Objective: To construct, express and purify ScFvl4/EGFP fusion proteins which containing Arg9, and to study their binding activities and internalization functions. Methods: Arg9 gene was recombined into 5' terminal, 3' terminal of ScFv/EGFP gene and between them respectively before they were cloned into the expression vector pET32a. After induced in E. coli BL21 and purified, their binding activities and internalization were respectively analyzed by indirect ELISA and indirect immunofluorescence analysis. Results: DNA sequencing and restriction endonuclease digestion proved that the four fusion genes were correctly constructed. SDS-PAGE analysis and Western blot showed that they were successfully expressed and purified. Indirect ELISA confirmed that the expressed products had antigen specific binding activities. Indirect immunofluorescence analysis revealed the fusion protein containing Arg9 at its N terminal had much better internalization function, but never internalized into the cells which do not express HBsAg. Conclusion: The four fusion genes were constructed, expressed and purified successfully. The purified fusion proteins maintained the binding activities to HBsAg and the fusion protein containing Arg9 at its N terminal had much better internalization effect.

OBJECTIVEIt has been demonstrated that when a high-speed handpiece stops rotating, negative pressure will form. Thus, contaminating fluid in which there are many kinds of bacteria and viruses from the external environment will retract into various compartments of the handpiece and the dental unit. The purpose of the study is to compare the preventing effect of antisuction designed handpiece and conventional handpiece on viral contamination at different rotating times.

METHODSTwenty handpieces with or without antisuction device (10 of each) were used in the study. Each handpiece was submerged into 10(-6) microg/microl HBV particle solution rotating 5 and 10 times respectively (every time rotating for 10 seconds). Samples were obtained from the water line and chip air line of the handpieces and examined by RT-PCR.

RESULTSAt the same rotating times, there was statistical significance of the viral concentration between the two kinds of handpieces (P < 0.05) . However, there was no statistical significance of the viral concentration between different rotating times in each group (P > 0.05).

CONCLUSIONContamination taking place in both water and air lines of dental handpiece was not enhanced by increasing the number of rotating times of the handpiece. The antisuction devices installed into the water line and chip air line were demonstrated to prevent viral contamination effectively.

Dental High-Speed Equipment ; virology ; Equipment Contamination ; prevention & control ; Suction

This work was mainly studied the effects of the four alkaloids from Coptidis Rhizoma on the mouse peritoneal macrophages in vitro and preliminarily discussed the regulating mechanisms. The effect of alkaloids from Coptidis Rhizoma on the vitality of macrophages was measured by the MTT assay. The effect of alkaloids on the phagocytosis of macrophages was determined by neutral red trial and respiratory burst activity was tested by NBT. The expressions of respiratory-burst-associated genes influenced by alkaloids were detected by qRT-PCR. The conformation change of membrane protein in macrophages by the impact of alkaloids was studied by fluorospectro-photometer. Results showed that the four alkaloids from Coptidis Rhizoma could increase the phagocytosis of macrophages in different level and berberine had the best effect. Berberine, coptisine and palmatine had up-regulation effects on respiratory burst activity of mouse peritoneal macrophages stimulated by PMA and regulatory activity on the mRNA expression of PKC, p40phox or p47phox, whereas the epiberberine had no significant influence on respiratory burst. Moreover, alkaloids from Coptidis Rhizoma could change the conformation of membrane protein and the berberine showed the strongest activity. The results suggested that the four alkaloids from Coptidis Rhizoma might activate macrophages through changing the conformation of membrane protein of macrophages and then enhanced the phagocytosis and respiratory burst activity of macrophages. Furthermore, the regulatory mechanism of alkaloids on the respiratory burst activity of macrophages may be also related to the expression level of PKC, p40phox and p47phox.
Alkaloids ; pharmacology ; Animals ; Cells, Cultured ; Coptis ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression ; drug effects ; Macrophages, Peritoneal ; drug effects ; Mice ; Phosphoproteins ; genetics ; metabolism ; Protein Kinase C ; genetics ; metabolism ; Rhizome ; chemistry

OBJECTIVETo investigate the impact of intravenous nutrition on plasma free amino acid spectrum and immune function for patients with sepsis.

METHODSForty severe sepsis patients were divided into two groups: Group B (amino acids + glucose + fat emulsion) and Group A (glucose + fat emulsion), 20 healthy individuals were enrolled as control group. The concentration of free amino acid and immune globulin were determined after 3 days.

RESULTSIn Group A, the levels of valine, leucine, isoleucine, alanine, serine, glutamic acid, histidine, proline and glycine were decreased; while the levels of threonine, cysteine, the ratio of phenylalanine and tyrosine (Phe/Tyr) were higher than those in control group. Meanwhile, peripheral blood IgM, complement C3 and C4 were decreased. In group B, all amino acid levels were improved, but the level of alanine, serine, glutamic acid, histidine and proline still lower than those in control group. Similarly, the levels of IgM, complement C3 and C4 in group B were increased.

CONCLUSIONIntravenous nutrition can support the basal requirement of amino acid and improve the immune function of patients with sepsis.

Adult ; Aged ; Amino Acids ; blood ; Complement C3 ; metabolism ; Complement C4 ; metabolism ; Female ; Humans ; Immunoglobulin M ; blood ; Male ; Middle Aged ; Parenteral Nutrition, Total ; Sepsis ; blood ; immunology ; therapy ; Time Factors

Objective To study the clinical characteristics of bulbar myasthenia gravis (MG). Methods Retrospective review was performed on 166 patients with bulbar type of myasthenia gravis, diagnosed at Tongji Hospital in the period of May 1983 through October 2005.Results Bulbar MG was a relatively rare type of MG,accounting for 5.7% (166/2888) of MG classifications.Females were more often affected than males (the ratio of male:female was 1:1.35).The peak of onset age was at 20—40 years.The incidence of myasthenia crisis in the group was 26.5% (44/166).Myasthenic crisis occurred in 10.8% (18/166) of the bulbar MG patients within 6 months after onset,resulting in a mortality rate of 6.0% (10/166) in the group.Out of the group,30 cases experienced puhnonary infections (18.1%). Thirty cases were initially misdiagnosed as other diseases such as nasopharyngeal disorders (33/166, 19.9%).The routine therapy was not very satisfactory.Median dose cyclophosphamide therapy appeared to be effective for ameliorating refractory MG.Thymectomy was performed in 25 patients,with optimistic efficacy rate up to 80.0% (20/25) in a 3-year follow-up.Conclusions The clinical analysis in the current study suggested that the bulbar MG had its own characteristics in such aspects as progression of the disease, complications,treatment and prognosis.The information of the clinical manifestations presented in this study may be useful in diagnosing and treating bulbar MG.

OBJECTIVETo explore the effect of low-flux polysulphone membranes on the serum levels of inflammatory cytokines, hyper-sensitivity C reactive protein (hsCRP), lipoprotein (a) [Lp (a)], and beta2 microglobulin (beta 2MG) in hemodialysis patients.

METHODSThe blood samples and dialysate samples were collected at pre-dialysis, 120 minutes later during the session, and post-dialysis in 27 stable hemodialysis patients. Variables determined included serum Lp (a), hsCRP, interleukin (IL)-1 beta, IL-6, and beta 2MG. The endotoxin levels were also detected.

RESULTSThere were no significant changes either in endotoxin level of dialysate or in all variables tested during one session (P > 0.05).

CONCLUSIONLow-flux polysulphone membrane has no effects on serum Lp (a), hsCRP, IL-1 beta, IL-6, and beta 2MG during one session in hemodialysis patients.

Adult ; Aged ; C-Reactive Protein ; metabolism ; Female ; Humans ; Interleukin-1beta ; blood ; Interleukin-6 ; blood ; Lipoprotein(a) ; blood ; Male ; Membranes, Artificial ; Middle Aged ; Polymers ; Renal Dialysis ; Sulfones ; beta 2-Microglobulin ; blood

OBJECTIVETo investigate the effects of Tpo and/or IL-11 gene modified stromal cells on the expansion of CD(34)(+) hematopoietic stem/progenitor cells in cord blood.

METHODSRetroviral vectors containing Tpo or IL-11 gene were constructed and used to transfect the stromal cell line HFCL. Tpo and/or IL-11 mRNA was assayed by Northern blot. Non-modified stromal cells were used, CD(34)(+) hematopoietic stem/progenitor cells from cord blood were expanded on gene-modified stromal cells for 7 days. The phenotype of CD(34)(+)CD(38)(-) primitive progenitors was detected by flow cytometry.

RESULTSHFCL expressed Tpo and/or IL-11 mRNA after transfected by the retroviral vectors. The percentages of CD(34)(+)CD(38)(-) primitive progenitors in the cultures of Tpo, IL-11 and Tpo + IL-11 modified HFCL were (1.8 +/- 0.24)%, (1.62 +/- 0.23)%, and (2.45 +/- 0.28)%, respectively, which were higher than that in the control [(0.8 +/- 0.23)%].

CONCLUSIONThe stromal cells modified by Tpo and/or IL-11 gene were able to enhance ex vivo expansion of CD(34)(+) and CD(34)(+)CD(38)(-) hematopoietic stem/progenitor cells from cord blood.

ADP-ribosyl Cyclase ; analysis ; ADP-ribosyl Cyclase 1 ; Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; physiology ; Humans ; Infant, Newborn ; Interleukin-11 ; genetics ; Membrane Glycoproteins ; Stromal Cells ; physiology ; Thrombopoietin ; genetics

Stem cells in the individual life are the cell population with high self-renewal capacity and multiple differentiation potential. At present, embryonic stem cells and tissue stem cells are the major objects for study in the field of stem cell engineering. At the same time, with the development of tissue engineering, cell replacement therapy became a new approach to treat some diseases. Tissue stem cells were tried to expand and committedly induce in vitro to some cells that are needed, then implanted them into patients to repair damage, replace regressive tissue and improve the function of hereditarily defect tissue. Based on recent progress of research on stem cells, this paper reviewed the biological characters and clinic application prospects of tissue stem cells.
Animals ; Cell Differentiation ; Humans ; Stem Cells ; cytology ; Tissue Engineering ; methods ; trends

OBJECTIVETo investigate the gene mutation in D-loop region of mitochondrial DNA (mtDNA) in oral squamous cell carcinoma (OSCC) tissue and to explore the role of the gene mutation in D-loop region in the OSCC tumorigenesis.

METHODSmtDNA was obtained from cancer, paracancerous and normal mucosa tissues of thirty patients with OSCC. The D-loop regions of mtDNA were amplified with PCR, sequencing and then analyzed by Chromas software and BLAST to identify the mutation site.

RESULTSMutation in the D-loop region was found in eight cases, with the mutation rate of 27%. There were nine mutations totally, including one point mutation, two base deletions, three insertion mutations, three heterozygous mutations. In these mutations, base deletions were different from each other and heterozygous mutations had no same mutation form, while the three insertion mutations were same, the insertion of base C. One case had T/A heterozygous mutation and base C insertion at the same time.

CONCLUSIONSThere were mutations in mtDNA D-loop in OSCC, but the relationship between occurrence of OSCC and mutation of mtDNA needs further study.

Adult ; Aged ; Carcinoma, Squamous Cell ; genetics ; DNA, Mitochondrial ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mouth Neoplasms ; genetics ; Mutation

Objective To investigate the effect of leptin on the tolerance of cultured rat brain astrocytes to ischemia and hypoxia.Methods The brain astrocytes isolated from neonatal SD rats,after purification and identification,were incubated in serum-and glucose-flee medium in the presence of 5%CO2+95%N2 for 90 min to induce isehemic and hypoxic injury. RT-PCR was performed to detect the expressions of the leptin receptors Ob-Ra and Ob-Rb in the cells, and colorimetry was used to measure the content of malonaldehyde(MDA) and lactate dehydrogenase(LDH) activity in the cell supematant.The expression level ofglial fibrillary acidicprotein(GFAP)in the cells was detected with fluorescence immunocytochemistry.Results Ischemic and hypoxic exposure of the cells induced obvious cell necrosis.Compared with the cells without the exposure,significantly decreased Ob-Rb expression(0.52±0.01 vs 1.32±0.01,P<0.05)and increased MDA,LDH and GFAP levels(709.68±47.16 vs 516.13±29.08,3.94±0.36 vs 1.81±0.21,and 0.122±0.016 vs 0.057±0.006,respectively,P<0.05) occurred after the exposure,whereas the expression level of Ob-Ra underwent no significant changes(3.87±0.13 vs 3.96±0.24,P>0.05). Compared with the exposed cells,the leptin-treated cells showed a significant reduction in MDA levels(3.94±0.36 vs 3.19±0.25,P<0.05) with significantly increased GFAP expression(0.057±0.006 vs 0.109±0.008, P<0.05)after the exposure, and the cells maintained basically intact cell morphology.Conclusion With neuroprotective effects against ischemic neuronal injuries,leptin canimprove the tolerance of rat brain astrocytes to ischemia and hypoxia.