Simultaneous ex vivo generation of cytomegalovirus pp65 and Epstein-Barr virus specific cytotoxic T-lymphocyte from human umbilical cord blood

AIM: To investigate the possibility of simultaneously ex vivo generating cytomegalovirus (CMV) pp65 and Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) from human umbilical cord blood (CB). METHODS: Mononuclear cell derived from CB (CBMC) was used to construct EBV-transformed B-lymphoblastoid cell lines (BLCL). Then BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. CBMC from the same CB donor were stimulated with pp65-expressing BLCL (BLCLpp65) weekly for 5~6 weeks. Chromium release assays (CRA) were performed to detect the specific cytotoxicity of the CTL against EBV and CMV. RESULTS: Western blot analysis and immunocytochemistry confirmed that BLCLpp65 could simultaneously express CMVpp65 and EBV antigen. CRA results showed that the generated CTL possessed specific cytotoxic against EBV and CMV, and the cytotoxicity was mediated by CD₈⁺ CTL. CONCLUSION: BLCLpp65 can be used as antigen-presenting cells to stimulate expansion of EBV and CMV specific CTL simultaneously from the predominantly native T cell population in CB.     

Relationship between persistent HPV infection and cervical carcinoma

Human papillomavirus (HPV) genital infection is a very common sexual transmitted disease. Mostly, the infection is transient and asymptomatic. The induction of effective immune responses usually allows the infection to be spontaneously cured. However, the infection sometimes can be responsible for an intraepithelial lesion, which may progress to be cervical cancer. Cervical cancer is one of the most common neoplastic diseases affecting women. It is clear that carcinogenic HPV infection is the necessary process for the development of cervical cancer. HPV infection is very frequent in young women aged less than 25 years and viral clearance lasts for 8 months in average. This clearance is the consequence of host immunity intervention, which leads to spontaneous regression of infection and the overwhelming majority of low-grade squamous intraepithelial lesions (more than 80% within a period of 2 years). The major factor, which permits the progression to high grade squamous intraepithelial lesions and invasive cervical cancer, is the persistent feature of HPV infection. HPV infection is usually transient, but several factors were identified as host factors (genetic, immunodepression, oral contraception and smoking) and viral factors (genotype, variants, viral load and integration) to increase the persistence. Cervical cancer is clearly the first virus-induced solid tumor discovered in human. Furthermore, it represents a woman death cause that can be avoided.     

Effects of TNFα and IL-10 on cytomegalovirus infection in human embryonic lung fibroblasts

AIM:To study effects of tumour necrosis factor alpha(TNFα) and interleukin-10(IL-10) on human cytomegalovirus AD169 (HCMV AD169) infection in human embryonic lung fibroblasts (HEL),and the ability of the infected HEL to produce TNFα. We have attempted to understand the effect of cytokines in the immune of HCMV infection. METHODS:TNFα、IL-10 were added separately with different concentrations before 24 h HCMV infection of HEL to study the effect of TNFα and IL-10 on multiplication of HCMV. HCMV was incubated with HEL, TNFα in culture supernatants were measured at 4, 24, 48, 72, 96 h postinfection. The level of TNFα was measured by enzyme-linked immunosorbent assay( ELISA). RESULTS:The addition of TNFα with the concentration of 10-100 μg/L or IL-10 with the concentration of 1-10 μg/L before 24 h HCMV infection of HEL could remarkably inhibited the multiplication of HCMV in HEL. Level of TNFα didn't increase in infected cell supernatants. CONCLUSION:TNFα and IL-10 play an important role in the immune of HCMV infection.     

Role of NF-κB in inducing HEL cell proliferation and apoptosis during human cytomegalovirus infection

AIM:To investigate whether human cytomegalovirus(HCMV) regulate human embryonic lung fibroblast(HEL) cell proliferation and apoptosis by activating NF-κB.METHODS:Immunohistochemistry and Western blot analysis were used to detect the NF-κB translocation and/or Bcl-2 and the levels of I-κBα during HCMV infection. Apoptotic cell were examined by flow cytometry, and the HEL cell proliferation was determined by MTT.RESULTS:The levels of NF-κB in the nucleus reached highly 48 h postinfection, and the levels of I-κBα were low 24 h postinfection. The activity of NF-κB was inhibited 120 h postinfection. The levels ofbcl-2was accorded with the activity of NF-κB. HCMV promoted HEL cells to proliferate before 72 h postinfection and induced apoptosis 120 h postinfection.CONCLUSION:NF-κB plays a role in HEL cell proliferation and apoptosis during HCMV infection, and it involves in the pathological mechanisms of diseases associated with HCMV infection.     

Relationship between apolipoprotein E gene polymorphism and sporadic Alzheimer's disease

AIM: To evaluate the association between apolipoprotein E(apoE) gene polymorphism and sporadic Alzheimer's disease (AD). METHOD: A case-control study was undertaken detecting the polymorphism of apoE by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP).RESULTS:(1)The frequencies of 3/4 genotype and 4 al ele in AD were significant ly higher than that in age-matched controls(P<0.05).(2)The frequency of G/G genotype for apoE IE1 in AD was significantly higher than that in age-mat ched control(P<0.05).(3)The apoE 4 al ele was associated with a tripling of the risk for AD compared with no 4 allele(odd ratio 2.932, 95%CI 1.379~6.226);Homozygosity of the G allele in IE1 was associated with adoubling of the risk for AD compared with the G/C and C/C genotypes(odd rat io 2.223, 95%CI 1.075~4.599).However, the IE1 G al ele is also closely associated with apoE 4.When the sample was split on the basis of apo Egenotype, the associat ion between IE1 G/G genotype and AD was no longer statistically significant.CONCLUSION: ApoE ε4 was a risk factor of AD, and the apparent association between IE1 G/G and AD is a consequence of the association between the ε4 and IE1 G/G genotype.     

Relationship between STEAP expression and hydrogen peroxide in human prostate tissues

AIM:To observe the STEAP expression and its function in human prostate tissues.METHODS:The expressions of STEAP mRNA and protein were detected by RT-PCR and Western blotting. H₂O₂ and SOD levels were detected by molybdic acid reduction and xanthine oxidation methods respectively.RESULTS:STEAP mRNA and protein were highly expressed in prostate cancer tissues. H₂O₂ and SOD levels in prostate cancer were obviously higher than those in normal prostate tissue.CONCLUSION:The function of STEAP gene is possibly to induce intracellular H₂O₂ and promote cell growth.     

干旱胁迫对杏脂氧合酶活性的影响

为了研究脂氧合酶(LOX)在植物干旱反应中的作用,以抗旱的山杏和抗旱性弱的龙王帽实生苗为试材,观察了自然干旱过程中叶片的生长状况以及测定了土壤含水量、新叶生长率、叶片脱落率、根和叶片的LOX活性。结果表明,在自然干旱过程中随着土壤含水量的降低,山杏和龙王帽的新叶增长率也下降。干旱后期山杏比龙王帽的新叶增长速率下降快、生长停止早。干旱使山杏比龙王帽叶片早脱落,而且落叶比率明显高于龙王帽。自然干旱时杏的LOX活性表现为叶片升高,在根中变化不大。而且山杏叶片的LOX活性增加幅度比龙王帽大,峰值出现比龙王帽早。因此,干旱条件下抗旱的山杏叶片脂氧合酶活性比龙王帽提早增加而且叶片易早脱落。山杏叶片LOX活性急剧升高出现在叶片萎蔫衰老之前,推测LOX活性增强可能与引发衰老有关。     

Relationship between ADH activity,ripeness and softness in six tomato cultivars

Fruit from six tomato cultivars were harvested at progressive stages of ripeness. Fruit softness was measured together with the activity of the alcohol dehydrogenase (ADH) enzyme in the pericarp tissue. The rate of increase in ADH activity in the ripening fruit was found to be strongly correlated (r=0.970) with the rate of softening of the fruit suggesting that induction of ADH activity in the fruit was possibly a function of the softening of the fruit rather than a direct function of ripening. As the ADH enzyme is involved in several aspects of flavour development in the ripening tomato fruit, in particular the regulation of accumulation of some aldehydes and alcohols, a correlation between fruit softening and activity of the enzyme has important implications regarding flavour development in tomato fruit of firm or soft varieties.     

Association between HBV infection and HLA-DPB1 gene in population of Guangzhou Chinese

AIM: To investigate the association between HBV infection and HLA-DPB1 gene in population of Guangzhou Chinese. METHODS: 58 unrelated patients (test positive of HbsAg, HBeAg, HbcAb) and 75 unrelated healthy control individuals were typed by sequencing based typing (SBT) method in their HLA-DPB1 gene. RESULTS: The phenotype frequencies of HLA-DPB1 alleles of patients and control have no significant difference. CONCLUSION: These results indicate that there is no association between HLA-DPB1 gene and HBV infection.     

Relationship between zinc and zinc transporter expression in human lung cancer cells

AIM: To study the changes of zinc transporter gene expression in A-549 cell line exposed to ZnCl2 and N,N,N’,N’-tetrakis 2-pyridylmethyl ethylenediamine (TPEN).METHODS: Human lung cancer cell line A-549 was exposed to different concentrations of ZnCl2 (0, 50, 100, 150, 200 μmol/L) and TPEN (0, 5, 10, 15, 20 μmol/L), respectively. Twelve hours later, the cell viability was measured by MTT (methyl thiazolyl tetrazolium) and levels of zinc transporter mRNA was detected by RT-PCR. Zinquin was used to estimate the intracellular zinc concentrations.RESULTS: A-549 cell viability rate was significantly decreased when exposed to ZnCl2 at concentrations of 150 and 200 μmol/L, and to TPEN. The intracellular zinc concentration was significantly increased when exposed to ZnCl2 and decreased when exposed to TPEN. Zinc transporter (ZnT-1) mRNA level was increased along with the increase in the concentration of ZnCl2 but decreased when exposed to TPEN. The expressions of ZIP-1 and ZIP-10 (Zrt-and Irt-like protein) were increased along with the increase in the concentration of TPEN but decreased when exposed to ZnCl2.CONCLUSION: ZnT-1 expression is induced by zinc supplement. ZIP-1 and ZIP-10 expressions are induced by zinc deficiency and repressed by zinc supplement.     

Relationship between SNP of TNFR gene and incidence/severity of pneumonia

AIM：To analyze the relationship between the single nucleotide polymorphism (SNP) of tumor necrosis factor receptor (TNFR) gene and the incidence and severity of pneumonia. METHODS：Total 132 Chinese individuals were enrolled in this study. There were 66 patients with pneumonia and 66 healthy subjects. The SNPs of TNFR gene including TNFR1+36A/G, TNFR1-609G/T, TNFR2+676T/G, TNFR2+1663T/G, TNFR2 +1668A/G and TNFR2 +1690C/T were genotyped by polymerase chain reaction-restriction fragment length polymorphism or gene sequencing for all subjects. Polymorphisms affecting pneumonia incidence and severity were calculated by SPSS. RESULTS：The frequencies of TNFR1-609G and T alleles in pneumonia patients were 40.9% and 59.1%, while those in healthy subjects were 53.8% and 46.2%. The frequency of TNFR1-609T in pneumonia patients was higher than that in healthy subjects (P<0.05). Besides, the frequencies of TNFR1-609G and T alleles in severe pneumonia patients were 25.0% and 75.0%, while those were 46.0% and 54.0% in non-severe pneumonia patients. The frequencies of TNFR2 +1690C and T alleles in severe pneumonia patients were 81.1% and 18.9%, while those were 61.0% and 39.0% in non-severe pneumonia patients. The frequencies of TNFR1-609T and TNFR2 +1690C in severity pneumonia subjects were higher than those in mild subjects (P<0.05). CONCLUSION：It appears that TNFR1-609T is associated with high incidence of pneumonia. TNFR1-609T and TNFR2+1690C are the risk factors of severity in pneumonia in Chinese.     

Relationship between polymorphism of angiotensin I converting enzyme gene insertion/deletion and ACE,PAI-1 activity in patients with myocardial infarction

AIM:To explore the relationship between polymorphism of angiotensin I converting enzyme gene insertion/deletion (I/D) and ACE, PAI-1 activity in patients with myocardial infarction (MI). METHODS:Ninety-three patients with MI and eighty-seven healthy controls were tested. ACE genomic DNA was amplified using the polymerase chain reaction (PCR). Serum ACE activity was measured by colorimetry, plasma level of PAI-1 activity was determined by spectrophotometric assay. RESULTS:① The frequency of ACE DD genotype and D alleles (32.3% and 54.3%) in MI group was significantly higher than those in control group (12.6% and 37.4%, P<0.01, respectively). ② The ACE activity in serum (216.00±58.26)U/L and plasma PAI-1 activity (0.85±0.19)AU/mL in MI group were significantly higher than those in control group (170.19±48.99)U/L, (0.66±0.20)AU/mL, P<0.01, respectively. The serum ACE activity was positively correlated with plasma PAI-1 activity both in MI group and control group (r=0.7108 and r=0.7829;P<0.01, respectively). ③ In MI group, the serum ACE activity and plasma PAI-1 activity showed a significantly higher level in subjects with DD genotype (251.64±57.76)U/L, (0.96±0.16)AU/mL than those with ID (211.47±51.87)U/L, (0.82±0.18) AU/mL and Ⅱ genotypes (179.84±52.65)U/L, (0.71±0.17)AU/mL. The serum ACE activity and plasma PAI-1 activity were significantly higher in subjects with ID genotype than those with II genotype (P<0.05). In control group, the serum ACE activity and plasma PAI-1 activity showed a significantly higher level in subjects with DD genotype (195.53±54.76)U/L, (0.78±0.20)AU/mL than the subjects with Ⅱ genotype (154.98±52.74)U/L, (0.59±0.17)AU/mL (P<0.05). CONCLUSION:The increased ACE activity caused by DD polymorphism may play an important role in elevating the level of plasma PAI-1. The DD genotype of ACE is associated with high PAI-1 level. The genetic variation of ACE contributes to the balance of fibrinolytic pathway, indicating the pathogenesis mechanisms linking to the ACE I/D genotype and MI.     

Relationship between thromboxane synthase gene and prostacyclin synthase gene expression and pulmonary hypertension induced by hypoxic hypercapnia

AIM: To study the effect of chronic hypoxic hypercapnia on gene expression of thromboxane synthase and prostacyclin synthase in pulmonary arterioles. METHODS: Sprague-Dawley rats were randomly divided into two groups: control group and hypoxic hypercapnic group. TXS mRNA and PGI₂-SmRNA were observed in pulmonary arterioles by in situ hybridization. RESULTS: mPAP, weight ratio of right ventricle (RV) to left ventricle plus septum(LV+S), contents of TXB₂ and 6-keto-PGF1_α in plasma and lung and TXS mRNAin pulmonary arterioles were much higher in rats of hypoxic hypercapnic group than those of control group. Differences of PGI₂-SmRNA in pulmonary arterioles were not significant in two groups. Light microscopy showed hypertrophy of vessel smooth muscle cells and vessel cavity straitness were found in hypoxic hypercapnic group. CONCLUSION: Changes of gene expressions of thromboxane synthase and prostacyclin synthase and imbalance of TXA₂/PGI₂ may play an important role in hypoxic hypercapnic pulmonary hypertension.     

Relationship between subclasses of serum HDL and LPL gene HindⅢ polymorphism in hyperlipidemia

AIM: To investigate lipoprotein lipase gene HindⅢ polymorphism and its relationship with serum lipids and apolipoprotein, serum HDL subclasses in patients with hyperlipoidemia. METHODS: Lipoprotein lipase gene HindⅢ polymorphism was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The subclasses of serum HDL in 152 hyperlipoidemia patients and 128 healthy subjects were determined by two-dimensional gel electrophoresis conjunction with immunodetection method. RESULTS: H+H+ genotype and allele H+ in hyperlipoidemia and control groups were both the highest. In hyperlipidemia group, H+H+ genotypes tended to be higher than that in control group, while H+H- and H-H- genotypes were significantly lower (P<0.05). In hyperlipidemia group allele H+ carriers' frequency tended to be higher than that in control group (P<0.05). In hyperlipoidemia group, the genotype of H+H+ showed higher serum TG, apoB100 levels, TG/HDL-C ratio, preβ1-HDL, HDL3b and lower HDL2a, HDL2b compared with H-H- (P<0.05). In control group, the genotype of H+H+ had higher serum TG，HDL3c and lower HDL2a compared with H-H- (P<0.05). CONCLUSION: The HindⅢ polymorphism at intron 8 of LPL gene is associated with the general shift toward smaller size of HDL particle size in hyperlipoidemia, and the change of HDL subclasses distribution profile may be closely related to the pathogenesis of atherosclerosis in Chinese patients with hyperlipoidemia.     

Polymorphisms of KIR gene and CMV-BKV DNA quantification in renal transplant recipients

AIM:To investigate the association of killer cell immunoglobulin-like receptor (KIR) genotype and cytomegalovirus (CMV)-BK virus (BKV) reactivation/infection in the first year after renal transplantation. METHODS:KIR genotypes were analyzed by PCR with sequence-specific primers (PCR-SSP) in 48 renal transplant recipients and KIR genotypes were grouped into AA if they contained only the canonical group A haplotype genes. The genotype containing additional KIR genes was referred to BX, as it contained at least 1 group B haplotype. Real-time quantitative PCR for CMV-BKV DNA was performed to test the viral load. The association of KIR genotype and CMV-BKV infection and the effect of KIR genotype on renal function were analyzed. RESULTS:No obvious difference in the concentration of immunosuppressant and the occurrence of CMV infection/reactivation in the first year after renal transportation was observed between KIR-AA and KIR-BX genotype groups. However, there was a significantly negative correlation between KIR-BX genotype and the cumulative incidence of BKV infection/reactivation. The average concentration of serum creatinine over 1~12 months after operation in KIR-AA genotype group was lower than that in KIR-BX genotype group. The levels of blood urea nitrogen and uric acid showed no obvious difference between the 2 groups. CONCLUSION:There is a significantly negative correlation between KIR-BX genotype and the cumulative incidence of BKV infection/reactivation, but not CMV infection/reactivation in the first year after renal transplantation.     

Key amino acids of interactions between interferon-induced protein N-Myc interactor and human cytomegalovirus tegument protein UL23

AIM:To investigate the key amino acids of interferon-induced protein N-Myc interactor (Nmi) that interacts with human cytomegalovirus (HCMV) tegument protein UL23. METHODS:According to the previous results, 10 groups of the truncated Nmi fragment were constructed on plasmid pGEX-4T-1. Recombinant plasmids were transformed into E.coli Rosetta (DE3) competent cells, and fusion proteins with GST tag were expressed and purified. The domains on Nmi that mediated the interaction with UL23 were identified by GST-pulldown test. Based on the results of GST-pulldown test, 3 groups of deleted Nmi fragments were inserted into the eukaryotic expression plasmid pcDNA4-Myc by homologous recombination. The plasmid with Flag-tagged UL23 plasmid and wild-type Nmi or deletion mutants were co-transfected into HeLa cells to reconfirm the key amino acids on Nmi that interacted with UL23 by the method of co-immunoprecipitation (Co-IP). RESULTS:The prokaryotic expression vectors of 10 groups of truncated Nmi mutants fused with GST gene were successfully constructed. Three groups eukaryotic expression vectors of Nmi deletion mutants fused with Myc gene were also successfully constructed. The results of GST-pulldown test indicated that the region of 192~202 aa located on Nmi was a key area to interact with UL23. The UL23 binding domain of Nmi in the amino acids 192~202 aa was confirmed by Co-IP, which was consistent with the GST-pulldown results. CONCLUSION:The 192~202 aa of Nmi are key amino acids in the interaction with UL23. This may provide the basis for clarifying the molecular mechanisms by which UL23 plays an important role in the latency of HCMV in host.     

Relationship between TNF-α gene polymorphism-308 and type 2 diabetes with periodontitis

AIM: To investigate the gene polymorphism-308 of tumor necrosis factor alpha (TNF-α) in the relation with the susceptibility to periodontitis combined with type 2 diabetes mellitus (DM) and its severity.METHODS: Human DNA samples were obtained from 240 DM patients with periodontitis (periodontitis group, n=120) and without periodontitis (control group, n=120). All patients were genotyped by PCR-RFLP analysis. The frequencies of genotypes and alleles were analyzed. Sulcus bleeding index (SBI) and probing depth (PD) in all patients were measured. The polymorphism-308 of TNF-α gene in the relation with the susceptibility to periodontitis combined with DM and its severity was analyzed.RESULTS: No significant difference in the frequency of genotype and allele was found between DM patients with mild periodontitis and DM patients without periodontitis (P>0.05). However, the frequencies of these genotypes and alleles in DM patients with moderate and severe periodontitis were significantly higher than those in DM patients without periodontitis (P<0.01). The findings showed that the level of TNF-α was associated with SBI and PD (P<0.01).CONCLUSION: TNF-α -308 G/A polymorphism is not associated with DM patients with mild periodontitis, whereas it may have a role in pathogenesis and prognosis of moderate and severe periodontitis combined with DM through TNF-α level.     

采后红富士苹果色泽变化与氧化酶的关系

供试红富士苹果在贮藏初期(1-4周)果皮的花青苷含量有一个再合成过程,且这种合成过程无需光照条 件。不同的贮藏温度条件下花青苷合成发生时期不一样。20℃条件下,果皮花青苷含量第2周增加了31.2％。从第3 周开始,果皮的花青苷含量急剧下降;0℃条件下,果皮的花青苷含量第4周有增加达到最高峰。不同贮藏温度条件 下果实的花青苷的降解速度也存在较大差异,在高温(20℃)条件下贮藏,SOD活性变化与苹果果皮内花青苷含量的 变化相吻合,表明SOD可减缓苹果果皮内花青苷的降解。POD存在2种调控机制:在贮藏前期,果实具有较高的 POD活性,能够清除H2O2,起保护反应。贮藏后期POD活性高促进了果实的衰老。而低温(0℃)条件下贮藏,除SOD 酶在贮藏初期增加外,其他相关酶活性在贮藏后期均保持较低水平,变幅较小,果实的花青苷含量在整个贮藏期间 变化不显著。     

Relationship between bone mineral density and polymorphism of vitamin D receptor gene in postmenopausal women in Guangzhou

AIM:To investigate the prevalence of vitamin D receptor (VDR) gene polymorphism in Guangzhou postmenopausal women and to study the relationship between bone mineral density (BMD) and VDR gene polymorphism.METHODS:The genotype of VDR gene of 203 postmenopausal women in Guangzhou was detected by polymerase chain reaction-restriction fragment length polymorphism. BMD of lumbar spine, femoral neck, troch and Wards triangle were measured by dual-energy X-ray absorptiometry. RESULTS:The distribution of VDR in 203 subjects was BB genotype 17(8.3%), Bb 60(29.6%), bb(62.1%), respectively. The B allelic gene frequencies reached 23.05%. The distribution followed the Hardy-Weinberg equilibrium. The difference was found in lumbar spine BMD between bb and the other two genotypes (P<0.05), but no significant difference between Bb and BB genotype (P>0.05). There was no significant difference in BMD of the other region among three genotypes (P>0.05). CONCLUSION:Genotype of VDR is related to BMD, but there is no enough evidence to support genotype of VDR as a genetic marker in predicting the risk of developing osteoporosis in Guangzhou postmenopausal women.