2008 Vol. 21, No. 4
2008, 21(4): 271-279.
To test the validity of Working Group on Obesity in China (WGOC) reference in screening childhood obesity using obesity-related metabolic syndrome (MS) and its components as disease risk evidence. Methods A total of 2020 adolescents (1007 boys and 1013 girls) aged 14-16 years were sampled in Beijing, China. Anthropometxic and biochemical measurements, as well as blood pressure parameters were available. Prevalence of overweight/obesity and related MS risk factors were analyzed across different body mass index (BMI) categories. The sensitivity and specificity of the WGOC cut-offs were compared with those of National Central Health Statistics (NCHS). Results Significantly high prevalence of MS and its components were found both in the obesity and overweight groups, which were classified by the WGOC and NCHS references. Similar distribution pattern of MS risk factors existed among different BMI categories, hut the frequency and clustering of these factors in the obesity group classified by the NCHS were much higher. Owing to its irrelevant high cut-offs for overweight/obesity (especially for girls since the mid-adolescence), the NCHS reference had a high specificity but a low sensitivity. By contrast, the WGOC reference with a high sensitivity (90.1% for boys and 89.2% for girls) and a relative high specificity (96.4% and 92.8% for obese boys and girls, 78.1% and 68.9% for overweight boys and girls respectively) was more suitable to support the need for early screening, intervention, and treatment of childhood obesity in China. Conclusion High sensitivity is more important than specificity in choosing appropriate screening tools for childhood obesity. Validity test demonstrates that it is rational to use the WGOC reference, established on the basis of the Chinese own reference population as a uniform screening tool for childhood obesity, which can effectively overcome the unnecessary treatment and psychosocial implications of stigmatization caused by misclassification.
Intracellular Retention of Human Melanocortin-4 Receptor:A Molecular Mechanism Underlying Early-onset Obesity in F261S Pedigree of Chinese
2008, 21(4): 280-285.
To investigate how F261S mutation identified from Chinese obese patients affects the function of melanocortin 4 receptor (MC4R) and to analyze the obesity-related phenotypes in subjects carrying the F261S mutation. Methods F261S mutant of MC4R was generated by site-directed mutagenesis. Plasmids encoding wild-type or F261S mutant of MC4R were transfected into HEK293 and COS-7 cells to examine their functional characteristics. Signaling properties of F261S MC4R were assessed by measuring intracellular cAMP levels in response to α-MSH stimulation. Cell surface expression of F261S MC4R was compared with that of wild-type MC4R. Clinical examinations were performed in subjects carrying F261S mutation and in non-mutated controls. Results The α-MSH-stimulated reporter gene activity was significantly reduced in cells expressing F261S MC4R, with a maximal response equal to 57% of wild-type MC4R. The F261S mutation also led to a significant change in the EC<,50> value compared with the wild-type receptor (P<0.01). Immunofluorescent assay revealed a marked reduction in plasma membrane localization of the MC4R in cells expressing the F261S mutant receptor. The resting metabolic rate and fat composition of the mutant carriers were not significantly different from those of the non-mutated obese controls. Conclusions The decreased response to α-MSH due to the intracellular retention of MC4R may cause early-onset obesity in the F261S pedigree of Chinese.
2008, 21(4): 286-289.
To evaluate the oxidative stress in patients with colorectal cancer and to investigate the relationship between oxidative stress and colorectal cancer. Methods Seventy-six subjects were divided into two groups (36 colorectal cancerpatients as the study group and 40 normal healthy individuals as the control group). Their protein oxidation, DNA damage, lipid peroxidation and antioxidants, vitamin C, vitamin E, glutathione (GSH), and antioxidative enzymes in serum were detected. Results The levels of protein carbonyl and advanced oxidation protein products (AOPP) were significantly higher in the study group than in the control group (P<0.01). Serum 8-OHdG was significantly increased in the study group compared to the control group (P<0.01). However, the mean serum level of MDA and conjugated diene was lower in the study group than in the control group (P<0.01). The activity of antioxidative enzymes was significantly decreased in the study group compared to the control group (P<0.01). Serum vitamins C and E concentrations were significantly reduced in the study group compared to the control group (P<0.01). Conclusion Colorectal cancer is associated with oxidative stress, and assessment of oxidative stress and given antioxidants is important for the treatment and prevention of colorectal cancer.
2008, 21(4): 290-295.
To filtrate breast cancer resistance protein (BCRP)-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance. Methods MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography (HPLC) assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immununohistochemistry (IHC) were employed to investigate the BCRP expression in breast cancer tissue specimens. Results MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil (5-Fu) (P<0.05, n=3) in the cell model, while HPLC assay indicated that the intracellular retention dose of 5-Fu was significantly correlated with the expression of BCRP (r=-0.897, P<0.05, n=3). A total of 140 breast cancer tissue specimens were collected. BCRP-positive expression was detected in forty-seven specimens by both RT-PCR and IHC. As shown by MTT MTT-ssay subsequently, the resistance index (RI) of 47 BCRP-positive breast cancer tissue specimens to 5-Fu was 7-12 times as high as that of adjacent normal tissue samples. BCRP expression was related to 5-Fu resistance (R<'2>=0.8124, P<0.01). Condusion Resistance to 5-Fu can be mediated by BCRP. Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.
Development of Quantitative Real-time Polymerase Chain Reaction for the Detection of Vibrio vulnificus Based on Hemolysin (vvhA) Coding System
2008, 21(4): 296-301.
To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene (vvhA) coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles (Ct value) and fluorescence intensity enhancement (△Rn value) were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 10<'3> copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.
Disease Progression Patterns of SHIV-KB9 in Rhesus Macaques of Chinese Origin in Comparison with Indian Macaques
2008, 21(4): 302-307.
To develop a model of SHIV-KB9/Chinese origin rhesus (Ch Rh) macaques for vaccine research and to compare the pathogenesis of SHIV-KB9 in Ch Rh macaques with that reported in Indian rhesus (had Rh) macaques. Methods Seven mamu-A*01 negative Ch Rh macaques were inoculated intravenously with 1-10000 MID<,50> of SHIV-KB9. The monkeys were monitored for viral load, CD4, CD8, SHIV-specific antibody and virus genetic variation. The results were compared with those previously observed in Ind Rh macaques. Results As compared to that observed in Ind Rh macaques, SHIV-KB9 in Ch Rh macaques displayed three identical disease progression patterns. However, the primary pattern was not identical between the two subspecies. The level of plasma viremia differed in SHIV-KB9-infected Ch Rh macaques which exhibited different outcomes from those in Ind Rh macaques. Generally, the values of viral load and the maintenance of CD4<'+> T cells were associated with humoral responses. Otherwise, the viral genetic distances (divergence, diversity) were larger in animals (M419, M425) with their CD4<'+>T cells profoundly depleted. Conclusion The model of SHIV-KB9/Ch Rh macaques displays a relatively slow progression to AIDS compared with Ind Rh macaques, which may more accurately reflect the potential ofcandidate vaccines in humans.
Reliability and Validity of Addiction Severity Index in Drug Users with Methadone Maintenance Treatment in Guizhou Province, China
2008, 21(4): 308-313.
To evaluate the reliability and validity of the Chinese version of addiction severity index (ASl)-5th version (ASI-C-5), in illegal drug users receiving methadone maintenance treatment (MMT) in China. Methods One hundred and eighty-six heroin addicts (144 men and 42 women) receiving MMT at three clinics in Guizhou province, southwest China, were recruited. They were all interviewed with a questionnaire of ASI-C-5 and 35 were re-interviewed at an interval of seven days to assess its test-retest reliability. Results Cronbach's alpha for internal consistency of CSs varied from 0.60 to 0.81 in all domains. Test-retest reliability of composite scores (CSs) of ASI-C-5 were satisfactory (r=0.38-0.97). Based on item analysis and expert's suggestions, five items were deleted and one item was modified in ASI-C-5. Criterion validity of ASI-C-5 was found acceptable, as compared to addicts' self-rating anxiety scale (SAS) and self-rating depression scale (SDS) (r=0.59 and 0.45) except for social support rating scale (SSRS). Conclusions ASI-C-5 can be used for heroin addicts receiving MMT with acceptable reliability and validity.
2008, 21(4): 314-318.
To characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B'/C vires-infected ART-naive individuals. Methods HIV-1-specific CTL responses were analyzed by IFN-γ ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P<0.05. Results Tat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study. Conclusion Tat and Rev can serve as targets for HIV-1-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV-1 natural infection and also be useful for the design and testing of candidate vaccines.
2008, 21(4): 319-324.
To estimate the heavy metal content in soil and selected medicinal plants procured from environmentally different sites of the same city. Methods Soil and plant samples of Abutilon indicum, Calotropis procera, Euphorbia hirta, Peristrophe bycaliculata, and Tinospora cordifolia were collected from 3 environmentally different sites of the city: heavy traffic area (HTA), industrial area (IA), and residential area (RA). Pb, Cd, Cr, and Ni were estimated in soil and plant samples by inductively coupled plasma emission spectrometry and compared. Results The level of heavy metal was higher in soil than in plant parts studied. Accumulation of heavy metals varied from plant to plant. Pb was the highest in Calotropis procera root from HTA site and the lowest in Peristrophe bycaliculata whole plant from IA site. It was also lower in residential area than in heavy traffic area. Conclusion The level of heavy metal content differed in the same medicinal plant collected from environmentally different sites of the same city. Thus, it reiterates our belief that every medicinal plant sample should be tested for contaminant load before processing it further for medication.
Immunoregulatory Effects of Ethyl-acetate Fraction of Extracts from Tetrastigma Hemsleyanum Diels et. Gilg on Immune Functions of ICR Mice
2008, 21(4): 325-331.
To evaluate the effects of ethyl-acetate fraction (EAF) of extracts from Tetrastigma hemsleyanum Diels et. Gilg (TDG) on immune functions of ICR mice. Methods ICR mice were exposed to different doses of EAF for 15 or 30 days and then their immune functions were analyzed, including ConA-induced splenic lymphocyte transformation, SRBC-induced delayed type hypersensitivity response, serum hemolysin analysis, antibody-producing cells, peritoneal macrophage phagocytized chicken red blood cells, natural killer cell activity, and serum level of cytokines. Results EAF of extracts from TDG at different doses had various effects on immune functions of ICR mice. As compared with the controls, it increased the mouse spleen lymphocyte transformation induced by ConA, the left-hind voix pedis thickness and the number of plague forming cells (PFCs) at the dose of 1.82 mg/mL, 5.48 mg/mL, and 9.12 mg/mL, respectively; increased the ink clearance ability at the dose of 0.91 mg/mL, 1.82 mg/mL, 5.48 mg/mL, and 9.12 mg/mL, respectively; increased the phagocytosis index of mononuclear-macrophages and production of serum interferon-gamma (IFN-γ) at the dose of 5.48 mg/mL; and could promote the production of serum tumor necrosis factor-alpha (TNF-α) at the dose of 9.12 mg/mL. Conclusion EAF of extracts from TDG can regulate mouse immune functions in vivo.
Malignant Transformation and Abnormal Expression of Eukaryotic Initiation Factor in Bronchial Epithelial Cells Induced by Cadmium Chloride
2008, 21(4): 332-338.
To analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 (eIF3 p36) in human bronchial epithelial (16HBE) cells induced by cadmium chloride (CdCl<,2>). Methods 16HBE cellswere treated several dmes with different concentrations of CdCl<,2>. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdCl<,2>, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique (FQ-PCR). Results The 35th passage of 16HBE cells transformed by CdCl<,2> exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects (P<0.01). All Cd-induced transformed cell lines formed tumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl<,2> were elevated as compared with the non-transformed control (P<0.01), and the eIF3 expression increased with the degree of cell malignancy. Conclusion CdCl<,2> is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl<,2> and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.
2008, 21(4): 339-344.
To isolate an isogenic radioresistant cancer cell line after fractioned X-ray radiation and characterize the resistant cells. Methods D6 cells were exposed to repeated X-ray irradiation, and after a total dose of 5200 cGy in 8 fractions, a radioresistant monoclone D6-R was obtained. The radiosensitivity and drug sensitivity of the novel radioresistant D6-R cells, together with their parent D6 cells, were measured using clonogenic assay and MTT assay respectively. Cell cycle distribution was analyzed by flow cytometry. Fluorescence microscopy and flow cytometry were applied for apoptosis detection. Comet assay was used for the detection of DNA damage and repair. Results D6-R cells showed higher and broader initial shoulder (D<,0>=2.08 Gy, D<,q>=1.64 Gy, N=2.20) than the parent D6 ceils (D<,0>=1.84 Gy, D<,q>=0.34 Gy, N=1.20). They were 1.65-fold more radioresistant than D6 cells in terms of SF<,2>(63% vs 38%) and were more resistant to ADM (3.15-fold) and 5-FU (3.86-fold) as compared with the latter. It was found that D6-R cells had higher fractions of cells in S phase (53.4% vs 37.8%) and lower fractions of ceils in G<,1>(44.1% vs 57.2%) and G<,2>-M phase (2.5% vs 5%). There was no difference in radiation-induced apoptosis between D6-R and D6 cells. D6-R cells showed less initial DNA damage and increased capacity in DNA repair after irradiation, as compared with the parent cells. Conclusions D6-R cells have been isolated by exposing the parental D6 cells to repeated irradiation. The difference in cell cycle pattern together with the induction and repair of DNA damage might, at least partially, explain the mechanism of the radioresistance.
2008, 21(4): 345-352.
To investigate the heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) in sludge of twelve wastewater treatment plants (WWTPs) in Zhejiang province of China, and to assess their potential for land application. Methods Sludge was collected from 12 WWTPs within the province. GC-MS and AAS were used to measure PAHs and HMs contents in sludge. Results Concentrations of HMs in most of the sludge samples were below the regulatory limits for the sludge to be used in agriculture in China with the exception of Zn in 2 sludge samples and Cd in 1 sample. All 16 PAHs, targeted by the USEPA agency, were found in the sludge from the twelve plants with a total concentration ranging from 33.73 mg kg-1 to 82.58 mg kg-1 (dry weight, d.w.). The levels of ∑9 PAHs varied from 13.87 mg kg-1 to 61.86 mg kg-1 (d.w.) in the sludge, far exceeding the limitation value recommended by the Europe Union. The concentration and composition of PAHs in sewage sludge varied and depended mainl