AIMTo examine the effect of inducible nitric oxide synthase (iNOS) on tumour cells chemosensitivity to mitomycin C (MMC) analogue 5-aziridinyl-3-hydroxyl-1-methylindole-4,7-dione (629) in vitro, and elucidate the possible role of iNOS in the metabolism of 629.

METHODSHuman sarcoma cells (HT1080) and its iNOS gene transfected clones (iNOS9, iNOS12) were exposed to 629 at concentrations of 1 nmol x L(-1) - 100 micromol x L(-1). 3-[4, 5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) assay, agarose electrophoresis and flow cytometric analysis were used to determine cell sensitivity, deoxyribonucleic acid (DNA) damage and the change of cell cycle in above process, respectively. All experiments were performed both in air and under hypoxia parallelly.

RESULTS629 was more toxic than MMC, and enhanced cytotoxicity under hypoxia, which resulted in cell necrosis. Sixteen hours after treated with 629, HT1080 cells and related iNOS-transfected clone cells were obviously blocked in G2/M phase.

CONCLUSIONiNOS plays dual roles in 629 metabolism, enhancing or decreasing the cytoxicity of 629 depending on the intracellular oxygen pressure P(O2), which caused higher cytotoxicity to hypoxia cells of 629 with the increasing of iNOS activity.

Antibiotics, Antineoplastic ; pharmacology ; Aziridines ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; DNA Damage ; Fibrosarcoma ; metabolism ; pathology ; Flow Cytometry ; Humans ; Indoles ; pharmacology ; Mitomycin ; chemistry ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Transfection

To provide the profiles of metabolism of mitomycin C (MMC) by human liver microsomes in vitro, MMC was incubated with human liver microsomes, then the supernatant component was isolated and detected by HPLC. Types of metabolic enzymes were estimated by the effect of NADPH or dicumarol (DIC) on metabolism of MMC. Standard, reaction, background control (microsomes was inactivated), negative control (no NADPH), and inhibitor group (adding DIC) were assigned, the results were analyzed by Graphpad Prism 4. 0 software. Reaction group compared with background control and negative control groups, 3 NADPH-dependent absorption peaks were additionally isolated by HPLC after MMC were incubated with human liver microsomes. Their retention times were 10. 0, 14. 0, 14. 8 min ( named as Ml, M2, M3) , respectively. Their formation was kept as Sigmoidal dose-response and their Km were 0. 52 (95% CI, 0. 40 - 0.67) mmol x L(-1), 0. 81 (95% CI, 0. 59 - 1. 10) mmol x L(-1), 0. 54 (95% CI, 0. 41 -0. 71) mmol x L(-1) , respectively. The data indicated that the three absorption peaks isolated by HPLC were metabolites of MMC. DIC can inhibit formation of M2, it' s dose-effect fitted to Sigmoidal curve and it' s IC50 was 59. 68 (95% CI, 40. 66 - 87. 61) micromol x L(-1) , which indicated DT-diaphorase could take part in the formation of M2. MMC can be metabolized by human liver microsomes in vitro, and at least three metabolites of MMC could be isolated by HPLC in the experiment, further study showed DT-diaphorase participated in the formation of M2.
Antibiotics, Antineoplastic ; metabolism ; Chromatography, High Pressure Liquid ; methods ; Dicumarol ; pharmacology ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Humans ; Microsomes, Liver ; drug effects ; enzymology ; metabolism ; Mitomycin ; metabolism

OBJECTIVETo review the current advances on the role of uncoupling protein (UCP) in the pathogenesis and progress of nonalcoholic fatty liver disease (NAFLD).

DATA SOURCESA comprehensive search of the PubMed literature without restriction on the publication date was carried out using keywords such as UCP and NAFLD.

STUDY SELECTIONArticles containing information related to NAFLD and UCP were selected and carefully analyzed.

RESULTSThe typical concepts, up-to-date findings, and existing controversies of UCP2 in NAFLD were summarized. Besides, the effect of a novel subtype of UCP (hepatocellular down regulated mitochondrial carrier protein, HDMCP) in NAFLD was also analyzed. Finally, the concept that any mitochondrial inner membrane carrier protein may have, more or less, the uncoupling ability was reinforced.

CONCLUSIONSConsidering the importance of NAFLD in clinics and UCP in energy metabolism, we believe that this review may raise research enthusiasm on the effect of UCP in NAFLD and provide a novel mechanism and therapeutic target for NAFLD.

Animals ; Fatty Acids, Nonesterified ; metabolism ; Fatty Liver ; etiology ; metabolism ; Humans ; Ion Channels ; physiology ; Mitochondrial Proteins ; analysis ; chemistry ; physiology ; Non-alcoholic Fatty Liver Disease ; Uncoupling Protein 2

This study is to evaluate the cytotoxicity of mitomycin C (MMC) and its analogue 5-(aziridin-1-yl)-3-hydroxymethyl-1-methylindole-4,7-dione (629) as well as the effect of transfection of constitutive androstane receptor (CAR) on their biological effects. HepG2 cells were transfected with the plasmids mCAR1/pCR3 mediated by liposome. Vector pCR3 was used as control. Transfected cells were screened by G418 resistance and limiting dilution. The expressions of plasmid mCAR1/pCR3 and CYP2B6 mRNA were detected by RT-PCR; Cytotoxicities of MMC and 629 in vitro were evaluated in g2car cells and HepG2 cells by MTT method under anaerobic and aerobic conditions. mRNA expression of CAR and CYP2B6 can not be detected in HepG2 cells and HepG2/pCR3 cells but can in g2car cells. It is shown that plasmid mCAR1/pCR3 was transfected into g2car cells successfully and target CYP2B6 was transactivated by CAR. To compare with aerobic and anaerobic, the cytotoxicities of MMC and 629 to HepG2 cells and g2car cells had significantly enhanced (P < 0.05), and transfect CAR gene can improve the cytotoxicity of MMC (P < 0.05), but not 629 (P > 0.05). Furthermore, CYP2B6 is one master enzyme for the metabolism of MMC and not 629. Transfection of CAR can increase expression of CYP2B6 mRNA in HepG2 cells, and can affect cytotoxicities of MMC and 629.
Antibiotics, Antineoplastic ; pharmacology ; Aryl Hydrocarbon Hydroxylases ; biosynthesis ; genetics ; Aziridines ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Death ; drug effects ; Cell Hypoxia ; Cell Line, Tumor ; Cytochrome P-450 CYP2B6 ; Humans ; Indoles ; pharmacology ; Inhibitory Concentration 50 ; Liver Neoplasms ; metabolism ; pathology ; Mitomycin ; pharmacology ; Oxidoreductases, N-Demethylating ; biosynthesis ; genetics ; Plasmids ; RNA, Messenger ; metabolism ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Transfection

Unrelated umbilical cord blood units for 54 cases in 21 transplant centers were provided by Guangzhou Cord Blood Bank in China from 1998 to 2003. This study was aimed to identify HLA-DRB1 alleles by means of PCR sequencing based typing methods (SBT) and to analyze the correlation between HLA-DRB1 alleles and GVHD in unrelated umbilical cord blood transplantation (UCBT). 48 out of 54 patients received UCBT were followed up. DNA were extracted from cryopreservation blood of recipients/donors with UCBT, HLA-DRB1 alleles typing were done by SBT. Compared with low resolution results of HLA-DRB1 alleles, high resolution results were analyzed to see any correlation between HLA-DRB1 alleles and GVHD. by double-blind statistically analysis of HLA-DRB1 high resolution in 48 donor/recipient typings in UCBT. The results showed that the incidence of GVHD (25%) in patients who has HLA-DRB1 alleles matched with donors significantly lower (65.6%) than that in the patients with HLA-DRB1 alleles mismatched (P = 0.008). It is concluded that HLA-DRB1 by high resolution typing method is important in clinical application in UCBT.
Alleles ; Blood Donors ; Cord Blood Stem Cell Transplantation ; methods ; Graft vs Host Disease ; genetics ; immunology ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Histocompatibility Testing ; methods ; Humans ; Retrospective Studies ; Sequence Analysis, DNA ; methods

From June 1998 to July 2004, Guangzhou umbilical cord blood bank provided unrelated umbilical cord blood for 54 patients to more than 21 transplantation centers. HLA sequencing-based typing (SBT) was used to re-analyze the results of HLA antigens and alleles so as to investigate the relationship between HLA alleles and GVHD. The information about 48 out of 54 patients was obtained after 6 months of follow up. SBT was used to identify HLA-A, B, DRB1 alleles in 48 patients received the unrelated umbilical cord blood units, and the obtained results were compared with the results of HLA-SSP Low Resolution Typing. The results showed that the difference of GVHD incidence between less than 2 mismatched HLA sites and less than 3 sites was statistically significant (P < 0.05). In the results from single factor analysis and high-resolution typing of HLA-A, B and DRB1 alleles, the mismatch between HLA-B and HLA-DRB1 alleles was found to be a significant factor for the occurence of GVHD. It is concluded that SBT plays an important role in umbilical cord blood transplantation, and the incidence of GVHD is higher in the transplantation with HLA-DRB1 alleles mismatching.
Adolescent ; Adult ; Aged ; Alleles ; Child ; Child, Preschool ; Cord Blood Stem Cell Transplantation ; adverse effects ; Female ; Fetal Blood ; cytology ; immunology ; Graft vs Host Disease ; prevention & control ; HLA-A Antigens ; genetics ; immunology ; HLA-B Antigens ; genetics ; immunology ; HLA-DR Antigens ; genetics ; immunology ; HLA-DRB1 Chains ; Histocompatibility Testing ; methods ; Humans ; Leukemia ; therapy ; Male ; Middle Aged ; Sequence Analysis

OBJECTIVETo analyze epidemiological characteristics of burn inpatients in Hainan province over 8 years.

METHODSSix thousand and ninety-nine burn patients admitted to 6 hospitals of Hainan province from January 2002 to December 2009 were enrolled in the study. The clinical data of these patients were analyzed retrospectively, including age, gender, injury cause, wound position, burn area, ailment prior to admission, admission time, medical insurance, length of hospital stay, and mortality rate, relationship among inpatient distribution, admission time, and ambient temperature at the time of admission. Data were processed with SPSS 13.0 software.

RESULTSThere were more burn male patients than female, with ratio of 2.1: 1.0. Most patients were younger than 13 years (57.2%, 3488/6099). The most common burn area was smaller than or equal to 10% TBSA (67.4%, 4108/6099), and the fewest patients had burn areas of over 50% TBSA (2.0%, 121/6099). The main causative agents were hot liquid and flame, accounting for 71.5% (4358/6099), 17.9% (1092/6099), respectively. Most patients had injuries of more than two body areas (60.7%, 3705/6099), and lower extremity injury (17.1%, 1042/6099) was predominant in wound of single body area. Among 703 cases who had other ailments prior to admission (11.5%), the highest rate of prior ailments was found in patients older than 60 years (18.5%, 48/260), it was lowest in children younger than 1 year (8.0%, 32/398). The length of hospital stay was 1 to 375 day, and the admission time was 10 minutes to 90 days after burn. Total mortality rate was 0.4% (26 cases). The number of inpatients aged from 19 to 59 was obviously higher in months with high ambient temperature (from June to August), and for inpatients younger than 13 years the incidence of burn injury showed no obvious seasonal change. The inpatients who had medical insurance accounted for 10.9% (66/603) to 19.5% (121/619) from 2002 to 2005, which increased to 46.0% (372/808) in 2007 and 79.1% (869/1098) in 2009.

CONCLUSIONSFor burn inpatients in Hainan province, the main injury cause of burn injury is hot liquid, the number of burn adults aged from 19 to 59 seems to increase in months with high ambient temperature, while the incidence of burn in children showed no obvious seasonal change. The number of inpatients and those with medical insurance showed a tendency of increase from 2005 to 2009 in Hainan province.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Burns ; epidemiology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Incidence ; Infant ; Inpatients ; Male ; Middle Aged ; Retrospective Studies ; Young Adult

OBJECTIVEFrom December 1998 to April 2004, 3960 umbilical cord blood units were stored in Guangzhou cord blood bank, which provided 100 umbilical cord blood units to 25 transplant center for 83 patients with malignant or non-malignant diseases. To study the related factors affecting unrelated umbilical cord blood stem cell transplantation, the authors analyzed retrospectively the results of transplantation of unrelated umbilical cord blood stem cells for 65 patients.

METHODSALL (acute lymphocytic leukemia) cord blood units were obtained from full term normal vaginal and cesarean deliveries in Guangzhou Women and Infants Hospital. The fractionation, cryopreservation and thawing of the cord blood were performed according to the regulations of New York umbilical cord blood bank and pertinent literature. The selection of cord blood was based on HLA typing and the number of nucleated cells. The sex and HLA antigens of donors were defined as the evidence of engraftment. Time to engraftment was recorded when the absolute number of neutrophil ANC (absolute neutrophil count) was higher than 5.0 x 10(8) for three days. Event-free survival and graft versus host disease (GVHD) were provided by transplant centers.

RESULTSOut of 65 patients who received unrelated cord blood stem cell transplant, 49 patients were diagnosed as having malignant diseases [including 23 with ALL, 16 with AML (acute myeloid leukemia), 7 with CML (chronic myelogenous leukemia), 3 with lymphoma and one with MDS (myelodysplastic syndrome)], 16 patients had non-malignant disease. The 65 transplanted patients (42 male, 23 female) had a median age of 10 years (range 1 - 33 years) and a median body weight of 27 kg (range 10 - 67 kg). The patients received cord blood stem cells from unrelated 0-locus (n = 9) or 1-locus (n = 43) or 2-locus (n = 13) HLA mismatched donor. The median dose of infused cells was: total neutrophil count (TNC) 5.7 x 10(7), CD(34)(+) 5.1 x 10(5), CFU-GM 3.8 x 10(4). Fifty of 65 (77%) patients had engraftment. GVHD occurred in 41 patients (63%), including acute grade I - II GVHD in 31 patients (76%), acute grade III - IV GVHD in 8 patients (20%) and chronic GVHD in 2 patients (5%). Fifty patients had engraftment (ANC > 5.0 x 10(8)) after a median time of 17 (range 7 to 44) days after transplant, while an autologous hematopoietic reconstitution was observed in 6 patients; 24 patients died of severe pneumonia (n = 8), acute GVHD (n = 4), or sepsis (n = 12) and the disease-free survival probability was 61%.

CONCLUSIONSUnrelated allogeneic umbilical cord blood transplantation may be a good substitution for unrelated allogeneic bone marrow transplantation with a good prospect.

Adolescent ; Adult ; Child ; Child, Preschool ; China ; Cord Blood Stem Cell Transplantation ; adverse effects ; methods ; Disease-Free Survival ; Female ; Graft vs Host Disease ; etiology ; mortality ; Humans ; Infant ; Leukemia ; mortality ; therapy ; Male ; Retrospective Studies ; Survival Rate ; Transplantation, Homologous ; Treatment Outcome ; Young Adult

Objective To investigate effects of 5-Aza-2′-deoxycytidine (5-Aza-CdR) on proliferation of human breast cancer cell line Hs578T,and the methylation status of PRDM10 gene in vitro in this cell line.Methods The human breast cancer cell line Hs578T was cultured with 1,3 and 5 μmol/L DNA methylation inhibitor 5-Aza-CdR respectively, and untreated cells were used as control.Cell proliferation was detected by MTT assay.Methylation-Specific PCR(MSP)was used to detect the methylation status of PRDM10 gene. The mRNA and protein expression levels of PRDM10 gene were detected by RT-PCR and Western blot assay. Results MTT results showed that the higher the concentration of 5-Aza-CdR,and the longer the treatment time,the more significant inhibitory effect on the proliferation of Hs578T cells.Compared with the control group(0 μmol/L),the proliferation of Hs578T was significantly inhibited after the treatment for 72 h in the 1 μmol/L group, and for 48 h in the 3 μmol/L and 5 μmol/L groups (P<0.05). MSP results showed that the higher the concentration of 5-Aza-CdR,the more significant demethylation of PRDM10.Results of RT-PCR and Western blot showed that the higher the concentration of 5-Aza-CdR, the higher the expression levels of mRNA and protein in PRDM10 (P<0.05).Conclusion 5-Aza-CdR could inhibit the cell proliferation of Hs578T,which might be related to the demethylation of PRDM10 gene in the cells.

Objective To assess the adherence,immunologic and survival responses in HIV-infected patients receiving free antiretroviral therapy (ART). Methods All adult HIV-infected patients in Wenxi county who started antiretroviral treatment (ART) between 01 July 2001 and 31 December 2006 and aged above 18 years were included in this study. Epidemiological survey and laboratory tests were performed before,0.5 months after, 1 months after, 2 months after and every 3 months after initiation of ART to recognize the adherence, efficacy (CD4+ T cell counts) and survival to the regimens. Results The median follow-up time period was 16.5 months (Interquartile: 15.5-20.8 months). At baseline, the median of CD4+ T cell counts were 154 cells/μl (Interquartile: 81-212 cells/μl). Treatment was effective in most of the patients, the CD4+ T cell count of patients increased after the initiation of ART. The maximum increase was recorded at month 3, from the median of 154 cells/μl to 220 cells/μl (P<0.001) ,and thereafter the count remained stable. When comparing with patients with baseline CD4+ T cell count≥100 cells/μl, those with baseline CD4+ T cell count < 100 cells/μl showed a higher mean increase in the first three months of treatment. The cumulative probability rates of remaining alive were 0.94,0.88 and 0.87 at 3,12,24 months, respectively. In multivariate Cox's proportional hazard models, after adjustment for the type ofinitial regimens (NVP vs. EFV/IDV), CD4+T cell count of less than 50 cells/μl (vs. 50 cells/μl or more) was strongly associated with death hazard ratio 0.21 (95% CI:0.06-0.68). Conclusion Our data showed that ART was effective for improving immunologic response of adult patients with HIV/AIDS. CD4+ T cell count at initiation was associated with survival time in patients starting ART,suggesting that monitoring of CD4+ T count should be strengthened to early initiate antiretroviral therapy for HIV-infected patients.