广州管圆线虫感染小管福寿螺相关基因定量分析及 α-tubulin基因组织表达分析

目的　探究小管福寿螺感染广州管圆线虫后部分基因表达情况,初步了解广州管圆线虫与其中间宿主小管福寿螺之间的相互作用关系,为防治广州管圆线虫病提供基础数据。方法　取含有广州管圆线虫Ⅰ期幼虫的大鼠粪便喂食福寿螺,分别于感染后1、10、20 d各取3～5只小管福寿螺,采集其血淋巴、肝胰腺、肾脏、肠道、头足和鳃组织,以未感染的小管福寿螺为空白对照组,提取不同感染时期小管福寿螺各组织总RNA,逆转录为cDNA。基于前期转录组测序结果,挑选10个涉及免疫防御、信号转导、细胞生长和代谢、应激反应等方面的基因,对小管福寿螺感染广州管圆线虫1、10、20 d的血淋巴进行基因荧光定量表达分析,并对α?微管蛋白（α?tubulin）基因在感染广州管圆线虫后的福寿螺肝胰腺、肾脏、头足部、肠道、鳃组织中的表达水平进行分析。结果　与空白对照组相比,CELA1基因在感染广州管圆线虫后1、10、20 d的福寿螺中表达量上调（t = 12.32、23.51、34.92,P均 < 0.05）,且表达量随感染时间延长而上升;GST基因表达量在感染后1 d为空白对照组的（7.26 ± 1.80）倍,在感染后10、20 d表达水平低于空白对照组（t = 23.89、19.83,P均 < 0.05）;ferritin基因在感染后10 d较空白对照组显著上调（t = 32.76,P < 0.05）。CRT基因表达水平在感染后1、10、20 d均较空白对照组上调（t = 7.23、5.78、6.32,P均 < 0.05）。α?tubulin基因在小管福寿螺肝胰腺组织中的表达水平最高（F = 17.58,P < 0.05）;在小管福寿螺感染广州管圆线虫后,该基因在各组织中的表达量均有不同程度变化,其表达量以肝胰腺组织中下调最为明显（P均 < 0.05）。结论　小管福寿螺感染广州管圆线虫后,血淋巴中多个基因表达水平发生改变,α?tubulin基因表达水平在多个组织中受到抑制。该研究为深入阐明小管福寿螺和广州管圆线虫的相互作用关系奠定了基础。     

广州管圆线虫感染小管福寿螺相关基因定量分析及 α-tubulin基因组织表达分析

目的　探究小管福寿螺感染广州管圆线虫后部分基因表达情况，初步了解广州管圆线虫与其中间宿主小管福寿螺之间的相互作用关系，为防治广州管圆线虫病提供基础数据。方法　取含有广州管圆线虫Ⅰ期幼虫的大鼠粪便喂食福寿螺，分别于感染后1、10、20 d各取3～5只小管福寿螺，采集其血淋巴、肝胰腺、肾脏、肠道、头足和鳃组织，以未感染的小管福寿螺为空白对照组，提取不同感染时期小管福寿螺各组织总RNA，逆转录为cDNA。基于前期转录组测序结果，挑选10个涉及免疫防御、信号转导、细胞生长和代谢、应激反应等方面的基因，对小管福寿螺感染广州管圆线虫1、10、20 d的血淋巴进行基因荧光定量表达分析，并对α?微管蛋白（α?tubulin）基因在感染广州管圆线虫后的福寿螺肝胰腺、肾脏、头足部、肠道、鳃组织中的表达水平进行分析。结果　与空白对照组相比，CELA1基因在感染广州管圆线虫后1、10、20 d的福寿螺中表达量上调（t = 12.32、23.51、34.92，P均 < 0.05），且表达量随感染时间延长而上升；GST基因表达量在感染后1 d为空白对照组的（7.26 ± 1.80）倍，在感染后10、20 d表达水平低于空白对照组（t = 23.89、19.83，P均 < 0.05）；ferritin基因在感染后10 d较空白对照组显著上调（t = 32.76，P < 0.05）。CRT基因表达水平在感染后1、10、20 d均较空白对照组上调（t = 7.23、5.78、6.32，P均 < 0.05）。α?tubulin基因在小管福寿螺肝胰腺组织中的表达水平最高（F = 17.58，P < 0.05）；在小管福寿螺感染广州管圆线虫后，该基因在各组织中的表达量均有不同程度变化，其表达量以肝胰腺组织中下调最为明显（P均 < 0.05）。结论　小管福寿螺感染广州管圆线虫后，血淋巴中多个基因表达水平发生改变，α?tubulin基因表达水平在多个组织中受到抑制。该研究为深入阐明小管福寿螺和广州管圆线虫的相互作用关系奠定了基础。     

Glycogen storage disease type 1b due to a defect of glucose-6-phosphate translocase

Patients with glycogen storage disease (GSD) type 1b have shown normal activity of glucose-6-phosphatase (EC 3.1.3.9) as assayed in frozen liver, though their clinical and biochemical findings were similar to those of patients with GSD 1a (McKusick 23220) (Senior and Loridan, 1968). In 1978, we suggested that a basic defect of GSD 1b exists in the glucose-6-phosphate (G6P) transport system (Narisawa et al., 1978; Igarashiet al., 1979). Since then, there have been reports confirming our observation (Beaudetet al., 1980; Langeet al., 1980; Corbeelet al., 1981; Schaubet al., 1981). Recently, it was postulated that the G6Pase system contains a phosphate translocase which mediates the efflux of phosphate, in addition to a G6P translocase and a non-specific phosphohydrolase (Arionet al., 1980). Therefore, it is possible that GSD 1b is caused by a defect of phosphate translocase. In this paper, the basic defect in GSD type 1b was investigated in two patients; one with severe, the other with mild, clinical symptoms.     

Quantitative evolutionary design of glucose 6-phosphate dehydrogenase expression in human erythrocytes

Why do the activities of some enzymes greatly exceed the flux capacity of the embedding pathways? This is a puzzling open problem in quantitative evolutionary design. In this work we investigate reasons for high expression of a thoroughly characterized enzyme: glucose 6-phosphate dehydrogenase (G6PD) in human erythrocytes. G6PD catalyses the first step of the pathway that supplies NADPH for antioxidant defense mechanisms. Normal G6PD activity far exceeds the capacity of human erythrocytes for a steady NADPH supply, which is limited upstream of G6PD. However, the distribution of erythrocyte G6PD activity in human populations reveals a selective pressure for maintaining high activity. To clarify the nature of this selective pressure, we studied how G6PD activity and other parameters in a model of the NADPH redox cycle affect metabolic performance. Our analysis indicates that normal G6PD activity is sufficient but not superfluous to avoid NADPH depletion and ensure timely adaptation of the NADPH supply during pulses of oxidative load such as those that occur during adherence of erythrocytes to phagocytes. These results suggest that large excess capacities found in some biochemical and physiological systems, rather than representing large safety factors, may reflect a close match of system design to unscrutinized performance requirements. Understanding quantitative evolutionary design thus calls for careful consideration of the various performance specifications that biological components/processes must meet in order for the organism to be fit. The biochemical systems framework used in this paper is generally applicable for such a detailed examination of the quantitative evolutionary design of gene expression levels in other systems.     

Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.     

Hpt, a bacterial homolog of the microsomal glucose- 6-phosphate translocase, mediates rapid intracellular proliferation in Listeria

Efficient replication in vivo is essential for a microparasite to colonize its host and the understanding of the molecular mechanisms by which microbial pathogens grow within host tissues can lead to the discovery of novel therapies to treat infection. Here we present evidence that the foodborne bacterial pathogen Listeria monocytogenes, a facultative intracellular parasite, exploits hexose phosphates (HP) from the host cell as a source of carbon and energy to fuel fast intracellular growth. HP uptake is mediated by Hpt, a bacterial homolog of the mammalian translocase that transports glucose-6-phosphate from the cytosol into the endoplasmic reticulum in the final step of gluconeogenesis and glycogenolysis. Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA, which upon entry into host cells induces a set of virulence factors required for listerial intracellular parasitism. Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence in mice. Hpt is the first virulence factor to be identified as specifically involved in the replication phase of a facultative intracellular pathogen. It is also a clear example of how adaptation to intracellular parasitism by microbial pathogens involves mimicry of physiological mechanisms of their eukaryotic host cells.     

Cytokine gene expression in regenerating haematopoietic tissues of mice after cyclophosphamide treatment

The goal of the study was to investigate changes in expression of selected growth factors tentatively involved in regeneration of haematopoietic tissues (bone marrow and spleen) following cyclophosphamide (CY) damage in the mouse. The bone marrow (BM) and spleen were examined separately, since the regenerating pattern for haematopoietic progenitor cells (HPC) markedly differs in these two haematopoietically active organs after CY. Cytokines assumed to have a stimulatory effect on HPC - stem cell factor (SCF), fetal liver tyrosine kinase 3-ligand (flt3-ligand), thrombopoietin (TPO), stromal cell-derived factor 1 (SDF-1), oncostatin M (OSM) -, a suppressive effect on HPC proliferation - macrophage inflammatory protein-1alpha (MIP-1alpha), transforming growth factor-beta1 (TGFbeta1), tumour necrosis factor-alpha (TNFalpha) - and to be involved in migration of HPC (SCF, flt3-ligand, MIP1alpha, SDF-1) were examined at the level of mRNA expression by means of real-time RT-PCR. The expression of a particular cytokine appears to be similar in both BM and spleen of untreated mice. CY administration changed the expression pattern of the studied genes in BM and spleen. In BM, the levels of mRNAs for SCF and SDF-1 were increased and that for TGFbeta1 decreased at time intervals at which HPC are known to proliferate intensively during BM regeneration. In contrast, stimulated proliferation of HPC in spleen was accompanied by increased expression of flt3-ligand and oncostatin M. Upon mobilization of HPC from BM into blood after CY, the expression of SCF, TPO, SDF-1 and TGFbeta1 tends to decrease in BM. Accumulation of HPC in spleen is accompanied by increased mRNA for flt3-ligand and OSM. Our findings demonstrate that different cytokines may be involved in the proliferation and mobilization/homing of HPC during recovery after CY damage in BM and spleen.     

贵州西江苗族葡萄糖-6-磷酸脱氢酶基因突变分析

目的了解贵州西江苗族葡萄糖-6-磷酸脱氢酶(G6PD)缺陷症的基因频率、基因突变类型特点及分布特征，从分子水平揭示G6PD基因多态性。方法世代居住当地苗族男性431人，用四氮唑蓝(NBT)纸片定性法及G6PD／6PGD比值法作G6PD缺陷症筛查。确诊者用错配引物介导的聚合酶链反应／限制性酶切分析法(PCR—RE)进行中国人常见7种G6PD基因突变型分析，突变特异性扩增系统法(ARMS)验证其中3种突变。结果431人中检出G6PD缺陷28例，总检出率6．50％，缺陷症基因频率0．065。缺陷基因分析：检出突变基因cDNA95(A→G)6例，发生频率为0．214；cDNA1024(C→T)8例，发生频率为0．286；cDNA1376(G→T)1例，发生频率为0．036；cDNA1388(G→A)7例，发生频率为0．250，未知型突变6例；未发现cDNA487(G→A)、cDNA493(A→G)、cDNA592(C→T)突变。结论G6PD缺陷症在贵州西江苗族人群有着较高的的基因频率，其主要突变类型为G6PD基因cDNA1024(C→T)、cDNA1388(G→A)、cDNA95(A→G)3种。中国人中较常见的cDNA1376(G→T)突变在该人群中频率较低，与已有报道的贵州汉族及其他民族有一定差异。     

葡萄糖-6-磷酸异构酶mRNA在RA患者外周血单个核细胞中的表达及意义

目的探讨葡萄糖-6-磷酸异构酶（glucose-6-phosphate isomerase，GPI）mRNA在类风湿关节炎（RA）患者外周血单个核细胞（PBMCs）中的表达及其与RA疾病活动性的关系。方法对30例RA活动期患者、30例RA缓解期患者、30例其他风湿性疾病患者及30名健康体检者，采用反转录聚合酶链反应（RT—PCR）半定量检测PBMCs中GPImRNA的表达水平。结果RA活动期患者GPImRNA的表达水平明显高于RA缓解期患者、其他风湿性疾病患者及正常对照组（P〈0．05），在RA活动组和RA缓解组，差异亦有统计学意义（P〈0．05）。结论GPImRNA在部分RA患者中高表达，其可能在RA发病机制中起作用，并与疾病的活动性相关。     

The pattern of gene expression in human CD15+ myeloid progenitor cells

We performed a genome-wide analysis of gene expression in primary human CD15(+) myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes.     

Genotype and phenotype correlation in glucose-6-phosphate dehydrogenase deficiency

BACKGROUND AND OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common erythrocytic enzymatic disorder in Italy and is characterized by wide clinical, biochemical and molecular variability. We studied the clinical and hematologic data from 54 G6PD-deficient, unrelated males from the Apulia region. DESIGN AND METHODS: Analyses for enzymatic activity, G6PD electrophoresis and molecular typing were performed on all subjects. Thirty-nine subjects (72.2%) showed a severe G6PD deficiency (<10% residual enzymatic activity) and 15 subjects (27.8%) a moderate deficiency (10--60% residual activity). RESULTS: The Mediterranean variant was found in 48.2% of cases, the Seattle variant in 33.3%, the A- variant in 7.45% and the Montalbano variant in 3.7%; the variant was not identified in four subjects. Thirty-two patients (59.2%) were asymptomatic; of these, 37.04% demonstrated acute hemolytic crises induced mainly by ingestion of fava beans and 3.7% had had neonatal jaundice. Acute hemolytic anemia was found in 53.8% of subjects with the Mediterranean variant, in 5.5% with the Seattle variant, in 100% with the A-variant and 0% with the Montalbano variant. INTERPRETATION AND CONCLUSIONS: Enzymatic activity was shown to be a poor predictive parameter of acute hemolytic crises and was not correlated with clinical features. Subjects with Mediterranean or A- variants had a more severe clinical phenotype which was not related to enzymatic activity. The Seattle, and probably the Montalbano, variant appears to have a milder clinical expression.