To evaluate JAK2V617F point mutation in patients with polycythemia vera (PV) and its clinical significance, the point mutation was detected by allele specific polymerase chain reaction (AS-PCR), and the clinical and laboratory features of 50 PV patients with JAK2V617F positive and negative mutations were analyzed and compared each other. The results showed that among 50 patients, 31 patients (62.0%) had JAK2V617F point mutation; 12 patients (24.0%) showed thrombosis and microvascular disturbances; 3 patients had chromosome karyotype abnormalities. As compared with negative mutation group, the age and leukocyte count in patients with JAK2V617F point mutation were older (57.5 +/- 10.0 vs 45.6 +/- 14.9, p < 0.05) and higher (16.2 +/- 6.7 vs 9.0 +/- 5.2, p < 0.05) respectively. It is concluded that the frequency of the JAK2V617F point mutation is 62.0% in PV patients, the age and leukocyte count of patients with JAK2V617F point mutation are older and higher respectively than those in negative mutation group.
Adult ; Aged ; Female ; Humans ; Janus Kinase 2 ; genetics ; Leukocyte Count ; Male ; Middle Aged ; Point Mutation ; Polycythemia Vera ; genetics ; Young Adult

OBJECTIVETo investigate the relationship between the drug resistance of Pseudomonas aeruginosa (PA) isolated from burn patients wounds and its mobile genetic elements, including plasmid, transposon, and integron.

METHODSThirty-two strains of PA were isolated from wounds exudate of hospitalized burn patients in Ningbo No. 2 Hospital. PA drug sensitivity was determined using GNS-448 drug sensitivity card and K-B tests. The genetic markers of plasmid, transposon and integron including traA, traF, tnpA, tnpU, merA, int I 1 were amplified by PCR and verified by gene sequencing.

RESULTSDrug resistant rate of 32 PA strains to gentamicin, amikacin, cefoperazone/sulbactam, ciprofloxacin was 43.7%, 32.0%, 46.8%, 49.9%, respectively. PA drug resistant rates to piperacillin, cefotaxime, ceftazidime, cefepime, aztreonam, piperacillin/tazobactam, levofloxacin, imipenem and meropenem were all above 56.0%. Seventeen out of 32 PA strains were found to carry transposon and (or) integron genetic markers. One strain was positive for both tnpA and merA, 8 strains were positive for both merA and int I 1, 1 strain was only positive for tnpA, 2 strains were only positive for merA, and 5 strains were positive for int I 1 only.

CONCLUSIONSPA isolated from burn wounds of hospitalized patients in Ningbo No. 2 Hospital is seriously drug resistant, which may relate with its high positive rate of mobile genetic elements of transposon and (or) integron.

Anti-Bacterial Agents ; pharmacology ; Burns ; microbiology ; DNA Transposable Elements ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Integrons ; Microbial Sensitivity Tests ; Plasmids ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification

Objective To discuss the effects of different exercise frequency and diet guidance for fat loss on obese patients, and to develop a safe and effective multidisciplinary intervention program for obese patients. Methods A total of 140 members of Ningbo Slimming Club were recruited, with the age between 18-60 years, body mass index (BMI) greater than 28 kg/m2, and they were randomly assigned to low frequency group and high frequency group. In low frequency group, the intervention was complete 100 minutes aerobic exercise and 3 times a week. In high frequency group, the intervention was complete 50 minutes of aerobic exercise and 6 times a week. Each group followed the same diet weight loss program. By comparing each index before and after the intervention, the effect of fat loss was evaluated. Results After intervention, systolic blood pressure, diastolic blood pressure, body weight, BMI, waist circumference, body fat percentage, triglyceride, low density lipoprotein and total cholesterol in two groups were decreased, and high density lipoprotein were increased (P<0.05) . Compared between the low frequency and high frequency exercise group, there were no significant difference for the systolic blood pressure, diastolic blood pressure, body weight, waist circumference, BMI, triglyceride, high density lipoprotein, low density lipoprotein and total cholesterol levels (P>0.05), but body fat percentage in low frequency exercise group decreased significantly (P< 0.05) . Conclusion Diet intervention combined with exercise has a significant effect of fat loss on obese patients. Based on the same weekly aerobic exercise time, obese patients with low frequency of exercise had better fat loss than obese patients with high frequency of exercise.

OBJECTIVE: To establish a method for DNA examination on mouth mucosa exfoliative cells from toothbrush bristles. METHODS: Slough-off mouth mucosa exfoliative cells were collected by bristles extracted method and direct washing method. The DNA of exfoliative cells was extracted with Chelex-100 method and DNA IQTM kit. Then all the extracted DNA took PCR amplification and STR analysis. RESULTS: There was a significant difference between the groups with bristles extracted method and with direct washing method at the success rates of over 9 STR loci detected (P<0.05). However, there was no statistical significance between Chelex-100 method and DNA IQTM kit (P>0.05). CONCLUSION It is demonstrated that mouth mucosa exfoliative cells collected by bristles extracted method is more available than that with direct washing method.
DNA/analysis* ; Epithelial Cells/metabolism* ; Forensic Genetics ; Humans ; Microsatellite Repeats/genetics* ; Mouth Mucosa/cytology* ; Polymerase Chain Reaction ; Toothbrushing

Dimethyl sulfoxide (DMSO), a well-known differentiation inducer in several myeloid cells, induces G1 phase arrest in many cell lines. In this study, we investigated the possibility of using DMSO to arrest H18 hybridoma cells to the G1 phase and monitor whether the arrest improves antibody production. We showed that DMSO in concentration ranging between 0.3% and 0.6% efficiently arrested H18 hybridoma cells in G1 phase. In our experiment, > 80% of cells grown for 36h in presence of the 0.6% DMSO were arrested in G1. Furthermore, expression levels of P27 were up-regulated tow fold during the G1 phase. Higher concentration of DMSO at 0.9% leads to cytotoxicity. Herein we show a simple way, a two-stage process for antibody production, which consists of a proliferation phase leading to the desired cell density, followed by an extended production phase during which the cells remain at G1 phase. Our observation that the addition of DMSO results in increase antibody production is of significance in further use of hybridoma cells in high density large scale cell culture.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cell Proliferation ; drug effects ; Dimethyl Sulfoxide ; pharmacology ; G1 Phase ; drug effects ; Hybridomas ; drug effects ; immunology ; Mice ; Proliferating Cell Nuclear Antigen ; analysis

OBJECTIVETo focus on the study of the effect on proliferation and apoptosis of human aortic smooth muscle cells (ASMC) by adeno-associated virus (AAV) vector carrying antisense thrombin receptor (ATR) and p21 double gene co-expression system.

METHODSCultured human AMSC was infected with recombinant AAV containing ATR, p21 single gene and AP double gene respectively. The integration and expression of genes were confirmed by semi-quantitative RT-PCR. The anti-proliferation effect was determined by MTT assay. Cell cycle and apoptotic cell counts were measured through Flow Cytometry. The rate of apoptotic cells was examined with acridine orange/ethidium bromide(AO/EB) stain.

RESULTSRT-PCR indicated that the exogenous genes had been integrated into ASMC. The rates of cell survival were decreased by 16.67%, 21.60%, and 29.4% and the cell counts of G0/G1 phase were (61.8 +/- 2.9)%, (82.5 +/- 4.0)%, (80.4 +/- 6.1)% in ATR, p21 and AP group respectively after rAAV infected 4 days. The level and area of apoptotic peak were greater in AP double gene than ATR and p21 single gene. Cell stain indicated that apoptotic cells were (7.2 +/- 3.3)%, (10.7 +/- 5.6)%, and (18.3 +/- 2.7)% in each transgene group compared with (1.5 +/- 0.8)% in control group.

CONCLUSIONAP double gene co-expression system has powerful effect for inhibiting proliferation and inducing apoptosis ASMC than ATR and p21 single gene and that is a superior way for gene therapy to restenosis.

Adenoviruses, Human ; genetics ; Antisense Elements (Genetics) ; Aorta ; cytology ; Apoptosis ; Cell Division ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; biosynthesis ; genetics ; Fetus ; Genetic Vectors ; Humans ; Muscle, Smooth, Vascular ; cytology ; Receptors, Thrombin ; biosynthesis ; genetics

AIDS-Related Opportunistic Infections ; diagnosis ; Acquired Immunodeficiency Syndrome ; complications ; Diagnostic Imaging ; methods ; Humans ; Nervous System Diseases ; diagnosis ; etiology

BACKGROUNDExperimental studies and preliminary clinical studies have suggested that growth hormone (GH) treatment may improve cardiovascular parameters in chronic heart failure (CHF). Recombinant human GH (rhGH) has been delivered by a recombinant protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. The present study aimed to examine a new method for delivery of rhGH using genetically modified bioartificial muscles (BAMs), and investigate whether the rhGH delivered by this technique improves left ventricular (LV) function in rats with CHF.

METHODSPrimary skeletal myoblasts were isolated from several Sprague-Dawley (SD) rats, cultured, purified, and retrovirally transduced to synthesize and secrete human rhGH, and tissue-engineered into implantable BAMs. Ligation of the left coronary artery or sham operation was performed. The rats that underwent ligation were randomly assigned to 2 groups: CHF control group (n = 6) and CHF treatment group (n = 6). The CHF control group received non-rhGH-secreting BAM (GFP-BAMs) transplantation, and the CHF treatment group received rhGH-secreting BAM (GH-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: sham control group (n = 6) and sham treatment group (n = 6). The sham control group underwent GFP-BAM transplantation, and the sham treatment group underwent GH-BAM transplantation. GH-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats with ligation of the left coronary artery or sham operation was performed. Eight weeks after the treatment, echocardiography was performed. hGH, insulin-like growth factor-1 (IGF-1) and TNF-alpha levels in rat serum were measured by radioimmunoassay and ELISA, and then the rats were killed and ventricular samples were subjected to immunohistochemistry.

RESULTSPrimary rat myoblasts were retrovirally transduced to secrete rhGH and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 1 to 2 microg of bioactive rhGH per day. When implanted into syngeneic rat, GH-BAMs secreted and delivered rhGH. Eight weeks after therapy, LV ejection fraction (EF) and fractional shortening (FS) were significantly higher in CHF rats treated with GH-BAMs than in those treated with GFP-BAMs ((65.0 +/- 6.5)% vs (48.1 +/- 6.8)%, P < 0.05), ((41.3 +/- 7.4)% vs (26.5 +/- 7.1)%, P < 0.05). LV end-diastolic dimension (LVEDD) was significantly lower in CHF rats treated with GH-BAM than in CHF rats treated with GFP-BAM (P < 0.05). The levels of serum GH and IGF-1 were increased significantly in both CHF and sham rats treated with GH-BAM. The level of serum TNF-alpha decreased more significantly in the CHF treatment group than in the CHF control group.

CONCLUSIONSrhGH significantly improves LV function and prevents cardiac remodeling in rats with CHF. Genetically modified tissue-engineered bioartificial muscle provides a method delivering recombinant protein for the treatment of heart failure.

Animals ; Bioartificial Organs ; Echocardiography ; Heart Failure ; therapy ; Human Growth Hormone ; administration & dosage ; Myoblasts, Skeletal ; metabolism ; Myocardial Infarction ; pathology ; physiopathology ; therapy ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; administration & dosage ; Tissue Engineering ; Tumor Necrosis Factor-alpha ; blood ; Ventricular Function, Left

OBJECTIVETo study the specific binding of the artificial clonal aryl hydrocarbon receptor translocator (ARNT) with the natural aryl hydrocarbon receptor (AhR) and the recolonization by polyclonal antibody. The dose-response relationship with tetrachlo-rodibenzo-dioxin (TCDD) was also studied to develop TCDD detection method and the binding degree related to dose response.

METHODS(1) The target genes including AhR-PAS, AhR-C and ARNT-PAS were amplified by RT-PCR by using the total RNA purified from the liver cells of C57BL/6J mice as templates to construct pGEX-5X1 recombinants. The recombinant plasmids were expressed in E. coli. (2) The rabbits were immuned by the clonal fusion proteins: AhR-PAS, AhR-C to prepare the polyclonal antibody. (3) The natural AhR from the hepatic cytosol of C57BL/6J mice was extracted. The artificial cloning expressed fusion protein:GST-ARNT-PAS and the natural AhR were incubated in different dose of TCDD. The quantity of the heterodimer through affinity adsorption and Western blots were measured.

RESULTS(1) The target proteins including AhR-PAS, AhR-C and ARNT-PAS were successfully cloned and expressed in E. coli. (2) The detection limit of polyclonal antibody AhR-PAS and AhR-C were 5 ng and 1 ng, respectively. (3) The total protein concentration prepared from the liver cells was 60.5 mg/ml. The artificial clonal protein ARNT-PAS could specifically bind to the natural AhR complex with the existence of TCDD. The detection limit of TCDD was 0.25 pmol which was 80 pg approximately.

CONCLUSIONA TCDD detection method based on the aryl hydrocarbon receptor system was established and the detection limit might reach pg grade.

Animals ; Cells, Cultured ; Limit of Detection ; Liver Extracts ; chemistry ; Mice ; Mice, Inbred C57BL ; Polychlorinated Dibenzodioxins ; analysis ; Rabbits ; Receptors, Aryl Hydrocarbon ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Objective To understand the epidemiological features of two rabies cases in Baoshan city year 2006 and 2007 and to analyze its source of infection.Methods Questionnaires were used to do the epidemiologieal survey on each of the rabies cases.Brain timue samples of rabies patients were collet to detect the rabies virus by direct immunofluoreseence assay(DFA)and RT-PCR assay.Homology and phylogenetic tree were analyzed.based on the whole nucleotide and deduced amino acid sequence of P,M and N gene of rabies virus followed by molecular epidemiological analysis.Results In July 2006,one human rabies case was identified in Longyang district,and another one in Tengchong county in Baoshan city in 2007.The degrees of exposure of these two patients was all at degreeⅢ.Two brain tissue samples among the dead patients(No.CYN0601H and CYN0701H)were confirmed positive by both DFA and RTPCR assay.The homology analysis of P,M and N gene sequences among CYN0601H,CYN0701H and other rabias strains isolated from other provinces and other counties.showed that the samples in Baoshan city shared the highest homology with the strains in Thailand.Phylogenetic analysis indicated that the two samples were very dose and all belonged to genetype 1 Lyssavirus,with the closest relationship between samples in Baoshan city and strains in Thailand.Conclusion It Was confirmed on the virus molecular level that the two patients in Baoshan city were both suffered from rabies.The prevalent strains in Baoshan city WaS probably imported from foreign country,suggesting that prevention and control measures on rabies virus in the boarder areas of Yunnan should be strengthened.