Objective To assess the blood safety under the current sero-screening program and gain experience on how to implement NAT as a routine assay for blood screening in China. Methods Sera from 103,539 non-remunerated donors of Shanghai blood center were tested by Chiron's Procleix(TMA HIV-1/HCV Assay in 2 study stages with 8 and 24 sample-pool respectively. Results 5 NAT positive samples were found to be Anti-HCV EIA-2 test positive at the same time. No window period cases of HIV-1/HCV were found in this donor group. But 275 (0.27% ) anti-HCV and 107 (0. 10%) anti-HIV false positive samples were identified by the second sero-screening. Conclusion The blood quality of Shanghai city was high to be close to the level of developed countries and the window period risk of HIV-1 and HCV was less than 1:100,000. With its high sensitivity and specificity,Procleix( TMA HIV-1/HCV Assay can be used in routine blood screening in China.

The biotechnology of glycolipid fermented b y a bacillus coagulans was studied and the fermentation pro cess in 10L bioreactor was conducted.Suitable medium contained 6% bean oil as ca rbon source,3 5g/L NaNO 3 as nitrogen source,0 75g/L yeast extract and some i no rganic salts.The fermentation temperature of 30℃,initial pH of 8 5,strring rev olution of 150～240r/min and fermentation period of 96h proved to be optimal.The yield of glycolipid in 10L bioreactor was 7 073g/L.

To establish neonatal rat cardiac fibroblast inflammatory secretion model by using LPS 100 microg x L(-1) combined with ATP 5 mmol x L(-1), in order to study the inhibitory effect of quercetin on the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts, further investigate the effect of quercetin on the protein expression of p-NF-kappaB p65 (S276) and p-Akt (S473) by western blot, and discuss the inhibitory effect of quercetin on the inflammatory secretion of cardiac fibroblasts. According to the findings, quercetin with the concentrations between 51.74 micromol x L(-1) and 827.81 micromol x L(-1) had no significant effect on the activity of cardiac fibroblasts. Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of TNF-alpha and IL-1beta induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.01 or P < 0.05). Quercetin with the concentrations of 82.78, 41.39 micromol x L(-1) could notably inhibit the increase of IL-6 induced LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.05), without any notable effect of quercetin with the concentration of 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20. 70 micromol x L(-1) could notably inhibit the NF-kappaB p65 (S276) activation induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 15 min, with the most significant effect in 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of p-Akt(473) expression induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 240 min (P < 0.05). Therefore, this study believes that quercetin could attenuate the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts by inhibiting the activation of NF-kappaB p65 (S276) and Akt (473).
Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; administration & dosage ; Endomyocardial Fibrosis ; drug therapy ; genetics ; immunology ; Female ; Fibroblasts ; drug effects ; immunology ; Heart ; drug effects ; Humans ; Interleukin-6 ; genetics ; immunology ; Male ; Proto-Oncogene Proteins c-akt ; genetics ; immunology ; Quercetin ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; genetics ; immunology

OBJECTIVETo clone and screen genes related to multidrug resistance (MDR) in leukemia.

METHODSSuppression subtractive hybridization (SSH) was performed to profile differentially expressed genes between a MDR leukemia cell line (K562/DOX, as tester) and its parent cell line (K562, as driver). Reverse Northern dot blot was carried out to further screen the subtracted cDNA library. The overexpressed cDNA fragments in K562/DOX cells were sequenced and compared with known genes in Genbank. RT-PCR and Northern blot were employed to confirm the differential expression of some identified genes.

RESULTSEleven genes were identified being overexpressed in K562/DOX, including S3 ribosomal protein (S3rp) gene, NADH dehydrogenase subunit 2 (ND2) gene and My023 gene, which have not been reported to be related to MDR in cancer.

CONCLUSIONSeveral genes, which might be involved in MDR were identified, indicating novel mechanisms of MDR in leukemia.

Blotting, Northern ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Gene Library ; Genes, MDR ; genetics ; Humans ; K562 Cells ; Leukemia ; genetics ; NADH Dehydrogenase ; genetics ; Nucleic Acid Hybridization ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosomal Proteins ; genetics

OBJECTIVETo investigate the expression of ghrelin and its receptor, growth hormone secretagogue receptor (GHS-R), in the hypothalamus and gastrointestinal tract in rats with chronic renal failure (CRF) and explore their relationship with the disorder of gastrointestinal tract motility.

METHODSSD rats were randomly divided into sham-operated group (n=8) and CRF group (n=16), and in the latter group, the rats were subjected to 5/6 nephrectomy to induce CRF. Real-time PCR and immunohistochemical staining were used to detect the distribution of mRNA and protein of ghrelin and GHS-R in the gastric fundus, duodenum, and hypothalamus.

RESULTSThe rats in the CRF group showed a significantly higher expression of ghrelin mRNA and protein in the gastric fundus but a lower expression in the hypothalamus than those in the sham-operated group (P<0.01), but the expression in the duodenum was similar between the two groups (P>0.05). The expression of GHS-R mRNA and protein in the gastric fundus was significantly higher in the CRF group than in the sham-operated group (P<0.01), while in the hypothalamus and duodenum, the expression was significantly lower in the CRF group (P<0.01).

CONCLUSIONThe different distribution patterns of ghrelin and GHS-R in the tissues may be an important pathological basis of gastrointestinal motility disorder in CRF.

Animals ; Gastrointestinal Tract ; metabolism ; Ghrelin ; genetics ; metabolism ; Hypothalamus ; metabolism ; Kidney Failure, Chronic ; metabolism ; Male ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Ghrelin ; genetics ; metabolism

This study was purpose to establish the transgenic mouse models of the truncated platelet integrin β3 by retrovirus-infected hematopoietic stem cells (HSCs) transplantation and to provide the basis for further study of the role of integrin β3 cytoplasmic domain in platelet bi-directional signaling pathways. Wild-type β3, β3-Δ759 (R(760) GT(762) truncated β3) and β3-Δ754 (T(755) NITYRGT(762) truncated β3) cDNAs were subcloned into MSCV MigR1 retroviral vector bearing a GFP gene and packaged into infective retrovirus with BOSC23 cell strain. The bone marrow HSCs of the β3 deficient mice were infected by the retroviruses, and transplanted into lethally-irradiated wild type C57BL/6 mice. GFP positive rate and surface β3 expression of the recipients' platelets at 6 to 8 weeks after transplantation were detected by flow cytometry to evaluate the transgenic efficiency. The results showed that four kinds of transgenic mouse models including vector, wild-type β3, β3-Δ759 and β3-Δ754 were established successfully. GFP positive rates of transgenic mouse platelets ranged from 18% to 66% and the β3 expression of transgenic mouse reached heterozygote (β3(+/-) level of mouse). It is concluded that establishment of transgenic mouse models mediated by retrovirus-infected HSCs transplantation is a feasible, fast, and high throughput transgenic approach and laid a solid foundation for further research on the role of integrin β3 cytoplasmic domain for bi-directional signaling of platelets in vivo, and for the gene therapy of platelet disorders.
Animals ; Blood Platelets ; metabolism ; Genetic Vectors ; Hematopoietic Stem Cell Transplantation ; Integrin beta3 ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Retroviridae ; genetics

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A novel fibrinolytic enzyme was produced from Rhizopus chinensis 120, which was screened from the starter for brewing rice wine in the South of China, by solid fermentation, and purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange and gel filtration chromatographies. The purified enzyme hydrolyzed fibrin, it cleaved the alpha-, beta- and gamma-chains of fibrinogen simultaneously, and it also activated plasminogen to plasmin. The enzyme hydrolyzed N-Succinyl-Ala-Ala- Pro-Phe-pNA, and Km was 0.23 mmol/L and Kcat 16.36 s(-1). The optimal temperature of the enzyme for hydrolying fibrin was 45 degrees C, and the optimal pH range of 6.8 - 8.8. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5 +/- 0.1. The enzyme was a glycoprotein. EDTA, PCMB, PMSF inhibited the activety of the enzyme, and SBTI, Lys, TPCK, Aprotinine had none obvious inhibition, which suggested that the activity centre of the enzyme had hydrosulfuryl, metal and serine. The first 12 amino acids of the N-termimal sequence of the enzyme were NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly, and had none homology with that of other fibrinolytic enzyme from other microbes. The novel fibrinolytic enzyme from Rhizopus chinensis 12# has potential to become a therapeutic agent in thrombosis.
Enzyme Stability ; Fermentation ; Fibrinolysin ; metabolism ; Fibrinolysis ; Fibrinolytic Agents ; chemistry ; Humans ; Plasminogen ; metabolism ; Rhizopus ; enzymology

Objective To investigate the effect of telmisartan on the expression of peroxisome proliferator activated receptor gamma(PPARγ)and its regulation in myocardial remodeling in spontaneously type 2 diabetic male Otsuka Long-Evans Tokushima Fatty(OLETF)rats fed with high-fat diet.Methods Twenty-eight four-week-old male OLETF rats were fed with high-fat diet. From 22-week of age, the pre-diabetic OLETF rats were randomly assigned to three groups:telmisartan-treated group[O-T group,5 mg/(kg·d),n=10],pioglitazone-treated group[O-P group,10 mg/(kg·d),n=8]and untreated group ( O-C group, equal volume of normal saline, n=10), continuously administration for 22-week. Twelve sex and age matched Long-Evans Tokushima Otsuka(LETO)rats were used as control(LETO group).At 22 and 48-week of age, the glucose tolerance of the rats was assessed by the oral glucose tolerance test(OGTT).At 48 weeks of age,five rats were randomly selected from each group,and clamp experiments were carried out.The glucose infusion rate from 60 min to 120 min(GIR60-120)was measured.All rats were sacrificed and the myocardial tissues were dissected.The ratio of heart weight to body weight(HW/BW)was calculated.Blood samples were collected,and serum PPARγ,tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6)and adiponectin were measured using ELISA and radioimmunoassay.The mRNA expressions of IL-6,PPARγ1 and PPARγ2 were measured by real-time PCR.The protein expression levels of PPARγ,adiponectin,IL-6 and NF-κB were determined by Western blot assay.The myocardial pathological changes were observed under light microscope (HE staining, Masson staining and PAS staining), and ultrastructural changes were observed under transmission electron microscope.Results At 22-week of age,neither type 2 diabetes mellitus(T2DM)nor IGT were found in three groups.At 48-week of age,T2DM was found in seven rats of O-C group,and IGT was found in two rats.T2DM was found in one rat of O-C group,and IGT was found in three rats.Neither T2DM nor IGT was found in the other two groups.GIR60-120and HW/BW were all significantly lower in the O-P group and O-T group than those of the O-C group at 48-week of age (P<0.05). Systolic blood pressure(SBP)and diastolic blood pressure(DBP)were significantly lower in O-T group than those in other three groups(P<0.05).Compared with the O-C group,the serum levels of PPARγ and adiponectin were significantly up-regulated,whereas the serum levels of IL-6 and TNF-α were down-regulated by telmisartan administration in O-T group (P<0.05).There were no significant differences in the above indexes between O-P and O-T groups.The results of real-time PCR and Westen blot assay showed that the mRNA expression of PPARγ1 was increased,and IL-6 expression decreased in O-T group compared with those of O-C group. The protein expressions of PPARγ and adiponectin were increased, and protein expressions of NF-κB and IL-6 were significantly decreased in O-P group and O-T group compared with those of O-C group(P<0.05).In the O-C group,the arrangernent of myocardial cells was irregular,myocardial fibers were swollen,a large amount of fibrotic tissue in the myocardial interstitium, and glycogen accumulation under light and electron microscope. Besides, myofibril breakage and perinuclear space expansion, myocardial mitochondria were apparently damaged or even dissolved. Compared with the O-C group, myocardial fibers arranged neatly, no obvious glycogen deposition and the ultrastructural changes of myocardium were obviously reduced in O-T group and O-P group. Conclusion Telmisartan can increase the expression level of PPARγ in the serum and myocardial tissue, reduce myocardial fibrosis and alleviate cardiac remodeling in the high-fat-diet OLETF rats.

Objective To understand the awareness of HIV/AIDS-related knowledge and characteristics of sexual behaviors among men who have sex with men (MSM) in Changsha City. Methods By a snowball sampling method, volunteers were recruited in two social public welfare organizations in Changsha (Qingcai and Zhongda Sunshine) and interviewed by anonymous electronic questionnaires. Data were analyzed using software SPSS 19.0. Results Among 150 MSM, the overall awareness rate of HIV/ AIDS-related knowledge was 86.0% (129/150). For different demographic characteristics, higher age group, higher education level and higher income groups had significantly higher rates of awareness about HIV/AIDS related knowledge, compared with the reference groups, respectively. For the sexual behaviors, 32.7% of the investigated MSM population had their first MSM sexual intercourse at age of <18 years old, the rate of ≥18 years old group was significantly higher than the <18 years old group (2=4.315, P=0.038), 46.7% of the MSM population had more than 1 sexual partner during the past six months, the ratio of MSM used condoms in the sexual intercourse occasionally or never was 29.3% and 6.7%, respectively. Conclusions Young age, relatively low educational level and low income MSM in Changsha had a relative low awareness of HIV/AIDS-related knowledge. MSM in Changsha City had first MSM sexual intercourse at a very younger age. There is an urgent need to take well-targeted measures to improve the HIV/AIDS-related knowledge with special MSM population, and develop effective intervention measures for the high-risk sexual behaviors among MSM.

To construct gene vaccine of PPV and to investigate the effects of interleukin 2 (IL-2) as an adjuvant on immune responses in mouse, the recombinant expression plasmid of pCIneo-IL2-VP2 was constructed and transfected into PK-15 cells by lipofectamine, the expressed product was detected by immunofluore assay. To study the immune effects of DNA vaccine in vitro and in vivo, mice were used as the animal model. The recombinant plasmid pCIneo-IL2-VP2, the control plasmid pCI-neo and the PPV live vaccine were immunized by intramuscular injection. Anti-PPV antibodies were measured by ELISA, lymphocyte proliferation activity was detected using MTT method, and the specific killing activities of CTL were assayed too. The results show that the immunized mice produced PPV antibody after one week, and reached to highest after four weeks. Compared with the control group, the pCIneo-IL2-VP2 immunized group produced significant differences in the antibody titers, the lymphocyte proliferation activity and the specific killing activities of CTL. The pCIneo-IL2-VP2 induced humoral and cellular immunity responses similarly to that the live vaccine induced. These results manifested that the PPV DNA vaccine successfully induced humoral and cellular immunity response in mice with the IL-2 gene as an adjuvant.
Adjuvants, Immunologic ; genetics ; Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; genetics ; immunology ; Capsid Proteins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Immunization ; Interleukin-2 ; genetics ; immunology ; Mice ; Parvovirus, Porcine ; genetics ; immunology ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology