人红细胞葡萄糖-6-磷酸脱氢酶分离纯化方法的改进

葡萄糖－６－磷酸脱氢酶（Ｇ－６－ＰＤ）是磷酸戊糖途径的限速酶。传统的以ＮＡＤＰ－Ｓｅｐｈａｒｏｓｅ４Ｂ作为亲和吸附剂的纯化方法由于较昂贵的ＮＡＤＰ－Ｓｅｐｈａｒｏｓｅ４Ｂ用量多,且耗时长,为此,作者对此传统方法进行了改进。报道如下：１材料与方法１．１...     

甜杨葡萄糖-6-磷酸脱氢酶基因的电子克隆及其功能预测

［目的］对甜杨葡萄糖-6-磷酸脱氢酶（G6PDH）进行克隆及功能预测。［方法］采用电子克隆和RT-PCR相结合的方法首次从甜杨中分离了G6PDH基因,通过Blast和其他生物信息学软件进行功能预测。［结果］甜杨G6PDH基因的cDNA长1697bp,可编码510个氨基酸,cDNA序列及其推导的氨基酸序列均与其他植物G6PDH存在着较高的同源性,说明所获得的cDNA是甜杨G6PDH基因（PsG6PDH,AY445917）。同时,运用PCR扩增技术获得了甜杨G6PDH基因组序列,测序结果表明,基因组DNA长度为5 040bp,含有15个外显子和14个内含子。Southern杂交结果表明,G6PDH基因在甜杨的基因组中可能是单拷贝或者低拷贝。通过网络服务器平台进行PsG6DH的功能预测,结果显示,PsG6PDH为G6PDH的成员之一,含有NADP结合区和G6P结合区2个保守功能域,具有多个磷酸化位点和跨膜区域,但不含有信号肽,表明PsG6DH可能作为膜受体而起作用。另外,G6PDH的电子表达谱分析结果发现,G6PDH在多种组织和不同发育过程均表达,并涉及各种逆境胁迫应答反应,这也说明了G6PDH在植物生长反育中的重要地位。［结论］可为下一步研究PsG6PDH基因的分子功能提供参考。     

光疗对G—6PD缺陷新生儿高胆红素血症的副效应观察

目的：观察光疗对红细胞葡萄糖６－磷酸脱氢酶（Ｇ－６ＰＤ）缺陷高胆红素血症（高胆）新生儿的不良影响。方法：对８８例Ｇ－６ＰＤ缺陷高胆儿和２１２例非Ｇ－６ＰＤ缺陷高胆儿行光疗的退黄时间、反跳及继续溶血情况作了观察。结果：Ｇ－６ＰＤ缺陷组退黄时间明显长于非Ｇ－６ＰＤ缺陷组，反跳现象及光疗后进行性溶血倾向均明显高于非Ｇ－６ＰＤ缺陷患儿（Ｐ〈０．０１）。结论：Ｇ－６ＰＤ缺陷患儿对光疗的溶血效应可能更为敏感，应从严掌握光疗指征。     

桉树葡萄糖-6-磷酸脱氢酶（G6PDH）的全基因组分析与进化研究

[目的]对桉树基因组中葡萄糖-6-磷酸脱氢酶（G6PDH）进行全基因组分析与进化研究。[方法]利用巨桉（Eucalyptus grandsis）全基因组数据,采用BLAST等生物信息学软件分析桉树G6PDH的基因特性、蛋白序列、系统进化树以及启动子特征。[结果]桉树基因组内存在6个G6PDH基因,其编码蛋白中,1个为胞质型G6PDH,5个为质体型G6PDH,而且桉树G6PDH均含有保守基序motif1、motif2、motif3、motif7、motif9和motif11。此外,桉树G6PDH启动子序列中均存在TATA框、增强子与涉及光应答、激素应答、胁迫应答等调控元件。[结论]可为下一步研究桉树G6PDH家族基因的分子功能提供参考。     

桉树葡萄糖-6-磷酸脱氢酶(G6PDH)的全基因组分析与进化研究

[目的]对桉树基因组中葡萄糖-6-磷酸脱氢酶（G6PDH）进行全基因组分析与进化研究。[方法]利用巨桉（Eucalyptus grandsis）全基因组数据,采用BLAST等生物信息学软件分析桉树G6PDH的基因特性、蛋白序列、系统进化树以及启动子特征。[结果]桉树基因组内存在6个G6PDH基因,其编码蛋白中,1个为胞质型G6PDH,5个为质体型G6PDH,而且桉树G6PDH均含有保守基序motif 1、motif 2、motif 3、motif 7、motif 9和motif 11。此外,桉树G6PDH启动子序列中均存在TATA框、增强子与涉及光应答、激素应答、胁迫应答等调控元件。[结论]可为下一步研究桉树G6PDH家族基因的分子功能提供参考。     

红麻6-磷酸葡萄糖酸脱氢酶(6PDGH)基因克隆及RNAi载体构建

[目的]克隆红麻不育系和保持系6-磷酸葡萄糖酸脱氢酶(6PDGH)基因,并构建其RNAi载体,为进一步研究其在红麻花药发育中的功能奠定基础.[方法]根据红麻花药转录组高通量测序数据结果和生物信息学方法获得6PDGH基因全长cDNA拼接序列,分别以红麻不育系(P3A)和保持系(P3B)的花药反转录cDNA及总DNA为模板克隆红麻6PGDH基因全长.依据RNAi载体设计原则,选取红麻6PGDH基因cDNA序列中段389bp的特异片段做为RNA干扰片段,构建红麻6PGDH因的RNAi载体.[结果]克隆获得的红麻不育系(P3A)和保持系(P3B)花药6PGDH基因全长均为1751 bp,均包含1458bp的开放阅读框,编码485个氨基酸残基,无内含子;其碱基序列在不育系和保持系中仅存在两个碱基差异.该基因氨基酸序列(GenBank登录号KF964027)与拟南芥、玉米、大豆、苜蓿、三角叶杨、蓖麻的6PDGH同源性分别为75.07%、84.57%、86.50%、87.24%、91.19%和92.01%.成功构建了红麻6PGDH基因的RNAi载体,获得了干扰表达载体pART27-pKANNIBAL-R1+2.[结论]成功克隆获得红麻6PGDH基因全长序列,构建的干扰表达载体可用于红麻6PGDH基因的功能研究.     

黄瓜6-磷酸葡萄糖酸脱氢酶基因cDNA片段的克隆及表达分析

以黄瓜品种露丰为材料,根据已报道的6-磷酸葡萄糖酸脱氢酶基因(6PGDH)的保守氨基酸序列设计简并引物,利用RT-PCR技术获得黄瓜6PGDH的cDNA同源片段,命名为CSPG(登录号为EU815934)。该片段长度为1 207 bp,包含一个936 bp的开放阅读框(编码311个氨基酸)和271 bp的Poly A 3′非翻译区末端,无内含子;该基因编码的氨基酸序列与拟南芥、大豆、水稻、玉米、菠菜的6PGDH基因有75%以上的同源性。运用半定量RT-PCR技术对6PGDH基因的转录水平进行分析,结果表明:该基因在叶、根、茎中均有表达,高温胁迫下的表达量高于常温对照,说明6PGDH基因与热胁迫相关。     

脑干听觉诱发电位测定在新生儿重度高胆红素血症中的意义

目的了解脑干听觉诱发电位（BAEP）测定对新生儿重度高胆红素血症预后评估的临床意义。方法将120例高胆红素血症患儿分为A组（66例,总胆红素值为220-340 mol/L）和B组（54例,总胆红素值〉340 mol/L）,两组分别于入院后48 h内进行BAEP检测;并与40例健康新生儿（C组）进行对照分析。结果 A、B两组与C组比较,BAEP各波段的绝对潜伏期PL、峰间潜伏期IPL和V波反应阈值均显著升高,且B组升高更明显,差异均有统计学意义（P〈0.05或0.01）。结论BAEP异常是早期诊断新生儿胆红素脑病的重要指标,为其治疗及预后评估提供了重要依据。     

米曲霉6-磷酸葡萄糖酸脱氢酶基因(gnd)的克隆及生物信息学分析

采用PCR技术从米曲霉CICC2012菌株基因组中克隆6-磷酸葡萄糖酸脱氢酶基因(gnd),并利用生物信息学手段对其氨基酸序列、进化树、理化性质、蛋白质结构等进行分析.序列测定和分析结果表明.gnd基因序列长为1 723 bp.包含1个1 551 bp的开放阅读框,编码516个氨基酸;gnd基因编码的6PGDH氨基酸序列与黄曲霉6PGDH基因的同源性为99％,存在的丝氨酸、苏氨酸和酪氨酸磷酸化位点分别有11,2和6个;6PGDH蛋白分子量为57.3 kD,等电点为5.63; gnid基因编码蛋白二级结构α-螺旋区域占44.57％,β-折叠区域占12.79％.无规则卷曲区域占42.64％;氨基酸残基11～195位点为NADP+结合区域.     

大豆幼苗中葡萄糖－6－磷酸脱氢酶对干旱胁迫的生理响应

以2种抗旱性不同的大豆品种（ZD11、YD24）为试验材料,用聚乙二醇（PEG）模拟干旱胁迫条件,探讨不同干旱胁迫条件下葡萄糖－6－磷酸脱氢酶（G6 PDH）活性的动态变化及其在抗旱性中的可能作用。结果显示,干旱胁迫增强了2种大豆品种中G6PDH的活性,而且抗旱品种YD24比对照品种ZD11提高的幅度大,提示了G6PDH可能参与了大豆的抗旱性响应;进一步研究结果显示,G6 PDH抑制剂处理进一步提高了干旱胁迫下大豆根中相对电导率、丙二醛含量;另外,G6 PDH 抑制剂处理进一步抑制了主根的生长以及侧根的数量。这些结果表明,干旱胁迫下G6 PDH在增强大豆的抗旱性中可能起着重要的作用。     

Athens variant of glucose-6-phosphate dehydrogenase

A variant of glucose-6-phosphate dehydrogenase (G6PD), characterized by slower than normal electrophoretic migration and associated with mild deficiency of G6PD in the red cells, was detected in two unrelated Greek males. Electrophoretic, chromatographic, and enzymologic study indicated that the new mutant is structurally different from normal G6PD (B+) and from the Mediterranean variant associated with red-cell enzyme deficiency (B-). Convincing electrophoretic separation of the new variant from the normal B+ and the Mediterranean B- enzymes was achieved only by detailed electrophoretic study in different buffer systems and conditions.     

Alpha-glycerophosphate dehydrogenase and glucose-6-phosphate dehydrogenase in tissues of the Weddell seal

High activity of alpha-glycerophosphate dehydrogenase and low activity of glucose-6-phosphate dehydrogenase in adipose tissue of Weddell seals suggest that neutral fat may be assembled there from exogenous sources of fatty acids. Low activity of glucose-6-phosphate dehydrogenase in other tissues tested precludes assignment of the function of fatty-acid synthesis to any specific tissue and emphasizes uniqueness of adipose mass in seals.     

Erythrocyte acid phosphomonesterase and glucose-6-phosphate dehydrogenase deficiency in Caucasians

Caucasian patients with erythrocyte glucose-6-phosphate dehydrogenase deficiency also have a deficiency in erythrocyte acid phosphomonoesterase. This acid phosphomonoesterase deficiency is not present in Negroes with the glucose-6-phosphate dehydrogenase deficiency.     

Sex-linkage of erythrocyte glucose-6-phosphate dehydrogenase in two species of wild hares

Glucose-6-phosphate dehydrogenase specific to the erythrocytes of each of two wild hares found in Europe was discerned by starch-gel electrophoresis at pH 7.0 and pH 8.6. The single, sharp band of the dehydrogenase of Lepus europaeus was faster than that of L. timidus, at both pH levels. The sex-linkage of this enzyme was tested through reciprocal hybrids between the two species. Each male hybrid had a single band of enzyme identical with that of its mother, while both parental types of this enzyme coexisted in female hybrids. Thus, sex-linkage of glucose-6-phosphate dehydrogenase has been suggested not only in man and in the family Equidae, but now in the family Leporidae of placental mammals as well.     

Negro variant of glucose-6-phosphate dehydrogenase deficiency (A-) in man

Glucose-6-phosphate dehydrogenase in erythrocytes of the Negro type associated with enzyme deficiency (A(-)) was separated by chromatography on a carboxymethyl-Sephadex column from the electrophoretically indistinguishable Negro variant with normal enzyme activity (A(+)). Quantitative immunologic neutralization tests indicated that the A(-) enzyme had about the same enzymatic and serological activity as the A(+) and the normal (B(+)) enzymes. The enzyme activity of the A(-) variant in young erythrocytes was similar to that in young cells from normal individuals, although the activity of the A(-) variant in unfractionated red cells was 10 to 15 percent of normal. These data indicate that the basic defect in the variant enzyme (A(-)) is a structural mutation which causes more rapid degradation of the enzyme during erythrocyte aging.     

Glucose-6-phosphatase and the exchange of glucose with glucose-6-phosphate

An equation is presented which makes it possible to estimate the exchange activity of glucose-6-phosphatase, as a percentage of the hydrolytic activity, for a given concentration of substrate and acceptor. The quantitative significance of the exchange-inhibition phenomenon is discussed.     

Air lead: relation to lead in blood of black school children deficient in glucose-6-phosphate dehydrogenase

Forty-four black children at two elementary schools within 0.7 mile of a battery plant had significantly higher (P < .001) concentrations of lead in their bloods (34.1 +/- 9.7, micrograms per 100 milliliters, mean +/- standard deviation) than 122 students (26.3 +/- 7.1) at seven schools 1 to 3 miles distant; 5 months later there was a comparable difference between red cell lead values (54.1 +/- 18.5 versus 37.4 +/- 12.6). Among the blacks, those deficient in glucose-6-phosphate dehydro-genase had a higher (P < .005) concentration of lead in the blood after correction for anemia (32.9 +/- 9.7) than the nondeficient (25.7 +/- 8.8), and a higher concentration in the red cells (47.3 +/- 14.7 as compared to 35.6 +/- 15.8, P < .001); the enzyme effect was independent of geographic location.     

甜瓜属野生种Cucumis hystrix Chakr.胞质葡萄糖-6-磷酸脱氢酶基因的克隆与启动子结构分析

根据植物葡萄糖-6-磷酸脱氢酶(G6PDH,EC1.1.1.49)氨基酸保守区域,设计简并引物,采用RT-PCR技术克隆得到甜瓜属野生种Cucumis hystrix Chakr.(2n=24)G6PDH基因cDNA片段;随后基于该序列设计特异引物,PCR方法筛选野生种BAC文库,获得2个BAC阳性单克隆。测序后获得了G6PDH基因全序列及其上游启动子序列,GenBank登录号:JQ771576。序列分析显示:G6PDH基因全长约6.5 kb,由15个外显子和14个内含子组成;内含子序列均符合5’-gt-ag-3’结构。外显子拼接后获得了G6PDH基因的开放阅读框(ORF)序列,序列全长1 551 bp,编码516个氨基酸,与黄瓜基因组网站公布的G6PDH基因核苷酸序列和氨基酸序列同源性分别为98.71%和99.03%。野生种G6PDH氨基酸序列N端缺少转运肽序列,确定为野生种胞质G6PDH。氨基酸序列与烟草、马铃薯、荷兰芹、猕猴桃、拟南芥、大豆、小麦、玉米、葡萄、杨树等植物胞质G6PDH同源性高达77%以上。系统发育树分析发现,甜瓜属胞质G6PDH与茄科植物最先聚类,植物胞质G6PDH在分子进化水平上与物种进化相符。启动子结构分析表明:序列含有启动子基本元件TATA-box、CAAT-box,还含有丰富的光响应元件,与环境胁迫响应相关的不同顺式作用元件,促进高水平转录的5UTR Py-rich stretch元件和提高表达的CAT-box元件等。