Mitochondrial DNA 8 point mutations in patients with type II diabetes mellitus

AIM: To explore the relationship between various mitochondrial (mt) DNA tRNA Leu (UUR) and ND1 gene mutations and type 2 diabetes mellitus (T2DM) among Chinese in Hubei Province. METHODS: PCR restriction fragment length polymorphism (PCR-RFLP) analysis was used to screen point mutations of mtDNA (3 243, 3 256, 3 290, 3 316, 3 394, 3 421, 3 426, 3 460, 3 593) in 174 T2DM and 207 healthy controls. Then, DNA sequencing, reverse dot blot hybridization and Genchip were used to compare and confirm mutations. All mutations were analyzed by DNASTAR and Antherprot softwares. RESULTS: In diabetic group, there were 5 carriers (2.9%) of 3 316 G→A (Ala→Thr) mutation, 4 (2.3%) of 3 394 T→C (Tyr→His) mutation, 1 (0.6%) of 3 593 T→C(Val→Ala) mutation, and 1 (0.6%) of 3 618 T→C(Phe→Phe) mutation. Among 3 316 (G→A) mutations , there were more than 1 point mutations in 2 cases, one accompanied with 3 256 C→T(Arg→Arg) and 3 688 G→C (Ala→Pro) mutations, another accompanied with 3 606 A→G(Leu→Leu) mutation. 3 606 (A→G), 3 618 (T→C) and 3 688 (G→C) were novel mutations, GenBank accession number is DQ092356. In controls, only 3 316 (G→A) mutation was found in 1 subject (0.5%). There was significant difference between two groups for 3 394 (T→C) mutation frequencies (P<0.05). CONCLUSION: mtDNA ND1 gene mutations at np 3 316 and np 3 688, np 3 394 might contribute to the pathogenesis of T2DM with other genetic and environmental factors.     

深绿木霉T95和球毛壳ND35混用对土传病害的防治效果

用深绿木霉T95和球毛壳ND35两种拮抗菌株以及两者的混合菌株对由瓜果腐霉Pa和立枯丝核菌Rs引起的猝倒病和立枯病的防治效果进行了初步研究。平皿对峙结果表明,对瓜果腐霉Pa抑制作用最大的为ND35+T95混合拮抗菌,生长抑制率为73.36%,高于拮抗菌ND35对瓜果腐霉Pa的生长抑制率47.87%。而对立枯丝核菌Rs的拮抗作用三者之间差异不显著。盆栽试验结果表明,在菜豆上ND35+T95混合菌株对猝倒病的防治效果高于单个菌株深绿木霉T95,但在黄瓜、番茄上差异不显著。田间试验结果表明,混合拮抗菌株ND35+T95组合对立枯病和猝倒病的防治效果分别为49.05%和60.37%,均高于单一拮抗菌株深绿木霉T95的防治效果,但与对照化学药剂恶霉灵相比存在一定的差异。     

球毛壳ND35菌肥对苹果连作土壤微生物和平邑甜茶幼苗生物量的影响

以平邑甜茶（Malus hupehensis Rehd.）幼苗为试材,在连作土盆栽条件下探讨了内生真菌球毛壳（Chaetomium globosmn）ND35菌肥对其生长和土壤环境的影响。试验共设3个处理：空白对照、连作土壤施加2%菌糠和连作土壤中施加2%球毛壳菌ND35菌肥（即菌糠 + ND35菌株）。结果表明：与对照相比,施加2%菌糠和ND35菌肥对平邑甜茶植株的生物量均有不同程度的促进作用,其中菌肥效果最为明显,施加2%菌肥的7月植株干、鲜样质量分别增加了71.9%、71.6%,9月增加了155.9%、153.1%,9月施加2%菌肥的效果比菌糠更加明显。施加2%菌糠和ND35菌肥均增加了土壤中微生物数量,且随着处理时间的增长,土壤细菌和放线菌数量均呈增加趋势,9月最多。其中施加2%菌肥增幅最高,7、8、9月细菌数量分别比对照增加了245.9%、89.1%、216.3%,放线菌数量分别比对照增加了68.7%、124.7%、131.1%。两种处理均增加了土壤酶的活性,8月的土壤脲酶、蔗糖酶和磷酸酶活性最高,其中以施加2%菌肥的土壤酶活性增幅最为明显。施加2%菌肥还增加了平邑甜茶植株的根系活力,7、8、9月分别比对照增加了59.0%、50.2%、20.2%。与对照和施加菌糠处理相比,球毛壳ND35菌肥可以更好地改善苹果连作土壤微生物区系（microflora）,提高土壤酶活性,增加平邑甜茶幼苗根系活力和生物量。     

Cryopreservation of round-headed human sperm

AIM: To investigate the cryopreservation of round-headed (globozoospermia) human sperm. METHODS: Round-headed sperm samples (n=13) were obtained from 6 infertile men. Yolk-free cryoprotective medium and the two-step freezing were used in cryopreserving samples. Frozen sperm functions were examined by using motility evaluation, sperm head-tail membrane integrity test, and survival time test. RESULTS:The results showed that sperm motility, membrane integrity rates, survival time in vitro significantly lowered after thawing (n=13, P＜0.01). No acrosomal proteinase activity was detected in both fresh and frozen sperm groups (n=5). There were no significant difference in post-thawing motility between yolk-free cryoprotective medium and yolk medium, and between two-step freezing and multiple-step freezing (n=7, P＞0.05). CONCLUSION: Human round-headed sperm could undergo the freezing-thawing process. However, cryo-survival rates were low. Frozen sperm functions were significantly decreased. Yolk-free cryoprotective medium and two-step freezing could be used in the cryopreservation of round-headed sperm.     

Effects of aging on sperm maturation and fertility in mice

AIM: To investigate the effects of aging on sperm maturation and fertility in mice. METHODS: Sperm of caput epididymides and cauda epididymides were obtained from Kunming mice aged 6 months (n=15, as control) and 18 months (n=15). Sperm parameters including sperm density, viability, motility and normal morphological rate were recorded. Sperm of cauda epididymides was observed by transmission electron microscope. The fertility potential and embryo developmental competence were performed by in vitro fertilization and embryo culture. RESULTS: Sperm motility, density and normal morphological rate in aged mice were lower than those in control (P<0.05). The fertilization rate and embryo developmental rate of aged group were significantly lower than those in control (P<0.01). CONCLUSION: Aging influences spermatogenesis in testes and sperm maturation in epididymides. Mouse would be served as a good model for male reproductive aging research.     

One novel mutation and copy number variation of NF1 gene in Chinese patients with type 1 neurofibromatosis

AIM: To investigate the mutations and the copy number variation of neurofibromatosis 1(NF1) gene in 2 sporadic patients with type 1 neurofibromatosis in China. METHODS: All coding exons and exon-intron boundaries of NF1 were amplified by PCR. The PCR products were sequenced. The DNA samples from 50 normal subjects were also sequenced for control. Multiplex ligation-dependent probe amplification (MLPA) was also employed to detect the copy number variation of NF1 gene in these patients. Long range PCR was used for the identification of the breakpoint in the large deletion of the gene. RESULTS: The novel mutation, c. 6345_6346 ins G (p. Leu2116Alafs*4), was detected in patient S736. This mutation was absent in her parents and the controls, indicating a de novo mutation. It caused open reading frame shifting, introducing a premature stop codon and resulting in the truncation of the 721 amino acids at the C terminus of the wild-type protein. This truncation cut off part of the armadillo (ARM)-type fold domain in the wild-type protein. A 1.3~1.9 Mb deletion of the gene was also detected in the other patient S743. The deletion spanned the whole NF1 gene and part of the flanking regions in both ends, but the breaking point was still unknown. CONCLUSION: We have identified a novel mutation of NF1, c.6345_6346 ins G (p.Leu2116Alafs*4). We also first report the copy number variation of NF1 gene in Chinese patients. The investigation will be helpful for the molecular diagnosis and understanding the pathogenesis of the disease.     

Study on the association of the polymorphism at the position -418A/G and -384C/T in the Apo(a) promoter

AIM: To investigate two single nucleotide polymorphisms (SNP) in the apolipoprotein(a) promoter at positions -418 and -384 and to compare distributing difference of genotype frequencies of single nucleotide among different races and to explore the influencies of them on serum lipid level and their association with coronary heart disease (CHD). METHODS: Using PCR-RFLP (BsgI,BfaI) method, we determined genotypes of these two SNPs in 156 unrelated healthy controls of HanZu Chinese and 56 unrelated CHD patients of HanZu Chinese and 56 unrelated African Blacks, then cloned polymerase chain reaction (PCR) products into T-vector and sequenced it by M13 currency primer, correspondingly. RESULTS: (1) There was no polymorphism at position -418A/A and -384C/C in control group. Only one CHD patient's genotype determined was -418G/G, other were -418A/A and -384C/C in CHD patients. (2) Only two African Blacks' genotype determined was -418G/G, other were -418A/A and -384C/C in African Blacks. (3) However, the Apo(a) promoter sequence was in coincident with the sequence publicized in GenBank and the base at positions -418 was adenine (A) and -384 was cytosine (C). CONCLUSION: The mutation frequencies at position -418 and -384 were low in the Chinese Han Population of Hubei and perhaps no single nucleotide polymorphisms was at two positions. No association with serum lipid levels and CHD was observed. There were great variabilities to the SNPs in the Apo(a) promoter among different races.     

Effects of ethanol on HBV gene mutations in patients with chronic hepatitis B by gene chip

AIM: To study ethanol influence on gene mutations of HBV DNA and to offer testimony for clinical diagnosis and treatment of chronic hepatitis B. METHODS: 85 patients with chronic hepatitis B were divided into alcoholic group and non-alcoholic group. Gene chip technique was used to detect gene mutations located in Pre-C nt G1896A and nt A1814C, basal core promoter (BCP) nt A1762T and nt G1764A, P nt C528A and nt T552C. RESULTS: The mutation frequency on BCP nt A1762T and nt G1764A in alcoholic group was significantly higher than that in non-alcoholic group (P<0.05). No difference of mutation frequency on pre-c nt G1896A nt A1814C and P nt C528A nt T552C between alcoholic and non-alcoholic group was observed (P>0.05). CONCLUSION: Ethanol stimulates HBV gene mutations on BCP nt A1762T and nt G1764A, enhances HBV DNA replication and gene expression, deteriorates the state of the illness.     

氮离子注入黄瓜诱变遗传育种研究

研究了不同氮离子注入黄瓜干种子次数后诱变效应的变化。结果表明:经过氮离子注入处理后的黄瓜均出现了较明显的突变,并且经过2次氮离子注入诱变处理的突变率高于只经过一次氮离子注入诱变处理7.9个百分点,2个处理中黄瓜M1代出现多种类型明显变异植株,M2代在瓜形突变上出现较大分离,M3代密刺瘤变异和瓜条变短变异遗传稳定,最高遗传率达到99.3%,并且得到了黄瓜商品性状改变优良的突变系。     

EMS 诱变番茄自交系TTD302A 的突变表型鉴定和分析

为创制用于番茄遗传育种和基因功能研究的新种质,用0.7% 的甲基磺酸乙酯（EMS）浸泡处理TTD302A 种子 8 h,清水冲洗后催芽育苗,定植于塑料大棚中,单株观察、单株留种。对M2 群体进行株系和单株的系统观察,表型表现一致的株系混合留种,有表型变异的单株进行单株留种,获得M3 种子。另外选取了15 份出现典型变异性状的M2 材料进行SSR 检测。通过对M2 群体表型性状的观察,共发现373 个变异性状,297 个变异单株,总的单株变异频率为7.1%。叶、花、果实和植株的表型变异频率依次为1.5%、2.8%、1.3% 和2.4%。SSR 分析结果表明9 份材料在 DNA 水平上有变异。     

主干形和开心形桃树的生产潜力研究(初报)

调查了湖南红壤地区主干形和开心形幼龄桃树生长,结果状况及树体光照,夏季有效光合的情况。结果表明：主干形桃树树势易缓和,成花多,修剪量轻,早期产量高。在夏季,主干形比开心形有效光合高,上层叶幕的有效光合峰值出现在上午１０时,中下层出现在中午１２时;开心形上中层峰值出现在中午１２时,下层峰值出现在下午２时,尽管开心光照比主干形好,但夏季叶片的有效光合并未提高。     

Effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells

AIM: To observe the effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells. METHODS: Both hepatocellular carcinoma cells (H2M) and epidermoid carcinoma cells (A431) with methylation of TIMP-3 promoter gene were treated with 5-Aza-2'-deoxycytidine (5Aza-dc). Invasion ability and motility of the cells were detected by Transwell experiments. Expressions of TIMP-3 protein and mRNA were detected by Western blotting and RT-PCR, respectively. TIMP-3 gene promoter methylation was detected by methylation-specific PCR (MSP). RESULTS: ① Invasion ability and motility of H2M and A431 cells were declined after treatment with 5Aza-dc; ② After treatment with 5Aza-dc, the expression of TIMP-3 protein and mRNA were increased in H2M and A431 cells; ③ After treatment with 5Aza-dc, methylation of TIMP-3 promoter gene was not detectable in the cell lines. CONCLUSIONS: 5Aza-dc induces demethylation in TIMP-3 promoter gene, restores TIMP-3 gene and protein expression, and inhibits invasion ability and motility of the carcinoma cells.     

Relationship of subclasses of serum HDL and Apo A-Ⅰ gene polymorphism in hyperlipidemia

AIM: To investigate apolipoprotein A-Ⅰ gene (Apo A-Ⅰ) polymorphism and its relationship with serum HDL subclasses in patients with hyperlipidemia (HL). METHODS: Apo A-Ⅰ genotype was assayed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The subclasses of serum HDL in 118 patients with hyperlipidemia and 109 healthy subjects were determined by two-dimensional gel electrophoresis conjunction with immunodetection method. RESULTS: Both in HL group and the control group, G/G and C/C genotypes were the most frequent at －78 bp and ＋83 bp of Apo A-Ⅰ gene, respectively. The frequency of rare A allele at －78 bp in HL group was significantly higher than that in control group. In HL group, subjects with G/A mutation had higher serum levels of TG, Apo C-Ⅲ, pre β1-HDL and HDL3a, and lower levels of HDL2a and HDL2b compared to the subjects with G/G genotype. CONCLUSION: The G/A transition in the －78 bp position of the Apo A-Ⅰ gene promoter in patients with hyperlipidemia is associated with HDL subclasses. There is a general shift toward smaller sized HDL, which, in turn, indicates that HDL maturation might be abnormal.     

方斑网衣的分子鉴定和系统发育学分析

为鉴定方斑网衣中的隐存种,以新疆地区的方斑网衣为试验材料,用分子系统发育学方法,研究了方斑网衣核糖体DNA内转录间隔区(internal transcribed spacer,ITS)碱基组成信息、变异位点以及简约信息位点、种内的遗传距离、遗传距离聚类情况、ITS位点系统发育树分析以及基于条形码间隔自动检索鉴定方斑网衣隐存种类。结果表明:方斑网衣ITS基因片段长度为528 bp,其中62个为变异位点、43个为简约位点、460个为保守位点、G+C碱基组成占总含量的56.1%、G+C含量高于A+T含量、单倍型多样性(haplotype(gene)diversity,Hd)为0.993、核酸多样性(nucleotide diversity,Pi)为0.048 76;基于遗传距离构建的聚类树同系统发育树分支聚类情况一致,分为2个大分支;通过条形码自动检索法鉴定出方斑网衣可为2个种。通过综合分析鉴定出方斑网衣2个隐存种即方斑网衣原种(Lecidea tessellata var.tessellata)和方斑网衣蓝变种(Lecidea tessellata var.caesia)。     

盐胁迫对莴苣种子萌发和幼苗生长的影响

通过研究不同浓度(50、100、150、200 mmol·L-1)NaCl胁迫对莴苣种子萌发特性和幼苗生长的影响,结果表明:50 mmol·L-1NaCl胁迫下,华艺奶油莴苣的发芽率、发芽势和发芽指数比对照略有增加,随着盐浓度的升高,发芽率、发芽势、发芽指数和活力指数都有不同程度的下降.200 mmol·L-1 NaCl胁迫下种子萌发完全受到抑制.随着盐浓度的增加,根和芽的长度和鲜重NaCl胁迫也表现为不同程度的降低,且根的下降幅度比胚芽大,说明盐胁迫对根的抑制作用大于芽.     

Relationship between RUNX3 gene 364 locus C→T mutation and gastric cancer in Chinese

AIM: To study the frequency difference of RUNX3 gene 364 locus C→T mutation between normal people(controls) and gastric cancer (GC) patients, and mutation in gastric mucosa of subjects with H.pylori infection. METHODS: Genomic DNA was extracted from peripheral blood and gastric mucosal biopsy specimens of normal people and GC patients in lower or higher prevalence region. Gene mutation was analyzed by PCR-RFLP. RESULTS: The frequency of RUNX3 T/T genotype was no significant difference between controls and GC in lower (χ2=0.57, P>0.05) or higher prevalence region (χ2=0.16, P>0.05). A higher mutation rate in mucosal tissue infected with H.pylori was not discovered. CONCLUSION: RUNX3 gene C364T mutation may be not a genetic susceptibility to GC in Chinese. The mutation is impossibly involved in the pathway of H.pylori infection resulting in gastric carcinoma.     

以马铃薯ND2 mRNA为内对照双重RT PCR法检测马铃薯纺锤块茎类病毒

 根据马铃薯纺锤块茎类病毒（PSTVd）基因序列设计特异引物,采用反转录聚合酶链式反应（RT-PCR）技术进行PSTVd检测研究。为避免由于提取的RNA降解或者RT-PCR反应质量不高所造成的假阴性问题，在检测过程中引入马铃薯线粒体NADH脱氢酶ND2亚基基因mRNA为内对照，该内对照的一个引物跨越了该基因内含子区域，只对剪接后的mRNA进行特异扩增而不扩增其本身DNA。利用内对照引物和PSTVd特异引物进行双重RT-PCR检测，分别扩增出190 bp的内对照基因特异片段和360 bp的PSTVd特异条带，与预期引物设计大小一致。引入的内对照可以较好地监控PSTVd的RT-PCR检测过程。     

Mutation of ATP-sensitive K+ channels and neonatal diabetes iDEND syndrome

Activating mutation in the KCNJ11 gene encoding Kir6.2 subunit of adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channel gives rise to intermediate developmental delay, epilepsy and neonatal diabetes (iDEND) syndrome, a rare hereditary endocrine metabolic disorder characterized by neonatal diabetes accompanied by developmental delay and muscle weakness, but no epilepsy. The Kir6.2 Val⁵⁹→Met⁵⁹ (V59M) activating mutation is the common cause of iDEND syndrome (>50%). Activating mutation causes iDEND syndrome by inhibiting normal closure of ATP-sensitive K⁺ channel, which leads to reduce insulin secretion. Most of such patients are more sensitive to sulfonylurea. High blood-brain barrier permeability and sulfonylurea receptor 1 (SUR1)-specific drugs are expected to become a major therapy.     

超干燥处理对豌豆种子抗氧化系统酶及热稳定蛋白的影响

 将含水量为12.2% , 11.3% , 8% , 5% , 4% , 3%的碗豆种子置于50℃干燥箱中人工老化,定期测定老化种子生活力和活力指标, 抗氧化系统酶活性及热稳定蛋白。结果表明: 与对照相比, 含水量4%和3%超干燥种子的活力指数、根干样质量显著下降, 而含水量8% , 5%的种子活力指数、发芽率和根干样质量没有出现明显下降。POD活性和CAT活性与种子的发芽率、活力呈正相关, 当老化处理6个月后, 含水量3%和4%的POD活性和CAT活性低于对照, 但含水量5%和8%的高于对照。超干燥种子中没有发现新的热稳定蛋白谱带, 但含水量为8%和5%的种子其热稳定蛋白质的含量增加。     

Application of PCR-HRM in the study of relationship between leptin gene promoter polymorphisms and liver cirrhosis

AIM: To analyze the feasibility of PCR-high-resolution melting curve analysis (HRM) for detecting the site mutations of C2549 and G2548 in leptin gene promoter from the patients with liver cirrhosis, and to explore the relevance between mutant genotypes and physiological and biochemical indexes in liver cirrhosis patients. METHODS: Compared with the method of PCR-restriction fragment length polymorphism (RFLP), the present research used the method of PCR-HRM to analyze the site mutations of C2549 and G2548 in leptin gene promoter in control group (n=100) and liver cirrhosis group (n=100). The physiological and biochemical indexes of the patients were also detected and compared. RESULTS: Leptin gene promoter polymorphism was detected using PCR-HRM with effectiveness, high flux and accuracy. Preliminary results showed that the main mutation of the patients with liver cirrhosis was in C2549 site, but not found in G2548 site. Leptin, free leptin index (FLI), fasting insulin (FINS) and insulin resistance index estimated by homeostatic model assessment (HOMA-IR) in liver cirrhosis group were higher than those in control group. Insulin sensitivity index (ISI) and soluble leptin receptor (sOB-R) in liver cirrhosis group were lower than those in control group with significant difference except leptin level. Meanwhile, FLI showed positively correlated with FINS and HOMA-IR (r=0.45, r=0.53, P<0.05), and negatively with ISI (r=-0.34, P<0.05). In the patients with liver cirrhosis, C2549A heterozygous mutation was predominant. The indexes of HOMI-IR, leptin, sOB-R and FLI of C2549A homozygotes and heterozygotes were higher than those of the wildtypes, which showed significant difference except leptin and sOB-R levels (P<0.05). CONCLUSION: PCR-HRM can be more accurate for identifying leptin promoter polymorphism. The increase in the frequency of C2549A mutation may be closely related with liver cirrhosis. Existence of hyperinsulinemia and insulin resistance may be correlated with leptin level in the patients with liver cirrhosis.