ABSTRACT
@#To explore the improving effect and mechanism of staphylococcal nuclease(SNase)-mediated degradation of neutrophil extracellular traps(NETs)on 2, 4, 6-trinitro-benzene sulfonic acid(TNBS)-induced colitis in mice. The model of colitis in female BALB/c mice was established by intrarectal injection of 2. 5% TNBS solution, and SNase loaded by Lactococcus lactis(L. lactis)were orally administrated for 6 days. To investigate the effect of SNase-mediated degradation of neutrophil extracellular traps on colitis in mice, the experiment was divided into control group, TNBS model group, NZ900 group and L. lactis pCYT: SNase group. The daily body weight, stool consistency and bleeding of mice were observed. The pathological condition of HE in colon group was detected. The activity of MPO and the mRNA expression level of inflammatory cytokines in each group were measured, and the concentration of inflammatory factors in serum was detected. The expression of NETs level marker citH3 in colon tissue was determined by immunohistochemistry. The results showed that SNase loaded by lactis acid bacteria could alleviate the weight loss, disease activity index score, colonic length and pathological damage induced by TNBS in mice, and reduce the levels of inflammatory cytokines in serum and colonic tissue, inhibit the activity of MPO and the expression of Ly6G and citH3 in colon tissue. The preliminary mechanism showed that SNase could down-regulate the expression of inflammatory cytokines and reduce the content of NETs markers to alleviate colitis in mice.
ABSTRACT
In order to study the relationship between Staphylococcal nuclease(SNase) and diabetes mellitus,genetic engineering bacteria E.coli BL21/pET28a-His-SNase was constructed,the expression of soluble extracellular protein SNase was induced and the a preliminary research was made on it.An expression vector pET28a-His-SNase plasmid containing the His-SNase gene was constructed and transformed into E.coli BL21 (DE3) competent cells.The protein was induced by lactose and purified by ultrasound destruction and Ni-affinity chromatography,respectively.It was then analyzed by SDS-PAGE and Western blot.The enzymatic properties for SNase has been preliminary studied as well.Results indicated that the purity of the correctly expressed fusion protein HisSNase was over 85％.SNase showed good activity within a wide range of pH and good heat resistance.This experiment might be a foundation work for the further study on the relationship between SNase and with diabetes.
ABSTRACT
Objective To analyze the association of staphylococcal nuclease domain-containing protein 1(SND1) and T-cell intracellular antigen 1(TIA-1) on stress granules, and the regulation of SND1 on stress granules under stress stimuli. Methods The immunofluorescence assay and laser scanning confocal microscopy were used to observe the co-localization of SND1 protein and TIA-1 protein under stress stimuli, and the over-expression plasmids of pEGFP vector were transfected into HeLa cells and to verify which domain of SND1 co-localized with TIA-1 under stress stimuli. RNA interference-mediated knockdown of the expression of SND1 protein in HeLa cells was measured by Western Blotting assay. Then whether the knockdown of SND1 affected the recruitment of TIA-1 on stress granules was observed. Heat shocks under different times were used to identify whether there were dynamic changes in transportation of SND1 and TIA-1 on stress granules. Results SND1 co-localized with TIA-1 on stress granules under stress stimuli, and the associated domain of SND1 were SN domain. TIA-1 still can be recruited on stress granules but a large amount of stress granules were reduced even though the expression of SND1 protein was decreased. And the transportation of SND1 on stress granules was laged behind TIA-1 under different-times of heat shocks. Conclusion SND1 protein co-localizes with TIA-1 on stress granules, and which co-regulates the cellular stress response under stress stimuli.
ABSTRACT
Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.
Subject(s)
Bacteriolysis , Bacteriophage lambda , DNA , Chemistry , Escherichia coli , Genetic Vectors , Micrococcal Nuclease , Chemistry , Plasmids , Protein Sorting SignalsABSTRACT
Objective: This work aimed to construct stable MCF-7 cell sublines from which staphylococcal nuclease domain con-taining 1 (SND1) expression was interfered to analyze the effect of SND1 silencing on the proliferation and metastasis of MCF-7 cells. Methods: The lentivirus that could mediate SND1 silencing was prepared. MCF-7 cells were infected with the lentiviruses to construct stable sub-cell lines. Quantitative real-time polymerase chain reaction and Western blot analysis were employed to determine SND1 ex-pression level. MTS, wound healing, and transwell assays were applied to analyze the effect of SND1 silencing on the proliferation, mi-gration, and invasion of MCF-7 cells. Results: A lentivirus expression vector that contains sequences encoding shRNAs targeting SND1 and an shRNA negative control were successfully established. The lentiviruses (LV-SH1, LV-SH2, LV-SH3, and 和 LV-NC) were then collected and packaged. Stabilized MCF-7 sublines were prepared through infection with lentiviruses. The most efficient MCF-7 stable cell subline, MCF-SH3, was selected for SND1 silencing. Compared with the control cell, the proliferation, migration, and inva-sion potential of MCF-SH3 were significantly decreased. Conclusion: SND1 could promote the proliferation, migration, and invasion of breast cancer cells. Thus, silencing SND1 expression will inhibit such proliferation, migration, and invasion. These results indicated that the unusual expression of SND1 is associated with breast cancer and may participate in cancer progression by affecting prolifera-tion, migration, and invasion.