EPS from S. thermophilus KLDS SM cultivated under different carbon sources were correlated with the transcription levels of the genes encoding chain length determination protein and glycosyltransferase. Our findings provide a better understanding of the link between the genetic elements and the chemical conformation of EPS and a theoretical basis for producing tailor-made EPS through genetic and metabolic engineering approaches.

Complete Genome Sequence of Streptococcus thermophilus KLDS 3.1003, A Strain with High Antimicrobial Potential against Foodborne and Vaginal Pathogens.

Lactic acid bacteria play increasingly important roles in the food industry. Streptococcus thermophilus KLDS 3.1003 strain was isolated from traditional yogurt in Inner Mongolia, China. It has shown high antimicrobial activity against selected foodborne and vaginal pathogens. In this study, we investigated and analyzed its complete genome sequence. The S. thermophilus KLDS 3.1003 genome comprise of a 1,899,956 bp chromosome with a G+C content of 38.92%, 1,995 genes, and 6 rRNAs. With the exception of S. thermophilus M17TZA496, S. thermophilus KLDS 3.1003 has more tRNAs (amino acid coding genes) compared to some S. thermophilus strains available on the National Centre for Biotechnology Information database. MG-RAST annotation showed that this strain has 317 subsystems with most genes associated with amino acid and carbohydrate metabolism. This strain also has a unique EPS gene cluster containing 23 genes, and may be a mixed dairy starter culture. This information provides more insight into the molecular basis of its potentials for further applications in the dairy and allied industries.

Complete genome sequence of Enterococcus durans KLDS6.0930, a strain with probiotic properties.

Enterococcus durans KLDS6.0930 strain was originally isolated from traditional naturally fermented cream in Inner Mongolia of China. The complete genome sequence of E. durans KLDS6.0930 was carried out using the PacBio RSII platform. The genome contains a circular chromosome and two circular plasmids. Genome sequencing information provides the genetic basis for bioinformatics analysis of bile salt and acid tolerance, cell adhesion, and molecular mechanisms responsible for lipid metabolism.

Inhibition of angiogenesis by a novel neutralizing antibody targeting human VEGFR-3.

Vascular endothelial growth factor receptor 3 (VEGFR-3) is a receptor for the vascular endothelial growth factor C and D (VEGF-C and D) and plays a critical role in the development of embryonic vascular system and regulation of tumor lymphangiogenesis. In this report, we generated a novel panel of 17 monoclonal antibodies (mAbs) against human VEGFR-3 and determined their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. Furthermore, in chick embryo CAM angiogenesis experiments, the angiogenesis induced by recombinant glutathione-S-transferase-VEGF-D was decreased in the presence of antibody BDD073. These data suggest that this novel neutralizing antibody against human VEGFR-3 could be a tool for the investigations into the biology of VEGFR-3, and potentially a reagent for blocking VEGF-D-induced angiogenesis and lymphogenesis.

Trastuzumab signaling in ErbB2-overexpressing inflammatory breast cancer correlates with X-linked inhibitor of apoptosis protein expression.

Inflammatory breast cancer (IBC) patients show poor survival and a significant incidence of epidermal growth factor receptor-2 (ErbB2) overexpression. A distinct mechanism involving increased expression of X-linked inhibitor of apoptosis protein (XIAP) and survivin, key members of the inhibitor of apoptosis protein (IAP) family, was observed post-trastuzumab (an ErbB2 monoclonal antibody) treatment in an ErbB2-overexpressing, estrogen receptor negative, IBC cellular model, SUM190PT, isolated from a primary IBC tumor. In contrast, a decrease in the IAP expression was observed in the non-IBC, ErbB2-overexpressing SKBR3 cells in which trastuzumab treatment also decreased p-AKT and cell viability. Further, in SUM190PT cells, therapeutic sensitivity to GW583340 (a dual epidermal growth factor receptor/ErbB2 kinase inhibitor) corresponded with XIAP down-regulation and abrogation of XIAP inhibition on active caspase-9 release. Specific small interfering RNA-mediated XIAP inhibition in combination with trastuzumab caused decrease in inactive procaspase-9 and inhibition of p-AKT corresponding with 45% to 50% decrease in cell viability in the SUM190PT cells, which have high steady-state p-AKT levels. Further, embelin, a small-molecule inhibitor that abrogates binding of XIAP to procaspase-9, caused significant decrease in SUM190PT viability. However, embelin in combination with trastuzumab failed to affect SUM190PT viability because it has no direct effect on XIAP, which is induced by trastuzumab treatment. These data have identified a novel functional link between ErbB2 signaling and antiapoptotic pathway mediated by XIAP. Blockade of the IAP antiapoptotic pathway alone or in combination would be an attractive strategy in IBC therapy.

[Recombinant human VEGF-D induces the angiogenesis of the chick embryo chorioallantoic membrane].

Vascular endothelial growth factor-D (VEGF-D) and vascular endothelial growth factor receptor-2, -3 (VEGFR-2, -3) with their corresponding signaling pathway play significant roles in the development of the embryonic vascular system and pathological lymphangiogenesis. The study was aimed to express and purify the GST-VEGF-D fusion protein, and to explore the angiogenesis effect of VEGF-D. The total RNA was extracted from human fetal lung tissue, and the mature form of VEGF-D was expanded by polymerase chain reaction (PCR), then the plasmid pGEX-5X-1/VEGF-D was reconstructed and the GST-VEGF-D fusion protein expressed in transformed E.coli BL21-DE3. The results showed that the molecular mass of this fusion protein was 38 kD and compassed more than 15% of the total bacteria proteins. The fusion protein was recognized by anti-GST and anti-VEGF-D antibodies. The soluble GST-VEGF-D fusion protein could interact with VEGFR-3/Fc and was able to stimulate the proliferation of human erythroleukemia cell line (HEL) cells. The data of chick embryo chorioallantoic membrane (CAM) experiments indicated that GST-VEGF-D could induce the CAM angiogenesis. It is concluded that the GST-VEGF-D fusion protein with biological activity was successfully expressed, and which may provide an experimental model for the investigation of the VEGF-D-induced angiogenesis and lymphangiogenesis.

[Screening differentially expressed genes in the process of vascular smooth muscle cell calcification in vitro].

AIM: The process of vascular calcification involves various genetic alterations which may play a very important role in the vascular calcification. Vascular smooth muscle cells undoubtedly composed the main part of vascular cells, and are involved in vascular calcification. So bovine artery smooth muscle cell (BASMC) was used to investigate the gene changes during BASMC's calcification. METHODS: Bovine artery smooth muscle cells cultured in vitro was induced calcified by beta-Glycerophosphate (beta-GP). Using DD-PCR technique to screening differentially expressed genes and those differentially expressed bands were reexamined by reverse Northern blot. All the ESTs were sequenced and BLAST with GenBank. RESULTS: Total 65 cDNAs were isolated as differentially expressed genes and 40 of them were successfully reamplified. Using reverse-Northern blot, seven of these 40 cDNAs were reproducibly expressed differentially between the two cells. Three of them are new bands and have not been reported before. CONCLUSION: This is the first time using DD-PCR to screen differentially expressed genes of BASMC calcification. Seven related ESTs were identified relating to BASMC calcification.

Association of the phosphatidylinositol signal pathway with prolonged myocardial ischemia.

OBJECTIVE: To study the changes in activity of phosphatidylinositol 4 kinase (PI 4 kinase), phosphatidylinositol 4 phosphate 5 kinase (PIP 5 kinase) and protein kinase C (PKC) during myocardial ischemia and elucidate the relationship between phosphatidylinositol signal pathways and prolonged myocardial ischemia. METHODS: In vivo an ischemic rat model was used. Activity of PI 4 kinase, PIP 5 kinase and PKC were measured at different times in postischemic heart cells using isotope analysis. RESULTS: The activity of PI kinase, PIP kinase and PKC in the myocardium increased to peak at 1 hour postischemia, with activities 6.1, 3.0 and 4.0 fold over control levels, respectively. Their activities declined to normal levels with time. CONCLUSION: The phosphatidylinositol signal pathway is involved in prolonged myocardial ischemia, but its mechanism needs further study.