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An important index determination for clinical diagnosis of renal function is to assay the creatinine concentration in serum. In the analytical process applied with coupled-enzyme, the quality control of sarcosine oxidase (SOX) as a key enzyme is the first problem to be solved. In order to establish an efficient and laboratory-scale production of SOX, the recombinant sarcosine oxidase (r-SOX) gene was a high-level expression in E. coli induced with lactose on a large-scale fermentation in 300 L fermenter. The results suggested that the biomass concentration reached OD600 of 22 and the expression of recombinant sarcosine oxidase in E. coli accounted for about 25% of total soluble protein in culture after fermentation. The cell-free extract obtained from high pressure homogenizer was processed by selective thermal denaturation and then purified with Ni-Sepharose FF chromatography. The sarcosine oxidase with 97% purity, 25 U/mg specific activity and 92.4% activity recovery was obtained. The molecular weight with single peptide chain of 53 kD and 55 kD of recombinant sarcosine oxidase was assessed by SDS-PAGE in presence or absence of 2-mercaptoehanol and Sephacryl S-200 chromatography. This sarcosine oxidase was found to be a conjugated protein, yellow enzyme, which combined with FAD as prosthetic group by covalent linkage. The contaminant of catalase was not detected in the sample pool of this enzyme. In addition, a further test to the thermal stability of sarcosine oxidase was done. According to the above results, the development and utilization of this enzyme has been set up on a reliable foundation.
Subject(s)
Escherichia coli , Fermentation , Recombinant Proteins , Sarcosine OxidaseABSTRACT
<p><b>OBJECTIVE</b>To evaluate the therapeutic effect of ulinastatin combined with thymosin α1 in the treatment of severe sepsis in rats.</p><p><b>METHODS</b>Normal Wistar rats were subject to cecal ligation and puncture (CLP) to establish models of severe sepsis. The rats were then randomized into 4 groups for treatment with saline (control), ulinastatin, thymosin α1, or the combination of the latter two injected through the caudal vein or subcutaneously at 6, 24, 48 and 72 h after modeling. The mortality rate was recorded daily and the rats were executed at 24, 48, 72 and 96 h after CLP to harvest the heart, liver, spleen, lung, kidney and small intestines for pathological examination. The spleen of the rats were taken for detection of apoptosis of the spleen cells.</p><p><b>RESULTS</b>The mortality rate of the septic rats in the combined treatment group was decreased significantly (P=0.0325). The control group showed the most severe organ damage, which was moderate in single drug treatment group and the mildest in combined treatment group. Obvious spleen cell apoptosis was found in the control group, and was significantly ameliorated in the combined treatment group[(47.4∓10.9)% vs (39.3∓11.4)%, P=0.0000].</p><p><b>CONCLUSION</b>Combined treatment with ulinastatin and thymosin α1 can significantly improve the prognosis and ameliorate organ damage and spleen cell apoptosis in rats with sever sepsis.</p>
Subject(s)
Animals , Male , Rats , Apoptosis , Drug Therapy, Combination , Glycoproteins , Therapeutic Uses , Rats, Wistar , Sepsis , Drug Therapy , Pathology , Spleen , Cell Biology , Pathology , Thymosin , Therapeutic UsesABSTRACT
Objective To detect the changes of interleukin-18 (IL-18) in plasma of trauma patients and evaluate their value in early warning of organ dysfunction. Methods A prospective study was carried out in 54 trauma patients admitted in from March 2001 to September 2002, which were divided into low injury severity score(ISS) group (Group L-ISS) and high ISS group (Group H-ISS). ELISA was applied to measure the level of IL-18 of blood samples that were collected on arrival, at days 4, 7 and 14 following admission. In the meantime, IL-18 level of plasma samples from patients with systemic inflammatory response syndrome (SIRS), sepsis and multiorgan dysfunction syndrome (MODS) was retrospectively analyzed so as to calculate the critical value of IL-18 in predicting organ dysfunction. Results After trauma, IL-18 concentration of plasma reached peak at days 4 and 7, and decreased gradually at day 14, which was significantly related to SIRS, sepsis and MODS, respectively. The IL-18 level was high relatively in plasma from patients with organ dysfunction. The higher IL-18 level in plasma within seven days after trauma indicated the severer organ dysfunction. Conclusion IL-18 is sensitive in early warning of organ dysfunction after trauma.
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Objective To approach the directive function of time efficiency of first-aid to war wound and trauma. Methods To analyse the relationship between time and war wound or trauma death based on the statistical data of those wounds, then to define the time window of first aid aiming at the peak value of death. Results Bleeding and asphyxia should be virtually controlled in “emergency platinum 10 minutes”, resuscitation from shock should be within 30 minutes, at the same time, a definite operation to save life should be performed in “golden an hour”. Conclusions It is important to raise the time efficiency in each stage by training and improving the military affairs. To spread the first-aid knowledge may decrease cripples and mortality.
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Objective To research the possibility of the rat mesenchymal stem cells (MSCs) to differentiate into hepatocyte-like with hepatocyte growth factor plus E growth factor in vitro. Methods (1) Bone marrow in the femurs of Wistar rat was collected by flushing under sterile condition. MSCs were separated and cultured according to the direct anchoring method, identified by using immunocytochemical methods and flow cytometry. After MSCs were induced by HGF (20mg/ml) and EGF (10mg/ml), mRNA expression level of AFP、Alb、CK-18 in the MSCs were detected by RT-PCR. In addition, ultrastructures in the cells induced were observed using electron microscope. Results Anchored MSCs is single or cell clone is developed. Cell is uniform in the configuration and converged long fusiform after 7-10 days. Cultured cells are certified to be MSCs in the immunocytochemistry and flow cytometry. Afer 21 days, cultured cells show Hepatocyte-like characters in the configuration. RT-PCR:On day 7 mRNA of AFP was detected; mRNA of Alb and CK-18 were detected at the beginning, then strengthen on 21 day. At the same time, induced cells in the electron microscope results coincided with hepatic cells. Conclusions MSCs can be isolated, cultured and expanded easily in vitro. MSCs induced by HGF plus EGF in vitro can differentiate into hepatocyte-like cells. As a result, it can provide a new therapeutic method for liver failure.
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Objective To investigate the location and ultrastructure of hepatic stem cells in adult rat. Methods Proliferation of hepatic oval cell of the rat was accomplished. Identification was made with immunohistochemistry with CK18, 19 and CD34 as markers. Ultrastructure of hepatic stem cells was observed with transmission electron microscope. Results The majority of hepatic oval cells were localized in the Hering channel, but some of them were distributed in the hepatic lobules. Electron microscopy revealed three types of oval cells. TypeⅠ cell was small, 7?m in size, with large nucleus but small amounts of cytoplasm and organelles. The cell was recognized as primitive oval cell. Type Ⅱ cell was larger, measuring 8?m in diameter, containing more cytoplasm and organelles. Type Ⅲ cells were larger than both of above cells, and they contained more organelles. Conclusion Hepatic oval cells in adult rat are localized in Hering channel, and they may be stem cells hepatocytes.