Objective To determine the hemolytic activity of products of sphingomyelinase hemolysin encoding genes of Leptospira interrogaas, and the transcriptional level alterations in the infected host cells. Methods By using genomic DNAs of pathogenic L. interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai and serogroup Pomona serovar Pomona strain Luo, and non-pathogenic L. biflexa serogroup Sama-ranga serovar Patoc strain Patoc Ⅰ as templates, PCRs were performed to amplify entire sph1-sph4 genes. The amplified products were sequenced after T-A cloning. Prokaryotic expression systems of sph1-sph4 genes were re-spectively constructed, and the expressions of target recombinant proteins rSph1-rSph4 were examined by SDS-PAGE. Ni-NTA affinity chromatographic column was used to extract the expressed rSph1-rSph4. Hemolytic ac-tivities of rSph1-rSph4 on sheep blood agar plate were identified. Transcription alterations of sphl-sph4 genes in L. interrogans strain Lai after infected J774A. 1 cells were measured by real-time fluorescence quantitative RT-PCR. Results From genomic DNAs of both L. interrogans strain Lai and Luo, but not from that of L. biflexa strain Patoc Ⅰ , the target fragments of sph1-sph4 genes could be amplified. All the cloned sph1-sph4 genes had 100% nucleotide sequence identities compared to the corresponding reported sequences. The constructed pro-karyotic expression systems were able to efficiently express the target recombinant proteins rSph1-rSph4, respec-tively. All the rSph1-rSph4 had hemolytic activities, and among the four products rSph2 displayed the strongest hemolytic activity. After L. interrogaas strain Lai infecting J774A. 1 cells, the transcriptional levels of sph1-sph4 genes were remarkably up-regulated, especially for mRNA levels of sph2 and sph4 genes. Conclusion sph1- sph4 genes exist only in pathogenic L. interrogans species, and their products have hemolytic activity. The up-regulation of sph1-sph4 gene transcriptional levels in L. interrogans strain Lai after infected cells implies that the sphingomyelinase hemolysins may play important roles in the process of L. interrogans infection in hosts.

Bis-(3′,5′) cyclic di-guanylate (c-di-GMP) is an almost ubiquitous intracellular second messenger in bacteria.Now it is known to regulate complex physiological processes, including mobility, adhesion, virulence and biofilm formation.The level of c-di-GMP is regulated by diguanylate cyclases (DGCs) containing GGDEF domains and phosphodiesterases (PDEs) containing EAL or HD-GYP domains.Recent studies have demonstrated that HD-GYP domain protein is a novel phosphodiesterase, which is also involved in the regulation of c-di-GMP degradation.This review highlights recent advances in the structure and biochemical functions of HD-GYP domain proteins, which might help to further clarify the mechanism of c-di-GMP signal system.

In 4 602 subjects for routine check-up,blood uric acid,total cholesterol,triglyceride,high density lipoprotein-cholesterol,and low density lipoprotein-cholesterol were determined.Results showed that the overall prevalence of hyperuricemia in Nanning,Guangxi was 19.8％ (28.8％ in male,9.4％ in female).Blood uric acid and lipids in hyperuricemia group were higher than those in normal uric acid group(all P＜0.01).Serum uric acid had a positive correlation with total cholesterol,triglyceride,low density lipoprotein-cholesterol,but it was negatively correlated with serum high density lipoprotein-cholesterol.The prevalence of hypercholesterolemia was 30.8％,and that of hypertriglyceridemia was 22.2％.Logistic multi-factor regression analysis showed that men,high total cholesterol,triglyceride,and low density lipoprotein-cholesterol could be independent risk factors for hyperuricemia,and that high density lipoprotein-cholesterol was a protective factor.The prevalence of hyperuricemia in population of Nanning,Guangxi during health examination is high.Hyperuricemia is closely associated with dyslipidemia.Timely intervention of hyperuricemia can reduce the related diseases effectively.

Objective To determine the function of toxic protein VapC in toxin/antitoxin system of Leptospira species and the cytotoxicity to host cells of the toxic protein.Methods Using genomic DNA of pathogenic L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai as the template,several PCRs were performed to amplify entire vapB,vapC and vapBC genes.Subsequently,the prokaryotic expression systems of vapB,vapC and vapBC genes were constructed.Expression of the target recombinant proteins rVapB and rVapC was detected by SDS-PAGE and the expressed rVapB and rVapC were extracted by NiNTA affinity chromatography.Activity of rVapB and rVapC to lyse the DNAs or RNAs from L.interrogans strain Lai and THP-1 cells were then determined.The changes of transcription and expression of vapB and vapC genes of L.interrogans strain Lai before and after infection of THP-1 cells were detected by real-time fluorescent quantitative RT-PCR and Western blot assay.The eukaryotic expression vectors of the vapB and vapC genes were generated for transfection of host cells and CCK-8 agent was used to detect the effect of leptospiral VapB and VapC proteins on activity of host cells.Results The nucleotide and putative amino acid sequences of the cloned vapB and vapC genes were completely identical with the reported corresponding genes.The constructed prokaryotic expression systems could express rVapB and rVapC,respectively.rVapC displayed RNase avtivity but did not lyse DNA.When L.interrogans strain Lai infected THP-1 cells,the transcription and expression of vapB and vapC genes were upregulated and partial VapC protein was secreted from the leptospiral cells.The mass mortality was observed in HEK293 human renal tubular epithelial ceils containing the vapC gene through transfection.Conclusion VapC protein of L.interrogans strain Lai is a RNase and is secreted during infection of host cells with obvious cytotoxicity.

Objective To compare the sensitivity and specificity of Leptospira interrogans using loop-mediated isothermal amplification (LAMP) and real-time PCR technology, then looking for a rapid,sensitive and specific methods for the detection of Leptospira interrogans. MethodsIn accordance with lipL41 gene from Leptospira interrogans, primers for LAMP and real-time PCR were designed and used to detect Leptospira interrogans in cultured 15 reference strains of 15 serogroups in China, then compared the sensitivity and specificity of the two methods in the detection of Leptospira interrogans. ResultsThe LAMP reaction could be completed within 30 min, and whole process of it less than 60 min. The whole real-time PCR reaction could be finished at about 60 min. Both of them had the same detection sensitivity and specificity,the lower detection limits in the reactions was approximately 100 copies and there was no false positive occurred. ConclusionBoth LAMP and real-time PCR were time-saving and had the same sensitivity and specificity. But LAMP reaction could be done under a constant temperature conditions, and need not a special expensive equipment. Therefore, as a sensitive and specific method for quantifying Leptospira interrogans,the LAMP assay was more rapidly and convenient than conventional methods.

Objective To construct the T-cells and B-cells combined epitope peptide gene based on the LipL32,OmpL1 and LipL21 protein from Leptospira interrogans and E.coli expression system,and better understanding of the immunological activity of the recombinant protein. MethodsThe immunodomaint T- and B-cells combined epitopes of LipL32,OmpL1 and LipL21 were identified and used to synthetic a new gene and then construct its prokaryotic expression system.The expression of recombinant protein was determined by SDS-PAGE; MAT was used to determine the titer of the antiserum to L. interrogans standard strains of China ; Western blot and ELISA were used to identify the immunity activity of the recombinant protein.Results The synthetic gene was effectively expressed in E.coli BL21 ( DE3 ) strain and mainly presented in dissoluble protein.Western blot result showed that the expression protein react well with the antibodies from immunized rabbit by Leptospira or recombinant protein.ELISA and MAT results showed that the multiepitope protein could cross-react with different serogroup or serovar of Leptospira.Conclusion In this study,we successfully constructed the recombinant T- and B-cells combine epitope gene of leptospires and expressed it in E.coli.The recombinant protein had a good immune activity,and could cross-reacted with antibodies from different serogroups Leptospira infected patients.

Adult ; Aged ; Arsenic Poisoning ; drug therapy ; etiology ; Drugs, Chinese Herbal ; adverse effects ; Female ; Humans ; Male ; Middle Aged

Objective To investigate the effects of different cell cycles and their regulating genes on apoptosis of mononuclear-macrophages induced by Leptospira interrogans. Methods The diversity and alteration of cell cycles of murine mononuclear-macrophage line(J774A. 1 ) and human monocyte line(THP-1 ) before and after infected with L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai were detected using Cell Cycle Stain Kit plus flow cytometer. The cell cycle synchronized J774A. 1 and THP-1 cells were generated and then identified by using different cell cycle blocking agents and flow cytometer. By using Annexin V/PI Detection Kit combined with flow cytometer, the rates of early-apoptosis and late-apoptosis/necrosis in the synchronized and non-synchronized J774A. 1 and THP-1 cells after infection with L. interrogans strain Lai were determined. Several real-time fluorescence quantitative RT-PCRs were performed to the changes of mRNAs levels of p21, p27, p53, c-myc and cycA genes that associated with cell cycle and apoptosis in J774A. 1 and THP-1 cells before and after infected with L. interrogans strain Lai. Results There were G1, S and G2/M phases in both the non-infected normal J774A. 1 and THP-1 cells. On the contrast,the majority of infected J774A. 1 and THP-1 cells were stagnated at G1 phase, but the amount of S phase THP-1 cells was elevated while that of S phase J774A. 1 cells was not(P ＜0.05). No remarkable early-apoptosis in both the infected G1 phase J774A. 1 and THP-1 cells was found, whereas the rates of early-apoptosis and late-apoptosis/necrosis in the infected M phase J774A. 1 and THP-1 cells were significantly increased (P ＜0.05 ). Additionally, late-apoptosis / necrosis rate in the infected G1 phase THP-1 cells (P ＜ 0.05 )that not found in the infected G1 phase J774A. 1 cells. Compared to the non-infected cells, the p21 mRNA levels in the infected J774A. 1 and THP-1 cells were significantly elevated(P ＜0.05), and the c-myc and p27 mRNA levels in the infected J774A. 1 cells and the cycA mRNA level in the infected THP-1 cells were also higher than those in both the non-infected cells ( P ＜ 0.05 ). Conclusion Different cell cycles and their regulating genes have a role to affect the apoptosis of human and murine mononuclear-macrophages caused by L. interrogans with a diversity of cell line origins.

Objective To determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Methods Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A. 1 and PMA-activated human monocyte line THP-1 and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A. 1 and PMA-activated THP-1 cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated. Results The leptospires in J774A. 1 cells were located in phagocytotic vesicles while the leptospires in THP-1 cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A. 1 and THP-1 cells in dose-dependent manner. The numbers of leptospires in J774A. 1 and THP-1 cells that pre-blocked with 10 μmol/L or above MDC and 1 μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05＝. After J774A. 1 and THP-1 cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased ( P<0.05 ).Conclusion Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in human and mice after infected with L. interrogans.

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.