Objective To study the prevalence of the mtDNA A1555G gene mutation in Chinese population with nonsyndromic hearing impairment.Methods PCR-RFLP,directional sequencing of PCR products were applied in 325 patients with nonsyndromic hearing impairment and 50 normal controls.Results The mutation rate of the mtDNA A1555G was 14.5% (47/325),28 of 47 cases were homozygosis,19 of 47 cases were heterozygosis.The same mutation was not detected in the control subjects.Conclusion The mutation rate of the mtDNA A1555G is relatively high in the Chinese NSHI patients,the mutation type includes both heterozygosis and homozygosis.

Objective To determine the influence of protein fusion on the biological characteris- tics of hymidine kinase(TK)and human immunodeficiency virus(HIV)Tat recombinant protein. Methods By utilizing polymerase chain reaction(PCR)technique,different fragments containing two,four or six glycines(Gly)were inserted between the HIV Tat gene and TK,and cloned into PBK vector.After testified by sequencing,the vectors were transfected into E coli.After induced by iso- propyl thiogalactose(IPTG),bacilli were collected and destructed by ultrasonic,the fusion proteins were determined by monoclonal antibody against HIV protein.HepG2 cells were incubated in DMEM supplement with 10?g/mL HIV-Gly(n)-TK(n=0,2,4,6)fusion protein,TK-HIV Tat and only HIV Tat.HepG2 cells in different groups were detected by immunofluorescence assay 24 hours after transduction with HIV Tat monoclonal antibody.The rate of apoptosis after cells were incubated with gencilovir(10?g/mL)for 3 days was determined by cell flow cytometry,while survival cell ratio was recorded by trypan blue.The data were analyzed by statistics(t-test).Results The Tat-Gly(n)-TK (n= 0,2,4,6)recombinant genes were constructed and inserted into PBK vectors,which were expressed in E coli and then purified.Cells in different groups,which were incubated with Tat-Gly (n)-TK(n=0,2,4,6)fusion proteins,Tat-TK fusion protein,TK-Tat fusion proteins or only Tat proteins respectively,were detected by immunofluorescence assay.The intensities of fluorescence in different groups were almost same,but the ratios of cell survival or apoptosis were different.The highest ratio of cells apoptosis(14.77%)was in the group that cellular culture medium was mixed with Tat-Gly(4)-TK fusion protein,followed by the groups containing 6,2 glycines or no TK gene in genes(4.30%,12.69% and 1.03%,respectively).There were significant differences between each 2 groups among the all groups(t-test,P

The effects of the variables, including the concentrations of glucose, sucrose, corn steep liquor (CSL) and peptone, and the conditions of fed-batch culture, on CoQ10 fermentation by Rhizobium radiobacter WSH2601 were assessed. The results showed that the optimum concentrations of glucose, sucrose, CSL and peptone were 30 g/L, 40 g/L, 11 g/L and 16 g/L respectively. Addition of CSL and tomato juice stimulated the cell growth. CSL, L-methionine and isopentyl alcohol efficiently increased the biosynthesis of CoQ10. In a 7L fermentor, the fed-batch culture improved both cell growth and CoQ10 production compared to a batch culture; and suitable mixed feeding of CSL and sucrose enhanced CoQ10 yield to 52.4 mg/L. The DCW reached 26.4 g/L, an increase of 53% in comparison to that without feeding, and an increase of 24% to that feeding with sucrose only. The C/B value reached 2.38 mg/g(DCW), representing an increase of 33% to that of without feeding, and an increase of 26% to that of feeding with sucrose only.
Agrobacterium tumefaciens ; growth & development ; metabolism ; Culture Media ; metabolism ; Fermentation ; physiology ; Glucose ; metabolism ; Industrial Microbiology ; methods ; Sucrose ; metabolism ; Ubiquinone ; analogs & derivatives ; biosynthesis

Cell Line ; Cytochrome P-450 CYP1A1 ; drug effects ; metabolism ; Hepatocytes ; cytology ; metabolism ; Humans ; Microsomes, Liver ; drug effects ; metabolism ; Quercetin ; pharmacology

[Objective] To investigate the environmental pollution status and infectious diseases in kindergartens and nurseries in 2012 in Baoshan District . [ Methods] The questionaire was used in com-bination with data from monitoring of the disinfection effect of environment , monitoring analysis of routine infectious disease and others . [ Results] The overall pass rate was 97.91% for environmental disinfec-tion in kindergartens and nurseries , and it was higher in public kindergartens and nurseries than in private ones.The conventional infectious disease in kindergartens and nurseries was mainly hane-foot-and-mouth disease (HFMD), whose incidence was higher in private kindergartens and nurseries than in public ones . And in terms of regional distribution , the incidence of HFMD was higher in streets and towns with more in-floating population than those with less .And the pass rate for the disinfection effect on the kindergartens nurseries and the incidence rate of HFMD were negatively correlated . [ Conclusion] Kindergartens and nurseries in our district should strengthen the complete configuration of hardware and software , establish standardized system of disinfection and isolation , and develop daily disinfection system , so as to reduce in-fectious disease .

OBJECTIVETo explore the clinical application of the tubularized incised plate (TIP) in the surgical treatment of hypospadias.

METHODSThis study included 169 cases of hypospadias treated by TIP surgery from January 2007 to April 2009. The patients ranged in age from 1.5 to 12 years (mean 3.68 yr). The TIP technique was modified based on that described by Snodgrass, with the urethral plate longitudinally incised and a urethral stent kept in place. The patients were hospitalized for 10 days postoperatively, and followed up for an average of 2 years, ranging from 6 months to 3 years.

RESULTSComplications developed in 18 (10.6%) of the patients, most frequently meatal stenosis (9 cases, 5.3%) and urethrocutaneous fistula (8 cases, 4.7%).

CONCLUSIONThe TIP technique, as a surgical method, can be applied to most hypospadias cases. The accumulation of clinical experience and skills may help raise the success rate and reduce the complications of TIP surgery.

Child ; Child, Preschool ; Humans ; Hypospadias ; surgery ; Infant ; Male ; Treatment Outcome ; Urethra ; surgery ; Urologic Surgical Procedures ; methods

OBJECTIVETo study mitochondrial DNA (mtDNA) A1555G mutation in seven families with nonsyndromic hearing loss (NSHL).

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real time-amplification refractory mutation system-quantitative PCR (ARMS-qPCR) were applied to detect mtDNA A1555G mutation in seven NSHL families. Related clinical data were also collected and analyzed.

RESULTSThe mtDNA A1555G mutation was detected in members from the maternal side, including heteroplasmy and homozygosis, others were negative for this mutation. The copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G correlated well with the degree of deafness (R = 0.341, P = 0.022 and R = 0.85, P = 0.015, respectively).

CONCLUSIONThe mutation rate of the mtDNA A1555G is high in the NSHL patients, the mutation type include heteroplasmy and homozygosis. There is significant correlation between the mtDNA A1555G copy number and the severity of hearing loss.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child, Preschool ; DNA, Mitochondrial ; genetics ; Female ; Gene Dosage ; Hearing Loss ; genetics ; pathology ; Humans ; Infant ; Male ; Middle Aged ; Pedigree ; Point Mutation ; Young Adult

BACKGROUNDIt has been suggested that the ratio of mutant and wild type mitochondrial DNA may be related to its clinical phenotype. In this study, we developed a high sensitive real-time amplification refractory mutation system-quantitative polymerase chain reaction (RT-ARMS-qPCR) assay for quantitation of the mitochondrial DNA (mtDNA) with a mutated 1555 site, and explored the relationship between the ratio of mutated mtDNA and the severity of hearing loss of mitochondrial deafness (MD) patients of Fujian province in China.

METHODSAn amplified mtDNA fragment containing the 1555 site was ligated into a vector to construct a plasmid DNA standard. An RT-ARMS-qPCR system was used to measure the mtDNA copy number containing wild-type and mutant sequences in a cohort of 126 MD patients of Fujian province in China. Combined with the clinical data, we explored the relationship between the ratio of mutated mtDNA and the severity of hearing loss of MD.

RESULTSThe variation coefficient in the cohort was 1.21%, the interassay variation coefficient was 1.78%, and the linear range was 10(2) - 10(8) copies/µl for detecting a recombinant, wild-type plasmid. The primers amplified only the intended sequences. Mutation copy number correlated with the degree of deafness in sporadic cases with heteroplasmic mutations of mtDNA A1555G (R = 0.811, P = 0.003), but not in sporadic cases with homoplasmic mutations (R = 0.007, P = 0.989). The copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G in familial cases correlated with degree of deafness (R = 0.352, P = 0.023 and R = 0.90, P = 0.012, respectively).

CONCLUSIONSThe RT-ARMS-qPCR system is suitable for determining the copy number of mtDNA fragments containing the A1555G mutation. The ratio of mutated mtDNA correlates with the severity of hearing loss of MD.

Adolescent ; Adult ; Child ; Child, Preschool ; China ; DNA, Mitochondrial ; genetics ; Deafness ; genetics ; Female ; Hearing Loss ; genetics ; Humans ; Male ; Mutation ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

The aim of this study was to investigate the mechanism to reverse the drug resistance of leukemia cells in tetrandrine (Tet) alone or in combination with droloxifen (Drol) by using protein chips and to lay the theoretical basis for the clinical applications. Three monoclonal antibodies against P-glycoprotein (P-gp), the multidrug resistance-associated protein (MRP1) and the breast cancer resistance protein (BCRP) were immobilized onto the agarose gel film-coated glass slides. Protein chips were prepared respectively from K562/A02 cells cultured for 12, 24 and 48 hours with Tet alone or in combination with Drol. The results showed that Tet alone or in combination with Drol could decrease only the expression of P-gp in a time-dependent manner, the effect for 48 hours as follows: Tet + Drol 82.620 +/- 3.227; Tet alone 86.440 +/- 2.906; Drol alone 87.230 +/- 2.049; control 93.670 +/- 2.748 (P < 0.05). However, down-regulation of P-gp by K562/A02 cells cultured with Tet alone or in combination with Drol began at 24 hours (Tet + Drol 85.270 +/- 3.095; control 93.670 +/- 2.748, P < 0.05). The results were coincident with that of FCM. It is concluded that Tet and Drol can downregulate the expression of P-gp in the time-dependent way. There is a significant difference between Tet alone and Tet combined with Drol at 24 hours (P < 0.05). The expression of MRP1 and BCRP are not closely correlated with the reversal mechanism of Tet and Drol, and which may be involved in the mechanism of this combination to reverse multidrug resistance in leukemia.
ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; biosynthesis ; ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; Antineoplastic Agents ; pharmacology ; Benzylisoquinolines ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; drug effects ; Drug Synergism ; Humans ; K562 Cells ; Leukemia ; metabolism ; pathology ; Multidrug Resistance-Associated Proteins ; biosynthesis ; Neoplasm Proteins ; biosynthesis ; Protein Array Analysis ; Tamoxifen ; analogs & derivatives ; pharmacology

BACKGROUNDHuman epididymis secretory protein 4 (HE4) has been proved to be a promising novel biomarker for the detection of epithelial ovarian carcinomas. Compared with CA125, HE4 assay demonstrated an improved ability to discriminate between pelvic mass with malignant and benign disease. Though it is well known that HE4 is overexpressed in ovarian cancer, however, the role of HE4 in the carcinogenesis and progression of ovarian cancer remains unkown.

METHODSIn this study, we explored the role of HE4 in the carcinogenesis and progression of ovarian cancer. We screened nine ovarian cancer cell lines for HE4 expression, and using RNA interference (RNAi), we silenced HE4 gene expression in CaoV3 and SKOV3.ip1 ovarian cancer cell lines. We assessed the effect of HE4 gene silencing on the transformed phenotype by examining the cell cycle, apoptosis, proliferation and transwell migration/invasion in vitro.

RESULTSHE4 gene silencing induces G0/G1 arrest and blocks the progression from the G1 to S phase in CaoV3 and SKOV3.ip1 cells. HE4 knockdown also inhibited cell proliferation, migration and invasion in SKOV3.ip1 cells in vitro.

CONCLUSIONHE4 may be involved in the regulation of the cell cycle and promote ovarian cancer migration and invasion.

Biomarkers, Tumor ; analysis ; Cell Line, Tumor ; Disease Progression ; Epididymal Secretory Proteins ; analysis ; genetics ; physiology ; Female ; Gene Silencing ; physiology ; Humans ; Ovarian Neoplasms ; pathology ; RNA Interference