OBJECTIVEExpress and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.

METHODSThe expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.

RESULTSSuitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.

CONCLUSIONSWe have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.

DNA, Single-Stranded ; genetics ; metabolism ; DNA-Binding Proteins ; chemistry ; isolation & purification ; metabolism ; Hepacivirus ; Hepatitis C ; metabolism ; virology ; Humans ; Molecular Weight ; Protein Binding ; RNA, Viral ; genetics ; metabolism

Objective To understand the risk behavioral networks of newly reported HIV infections in Taizhou prefecture, Zhejiang province. Methods Newly reported HIV infections from May 2008 through March 2010 in Taizhou prefecture were invited to participate in a cross-sectional survey which requested numbers and contact information of individuals with whom they had had high risk contacts as well as risk behavioral acts with them. Persons having had risky contacts with HIV-infected cases were thereof approached to participate in this survey and to receive HIV testing.Those who tested positive for HIV were subject to further round of the surveys. Results A total of 267 HIV cases were newly reported during this study period. Among them, 191 participated in the survey and served as 'index cases', including 158 cases identified from routine HIV surveillance programs and 33 cases identified from the present survey. Heterosexual transmission was the primary transmission route (74.9%, or 143/191 ), followed by homosexual transmission ( 19.4%, or 37/191 )and injection drug use (5.8%, or 11/191 ). These 191 HIV cases reported a total of 1152 individuals with whom they had had risky contacts. They were able to provide contact information of 461 risk contacts. Of them, 129 received HIV testing and 61 (47.3%) tested positive for HIV. HIV prevalence was the highest among spouses or long-term sex partners of HIV cases (45.6%, or 47/103) and malesex partners of HIV-infected men having sex with men (MSM) (60.0%, or 12/20). Condom use wasvery low among them, with only 33.9% consistently using condoms for sex. Conclusion Newlyreported HIV infections in Taizhou prefecture reported a large and complicated risk behavioral networks and low condom use, suggesting a potential risk of HIV among these connected people,especially among MSM. Much efforts are needed to intervene these high risk subgroups and high risk behavioral networks.

Adult ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cerebral Ventricle Neoplasms ; metabolism ; pathology ; surgery ; Female ; Follow-Up Studies ; Fourth Ventricle ; Ganglioglioma ; metabolism ; pathology ; surgery ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; Magnetic Resonance Imaging ; Nerve Tissue Proteins ; metabolism ; Oligodendrocyte Transcription Factor 2 ; Rosette Formation ; Synaptophysin ; metabolism ; Young Adult

OBJECTIVETo study viruses infecting Pinellia ternata in China.

METHODSymptom observation, DAS-ELISA and RT-PCR detection were applied.

RESULT AND CONCLUSIONDuring a survey in early spring, SMV and CMV were both commonly distributed as main viruses infecting P. ternata collected from different areas in China. But DsMV was the virus which infected P. ternate in natural condition. The infection ratio of cultivated P. ternate by SMV and CMV were 71.4% and 14.3% respectively for 21 samples collected from Ningbo, Zhejiang province; 100% and 44.4% for 18 samples from Xiaoshan, Zhejiang province; 61.9% and 33.3% for 21 samples from Hebei province; 50.0% and 41.7% for 12 samples from Anhui province; 16.7% and 16.7% for 12 samples from Sichuan province; 31.3% and none for 16 samples from Beijing. And the infection ratio of 25 wild samples from different areas of China infected by SMV and CMV were both 20.0%.

China ; Cluster Analysis ; Cucumovirus ; genetics ; isolation & purification ; DNA, Complementary ; chemistry ; genetics ; Mosaic Viruses ; classification ; genetics ; isolation & purification ; Pinellia ; virology ; Plant Diseases ; virology ; Plants, Medicinal ; virology ; Sequence Analysis, DNA

OBJECTIVETo explore the change in the distensibility of large arteries and its influencing factors in elderly patients with essential hypertension.

METHODSAutomatic measuring system for pulse wave velocity (PWV) was applied to examine carotid-femoral PWV as an index reflecting distensibility of large arteries. 118 hypertensive patients aged 64 - 83 (mean age 67.12 +/- 10.26) years were included in the study. Of them, 87 were males and 31 were females.

RESULTSPWV of 118 hypertensive patients increased with increasing age (P < 0.001). Multivariate regressive analysis demonstrated that age and systolic blood pressure had the close relationship with PWV (P < 0.001).

CONCLUSIONHypertension of the elderly could cause reduction of distensibility of large arteries. Age and systolic blood pressure had the close relationship with distensibility of large arteries in elderly patients with essential hypertension.

Age Factors ; Aged ; Aged, 80 and over ; Blood Flow Velocity ; Carotid Arteries ; physiopathology ; Elasticity ; Female ; Femoral Artery ; physiopathology ; Hemodynamics ; Humans ; Hypertension ; physiopathology ; Male ; Middle Aged ; Pulse

Doxorubicin loaded micelles were prepared by film-hydration method using stearyl sulfadiazine (SA-SD) which is pH sensitive, methoxy (polyethylene glycol)-2000-1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (mPEG-DOPE) and transactivator of transcription (TAT) peptide conjugated PEG-DOPE. Mean diameter of the pH-sensitive micelles was about 20 nm with a (99.1 +/- 2.1) % drug entrapment efficiency at pH 7.4. Flow cytometry studies revealed that the simple TAT micelles was taken up rapidly at the same level at pH 6.8 and pH 7.4. However, the pH-sensitive micelles entered the tumor cell less at pH 7.4 and significantly increase at pH 6.8. After 1 h incubation at pH 6.8, the amount of the pH-sensitive micelles taken up by cancer cell 4T1 was almost similar to simple TAT micelles. The confocal microscopy indicated that the pH-sensitive micelles entered the 4T1 cells at pH 6.8 more than at pH 7.4. It was indicated that the pH-sensitive micelles could shield TAT peptide at normal pH 7.4 and deshield it at pH 6.8. Hence, TAT peptides lead the drug-loaded micelles into the tumor cells and killed them selectively. The pH-sensitive micelle may provide a novel strategy for design of cancer targeting drug delivery system.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; chemistry ; Cell Line, Tumor ; Cell-Penetrating Peptides ; chemistry ; Doxorubicin ; administration & dosage ; chemistry ; Drug Carriers ; Drug Compounding ; Drug Delivery Systems ; Female ; Gene Products, tat ; chemistry ; Hydrogen-Ion Concentration ; Mammary Neoplasms, Experimental ; pathology ; Mice ; Micelles ; Phosphatidylethanolamines ; chemistry ; Polyethylene Glycols ; chemistry ; Sulfadiazine ; chemistry

The aim of this study was to analyze the promoter methylation patterns of inhibitory killer cell immunoglobulin-like receptor (KIR) which gene expression and the effect of demethylation treatment were studied, and to explore the possible regulation mechanism of inhibitory kir gene expression. The promoter methylation levels of kir2DL1 and kir2DL2/kir2DL3 in NK-92MI cell line were detected by bisulfite sequencing technique. Then NK-92MI cells were treated with 5-azacytidine to induce the demethylation of CpG islands. The levels of gene expression of kir were determined by RT-PCR. The results demonstrated that the methylation frequencies of CpG dinucleotides surrounding the promoter regions of kir2DL1 and kir2DL2/kir2DL3 genes were 25% to 88% and 5% to 80% respectively. DNA-demethylating treatment with 5-azacytidine resulted in re-expression of kir2DL1 gene and increased expressions of kir2DL1, kir2DL2 and kir2DL3 genes in NK-92MI cells. In conclusion, the promoter DNA methylation participates in the regulation of kir gene expression in NK-92MI cells.
Cell Line ; DNA Methylation ; Gene Expression ; Humans ; Killer Cells, Natural ; metabolism ; Receptors, KIR2DL1 ; genetics ; metabolism ; Receptors, KIR2DL2 ; genetics ; metabolism ; Receptors, KIR2DL3 ; genetics ; metabolism

CD4+ T cells mainly interact with Sphingosine 1-phosphate (S1P) to regulate immune function through Sphingosine 1-phosphate receptor 1 (S1P1). This study was aimed to investigate the effects of recombinant human granulocyte-colony-stimulating factor (rhG-CSF) mobilization on S1P1 expression in CD4+ T cells of donor's peripheral blood. The CD4+T cells of peripheral blood were isolated by magnetic beads from 17 allo-hematopoietic stem cell transplantation (allo-HSCT) donors before and at fourth day of mobilization with rhG-CSF. The S1P1 expression was detected by real time quantitative PCR in the RNA extracted from CD4+ T cells collected before and after rhG-CSF mobilization. The results showed that the expression of S1P1 was found in CD4+ cells before and after rhG-CSF mobilization, but the expression level of SIP1 in CD4+ cells after rhG-CSF mobilization was significantly lower than that before rhG-CSF mobilization (p<0.01). It is concluded that the mobilization with rhG-CSF obviously down-regulates the expression of S1P1 in CD4+ T cells of donor's peripheral blood.
CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; methods ; Hematopoietic Stem Cell Transplantation ; Humans ; Receptors, Lysosphingolipid ; metabolism ; Recombinant Proteins

The study was aimed to investigate the mobilization effect of medium dose of granulocyte colony stimulating factor (G-CSF) in allogeneic peripheral stem cell transplantation and changes of T lymphocyte subgroup in PBMNC before and after mobilization. G-CSF was administered at 600 microg/d (i.e. 300 microg i.v. twice a day) for successive 5 days to 31 matched sibling or unrelated donors for the mobilization. Stem cells were harvested on the fourth day. FACS was used to analyze the NC, MNC and T lymphocyte subgroups. The results showed that the number of NC, MNC, CD34(+) cells and CFU-GM in dose of 600microg/d significantly increased (P < 0.05), compared with 300 microg/d; the time for hematological reconstruction was significantly shortened (P < 0.05); the ratio of adverse effects was not obviously increased (P > 0.05) and the median percentage of CD3(+) lymphocytes before mobilization was 46.96% [(32.36-57.45)%], but 40.94% [(25.31-48.9)%] after mobilization, while the ratio of CD4(+)/CD8(+) did not significantly changed. It is concluded that the administration of G-CSF 600 microg/d in allo-PBSCT has a good effect in the mobilization of PBSC with minor side effects, which can markedly promote hematopoietic reconstitution after transplantation. The relative amount of CD3(+) lymphocytes significantly decreased and the ratio of CD4(+)/CD8(+) remained unchanged, which may lead to alleviation of a GVHD after PBSCT.
Adolescent ; Adult ; Antigens, CD34 ; analysis ; Blood Donors ; Female ; Flow Cytometry ; Graft vs Host Disease ; prevention & control ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; methods ; Recombinant Proteins ; T-Lymphocyte Subsets ; cytology ; drug effects ; immunology ; Time Factors

To study immunophenotype and cytotoxicity of the immunocytes in bone marrow and peripheral blood after activation by combined cytokines, mononuclear cells (MNC) of bone marrow and peripheral blood were activated by IFN-gamma, IL-1, IL-2 and McAb-CD3 in vitro. The cell amount and morphology during culture were observed. Cytochemical staining and immunophenotype analysis were done before and after culture in two groups of MNC. Cytotoxicity was tested by MTT method. The results showed that the cell number of two groups increased obviously in culture (P < 0.05), while the peripheral blood mononuclear cells increased more markedly (P < 0.05). The cytochemical staining showed POX decrease, but PAS increase in two groups. The positive ratios of CD3(+), CD56(+) and CD38(+) cells in two groups increased obviously after culture (P < 0.05), but there was no significant difference between those two groups. CD3(+) CD56(+) cells increased obviously in peripheral blood mononuclear cells activated by cytokines (P < 0.05), but CD3(+) CD56(+) cells did not increase in bone marrow mononuclear cells. There was no significant difference between two groups' cytotoxicity. It was concluded that IFN-gamma, IL-1, IL-2 and McAb-C D3 increased cell number and cytotoxicity of both bone marrow and peripheral blood mononuclear cells that can be used in cell immunotherapy.
ADP-ribosyl Cyclase ; immunology ; ADP-ribosyl Cyclase 1 ; Antibodies, Monoclonal ; pharmacology ; Antigens, CD ; immunology ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; CD3 Complex ; immunology ; CD56 Antigen ; immunology ; Cell Count ; Cell Division ; drug effects ; Coculture Techniques ; Cytokines ; pharmacology ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; drug effects ; HL-60 Cells ; Humans ; Immunophenotyping ; Interferon-gamma ; pharmacology ; Interleukin-1 ; pharmacology ; Interleukin-2 ; pharmacology ; K562 Cells ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Membrane Glycoproteins ; Time Factors