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Objective To obtain the recombinant protein LytA (rLytA) of Streptococcus pneumoniae strain (ATCC49619) through prokaryotic expression system and to investigate their diagnostic value for patients with community acquired pneumonia (CAP).Methods The specific primers were designed according to LytA gene sequence of Streptococcus pneumoniae M66 strain recorded in Genbank.The recombinant plasmid pET32a(+)/LytA was constructed and transformed into BL21(DE3) to express LytA.The expressed protein LytA was purified by electroeluting of bag filter.Serum IgM of anti-LytA accordingly of patients with CAP were detected by ELISA.The results were evaluated by Chi-square test.Results The recombinant protein LytA was expressed and purified successfully with a relative molecular weight of 56 000.The IgM antibodies level of anti-LytA was significantly higher than the healthy control group (P=0.000).Diagnostic sensibility and specificity of LytA-IgM were 27.8% and 100.0%,while sensibility and specificity of sputum culture were 19.4% and 72.2%,respectively.The sensibility of LytA-IgM was equal to sputum culture(χ2=0.693,P=0.405),but the specificity was higher than it(χ2=14.316 P=0.000).Conclusions A rLytA-ELISA assay maybe has clinical value for diagnosis of pneumococcal infections.It is more rapid and objective than the culture method.
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Background and purpose: The previous work of this study has showed that the treatment of liver cancer cells with emodin could induce endoplasmic reticulum (ER) stress and apoptosis. Given the cross-talk between ER stress and autophagy, this study aimed to investigate whether blockage of autophagy, a defense mechanism against environmental stress, could improve the killing effect of emodin on liver cancer cells. Methods: The CYTO-ID auto-phagy detection kit and Western blot were used to determine autophagy in liver cancer cells. After combined treatment with chloroquine (CQ) and emodin, cancer cell survival was analyzed by ATPlite assay and clonogenic assay. Apoptosis was detected by both flow cytometry analysis and Western blot. Results: Autophagy could be induced in liver cancer cells after treatment with emodin. Inhibition of autophagy significantly increased growth-inhibitory effect of emodin on both HepG2 and Huh7 cancer cells. The combination treatment with CQ and emodin promoted remarkable apoptosis in liver cancer cells, evidenced by the increase in the percentage of cells in sub-G1 phase and the higher expression lever of cleaved caspase-3. Conclusion: Therapeutically targeting autophagy is capable of enhancing cytotoxicity of emodin in liver cancer cell lines.
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BACKGROUND:Mesenchymal stem cels can secrete a variety of cytokines and growth factors that promote the survival of surrounding cels and play a paracrine role. OBJECTIVE:To investigate the effect of human umbilical cord mesenchymal stem cels on the proliferation and apoptosis of ectopic endometrial cels. METHODS:After isolation and culture, human umbilical cord mesenchymal stem cels and ectopic endometrial cels were co-cultured as observation group, and ectopic endometrial cels cultured alone served as control group. At 24, 48, 72 hours of culture, the proliferation and apoptosis of ectopic endometrial cels were detected by MTT and flow cytometry, respectively; RT-PCR was used to measure the expression ofPTEN gene in ectopic endometrial cels. RESULTS AND CONCLUSION:At 24, 48 and 72 hours, the proliferation of ectopic endometrial cels in the observation was inhibited significantly as compared with the control group, and the hypodiploid peak ratio also increased significantly (P < 0.05). Over time, the cel inhibition rate was gradualy declined, and there were significant differences at different time points (alP < 0.05). Compared with the control group, the expression of PTEN gene was up-regulated significantly in the observation group (P < 0.05). In the observation group, the expression ofPTEN gene at 48 and 72 hours was significantly higher than that at 24 hours (P < 0.05). These findings indicate that in the human umbilical cord mesenchymal stem cels can inhibit the proliferation of ectopic endometrial celsin vitro and promote their apoptosis by up-regulation ofPTEN mRNA expression.
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Objective To construct the recombinant protein AtlM ( rAtlM) of Staphylococcal au-reus through prokaryotic expression system and to investigate its antibacterial activity in vitro.Methods The specific primers were designed according to atlM gene sequence of Staphylococcal aureus recorded in Gen-Bank, and atlM gene was amplified by PCR from the Staphylococcal aureus strain (ATCC25923).The re-combinant plasmid pET-32а(+)/atlM was constructed and transformed into Transetta ( DE3 ) to express AtlM after induced by IPTG .The expressed protein AtlM was further analyzed by SDS-PAGE and purified by electroeluting of bag filter.The minimal inhibitory concentrations (MICs) of rAtlM to ATCC25923 and oxac-illin-resistant S.aureus strain were determined by the broth microdilution method .S.aureus ATCC25923 strain and oxacillin-resistant S.aureus strain (final concentration of 5×105 CFU/ml) were exposed to rAtlM (50 μg/ml) respectively to test its antibacterial activity in vitro.Results The recombinant protein AtlM was expressed and purified successfully with a relative molecular weight of 80 ×103 and a concentration of 1.25 mg/L.The MICs of rAtlM to ATCC25923 strain and oxacillin-resistant S.aureus strain were 8 μg/ml and 64 μg/ml, respectively.In vitro test showed that rAtlM had inhibitory effects on the growth of ATCC25923 strain and oxacillin-resistant S.aureus strain after 1 h of intervention (P=0.004 and P=0.026, respectively), which lasted to 5 h for ATCC25923 strain (P=0.012) and 3 h for oxacillin-resistant strain (P=0.001).Conclusion This study shows that rAtlM has a certain antibacterial effects on S.aureus ATCC25923 strain and oxacillin-resistant S.aureus strain, suggesting a possibility of serving as antimicrobial agent.
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Objective To obtain the pneumococcal autolysin(LytA)and choline binding protein A(CbpA)by prokaryotic expression system and investigate their diagnosis for infection caused by Streptococcus pneumoniae.Methods The specific primers were designed according to lytA and cbpA of Streptococcus pneumoniae gene sequence.lytA and cbpA were amplified by PCR form the pneumococcus genome.After IPTG inducing,the recombinant proteins were purified by electroeluting of bag filter,detected by SDS-PAGE and Western blot.Serum lgG and IgM antibodies accordingly of BALB/c mice infected with Streptococcus pneurnoniae were detected by ELISA.Results The recombinant plasmid pET-32a(+)/lytA and pET-32a (+)/cbpA were constructed successfully.Fusion proteins LytA and CbpA were expressed and displayed expected antigenicity.IgM and IgG antibodies level anti LytA were significantly higher than the control group (infections with B Streptococcus group and healthy mice),(P0.05).Diagnostic sensitivity of CbpA was 83.3%(IgG)and 75.0%(IgM).Diagnostic specificity of LytA was 100%(IgG and IgM).Conclusion The synergistic use of specificity of LytA and sensitivity of CbpA may be worthy of serological diagnosis for Streptococcus pneumoniae infection,and may be used for further clinical test.
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Aim To observe the intervention effects of sodium aescinate on acute lung injury model of rats induced by oleate. Methods Fifty four male SD rats were randomly divided into five groups: normal control group, sodium aescinate control group (without oleate) , oleate model control group,medrol interventional group and sodium aescinate interventional group. Acute lung injury models of rats were made by injecting oleate (OA, 0. 1 ml · kg~(-1) ) through caudal veins, and then rats were observed and killed to detect correlated in-dice. The observation indice were the histomorphology of lung, the wet and dry weights of lung ( W/D), score of injury of lung under light microscope (IQA ) , partial pressure of oxygen in artery ( PaO_2) , the levels of SOD and MDA in blood plasma and lung tissue. Results ① Histomorphology of lung: Lung surface hyperemia relieved obviously and pink secretion from trachea of rats in sodium aescinate interventional group and medrol interventioal group decreased significantly compared with oleate model control group. Under light microscope , compared with oleate model control group, effusion of inflammatory cells in alveolar space of rats in sodium aescinate interventional group and medrol interventional group decreased. ② The wet and dry weights of lung ( W/D ) ; W/D of rats in oleate control model group increased obviously compared with those in normal control group, W/D of rats in sodium aescinate interventional group and medrol interventional group decreased obviously compared with those in oleate model control group. ③ Score of injury of lungs under light microscope (IQA) ; IQA of rats in oleate model control group advanced obviously compared with that in normal control group. IQA of rats in sodium aescinate interventional group and medrol interventional group lowered significantly compared with that in oleate model control group.④ Partial pressure of oxygen in artery (PaO_2) : PaO_2 of rats in oleate model control group lowered significantly compared with that in normal control group. PaO_2 of rats in sodium aescinate interventional group and medrol interventional group improved significantly compared with that in oleate model control group. ⑤ The levels of SOD and MDA in blood plasma and lung tissue:The levels of SOD in plasma and lung tissue of rats in oleate model control group lowered significantly compared with those in normal control group. SOD in plasma and lung tissue of rats in sodium aescinate in-terventional group and medrol interventional group increased significantly compared with that in oleate model control group. The levels of MDA in plasma and lung tissue of rats in oleate model control group lowered significantly compared with those in normal control group. MDA in plasma and lung tissue of rats in sodium aescinate interventional group and medrol interventional group increased significantly compared with that in oleate model control group. Conclusion Sodium aescinate can improve W/D, IQA and PaO_2 by adjusting oxidization of the acute lung injury model of rats, which may provide a possible path for treating acute lung injury in clinical practice.
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Objective To explore the features of immune responsed activated by polysaccharides from herbal medicines and provide basis for immunomodulation of polysaccharides. Methods The mice were immuned by polysaccharides of Angelica, Atractylodes and Giant typhonium rhizome respectively, then the corresponeding antibodies and cross-reactive antibodies in serum were detected by ELISA. Results Three kinds of polysaccharides from herbal medicines could all activated the mice to produce not only the corresponding antibodies, but also the cross-reactive antibodies, the ranges of non-specific antibodies activated by distinct polysaccharide were different. Conclusions Polysaccharides from herbal medicines may be a kind of non-specific and broad-spectrum immunomodulators.
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BACKGROUND: It is proposed that elevated serum homocysteine is an important independent risk factor for ischemic stroke (IS), and 5, 10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme for homocysteine metabolism. The relationship between genetic mutation of MTHFR and IS remains controversial.OBJECTIVE: To examine the association of hyperhomocysteinemia and two MTHFR gene polymorphisms with IS in Northwest Chinese population.DESIGN: Case-control study.SETTING: Department of Neurology, First Hospital Affiliated to Jilin University, and Department of Neurology, Xijing Hospital, Fourth Military Medical University of Chinese PLA.PARTICIPANTS: Ninety-seven consecutive patients with ischemic stroke (71 males and 26 females) treated between November 2001 and May 2002were recruited, who were diagnosed by CT scan or MRI in the Department of Neurology, Xijing Hospital, Fourth Military Medical University of Chinese PLA. The control group consisted of 94 subjects (58 males and 36 females) without history of ischemic stroke. All the subjects were free of intracranial hemorrhage, cancer, renal dysfunction, and none used multivitamins or estrogen.METHODS: Serum homocysteine was measured by fluorescence polarization immunoassay. Polymerase chain reaction-restriction length polymorphism (PCR-RFLP) method was employed to detect the genotype at the two sites of C677T and A1298C in MTHFR gene.MAIN OUTCOME MEASURES: Serum homocysteine levels and the genotypic frequency frequencies of the two mutations of MTHFR.RESULTS: The 677T allele frequency was 59.3% in IS patients and 44.7% in the controls, showing significant differences (P=0.006), but no difference in 1298C allele frequency was detected between the two groups (22.7% vs 19.7%, P ＞ 0.05). Homozygous 677TT genotype was closely associated with hyperhomocysteinemie (P ＜ 0.01). In multivariate logistic regression analysis,677T gene mutation and hyperhomocysteinemie were all associated with the IS, with an OR of 1.870 and 1.031 (P＜ 0.05), respectively.CONCLUSION: Hyperhomocysteinemie is a risk factor of IS, and C677T mutation significantly increases homocysteine levels, and serves also as an independent genetic risk factor of IS.
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Progress in Cocaine toxicology research,especially in i ts toxicological mechanisms on the central nervous and cardiac-cerebral vascular systems, was reviewed in the last 5 years.The main problems with the current resear ch work and the future research direction were pointed out.