Extraction optimization, purification and characterization of polysaccharides from the seed coat of black soybean.

In this study, the extraction of water-soluble polysaccharides from the seed coat of black soybean (BSCP) was investigated and optimized. A response surface methodology based on a Box-Behnken design (BBD) was used to optimize the extraction conditions as follows: extraction temperature, 100°C; ratio of water to material, 22.3 mL/g; and extraction time, 133.

High-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete Leptospira interrogans.

Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira that infects humans and a wide range of animals. By combining computational prediction and high-accuracy tandem mass spectra, we revised the genome annotation of Leptospira interrogans serovar Lai, a free-living pathogenic spirochete responsible for leptospirosis, providing substantial peptide evidence for novel genes and new gene boundaries. Subsequently, we presented a high-coverage proteome analysis of protein expression and multiple posttranslational modifications (PTMs).

Molecular characterization of the pL40 protein in Leptospira interrogans.

Leptospirosis is a widespread zoonotic disease caused by pathogenic leptospires. The identification of outer membrane proteins (OMPs) conserved among pathogenic leptospires, which are exposed on the leptospiral surface and expressed during mammalian infection, has become a major focus of leptospirosis research. pL40, a 40 kDa protein coded by the LA3744 gene in Leptospira interrogans, was found to be unique to Leptospira.

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e.

Granulocyte-macrophage colony-stimulating factor DNA prime-protein boost strategy to enhance efficacy of a recombinant pertussis DNA vaccine.

Aim: To investigate a new strategy to enhance the efficacy of a recombinant pertussis DNA vaccine. The strategy is co-injection with cytokine plasmids as prime, and boosted with purified homologous proteins.

Method: A recombinant pertussis DNA vaccine containing the pertussis toxin subunit 1 (PTS1), fragments of the filamentous hemagglutinin (FHA) gene and pertactin (PRN) gene encoding filamentous hemagglutinin and pertactin were constructed.

Characterization of the cheY genes from Leptospira interrogans and their effects on the behavior of Escherichia coli.

The motility and chemotaxis system are critical for the virulence of pathogenic leptospire, which enable them to penetrate host tissue barriers during infection. The completed genome sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroups Icterohaemorrhagiae (L. interrogans strain Lai) suggested that there were multiple copies of putative chemotaxis homologues located at its large chromosome.

Characterization of cheW genes of Leptospira interrogans and their effects in Escherichia coli.

The motility and chemotaxis systems are critical for the virulence of leptospires. In this study, the phylogenetic profiles method was used to predict the interaction of chemotaxis proteins. It was shown that CheW1 links to CheA1, CheY, CheB and CheW2; CheW3 links to CheA2, MCP (LA2426), CheB3 and CheD1; and CheW2 links only to CheW1.

Thrombocytopenia in the experimental leptospirosis of guinea pig is not related to disseminated intravascular coagulation.

Background: Thrombocytopenia is commonly observed in severe leptospirosis. However, previous studies on coagulation alterations during leptospirosis resulted in inconsistent conclusions. Some findings showed that the prominent levels of thrombocytopenia observed in severe leptospirosis did not reflect the occurrence of disseminated intravascular coagulation (DIC) syndrome, while the others reached the conclusion that the hemorrhages observed in leptospirosis were due to DIC.

Expression and comparative analysis of genes encoding outer membrane proteins LipL21, LipL32 and OmpL1 in epidemic leptospires.

Leptospiral outer membrane proteins (OMPs) are highly conserved in different species, and play an essential role in the development of new immunoprotection and serodiagnosis strategies. The genes encoding LipL21, LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospira interrogans serovar lai strain Lai and expressed in vitro. Sequence comparison analysis revealed that the three genes were highly conserved among distinct epidemic leptospires, including three major epidemic species Leptospira interrogans, Leptospira borgpetersenii and Leptospira weilii, in China.

[Cytoplasmic expression of VP1 gene of coxsackievirus B3].

Objective: To increase the immune effect of gene vaccine, T7 RNA polymerase was used to establish a system of cytoplasmic expression.

Methods: (1) The plasmid pT7 EMCVP1, including T7 promoter sequence, 5'-untranslated sequence of encephalomyocarditis (EMC) virus, VP1 sequence of coxsackievirus B3 (CVB3), was cotransfected with the plasmid pAR 3132, which codes for the T7 RNA polymerase, into HeLa cells and murine peritoneal macrophages. (2) The plasmid pT7 EMCVP1 and pAR 3132 were respectively transformed into the attenuated Salmonella typhimurium SL 7207.

[Attenuated Salmonella typhimurium as DNA delivery vehicle for DNA-mediated immunization].

Objective: To study whether the live attenuated AroA-auxotrophic mutant of Salmonella (S.) typhimurium (SL7207) could be used as DNA delivery vehicle to induce more efficient immune response by using the eukaryotic expression plasmid pCMV-beta as report gene.

Methods: Murine peritoneal macrophages were infected with SL7207(pCMV-beta) in vitro, then the expression of the beta-gal were detected by X-gal staining or RT-PCR.

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio.

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogans serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver.

Analysis of gene expression profile in gastric cancer cells stimulated with Helicobacter pylori isogenic strains.

To understand the biological processes within host cells induced by VacA, isogenic strains of Helicobacter pylori (NCTC 11638 or 11638-DeltavacA) were used to stimulate gastric cancer cells SGC7901, and differentially expressed genes in host cells were identified using cDNA microarray technology. More than 300 genes were found to alter their mRNA expression at different time points, among which 68 were related to the cytoskeleton, 87 were associated with cell cycle, cell death and proliferation, IL8 expression was also found to be up-regulated. Cells co-cultured with broth-culture supernatant (BCS) of NCTC 11638 showed more alteration in microtubule cytoskeleton morphology, as observed by laser scanning confocal microscopy, and a lower apoptosis rate, detected by flow cytometry, compared with those co-cultured with BCS of 11638-DeltavacA.

mRNA expression profiling reveals a role of Helicobacter pylori vacuolating toxin in escaping host defense.

Aim: To study the immune response of host to Helicobacter pylori VacA.

Methods: The monocyte/macrophage-like U937 cells were infected with Helicobacter pylori vacA-positive strain NCTC 11638 or isogenic vacA-negative mutant. Differentially expressed genes were identified at 2, 6, 10, and 24 h post-infection by cDNA microarray.

Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis.

Aim: To construct a vacA-knockout Helicobacter pylori mutant strain, whose only difference from the wild strain is its disrupted vacA gene.

Methods And Results: A clone containing kanamycin resistance gene used for homologous recombination was constructed in a directional cloning procedure into pBluescript II SK, and then transformed into vacA+ H pylori by electroporation. Colonies growing on the selective media containing kanamycin were harvested for chromosomal DNA extraction, and the allelic exchange was determined by polymerase chain reactions and sequencing.