用gFM31探针进行谷子品种的指纹分析

用高度多态性探针ｇＦＭ３１对５９个谷子（ＳｅｔａｒｉａｉｔａｒｌｉｃａＢｅａｕｖ．）品种（包括地方品种、育成品种及品系）进行ＤＮＡ指纹分析,共鉴别出５８种类型,而黑谷和黑粒１５１６给出完全相同的带型,二者有可能是同一材料。通过对ｇＦＭ３１ＤＮＡ序列分析,未发现其中有小卫星ＤＮＡ和微卫星ＤＮＡ序列。该探针虽在谷子品种中显示很高的多态性,但与禾谷类的其他物种的ＤＮＡ杂交信号微弱,表现出较强的谷子基因组特异性。     

胡杨甜菜碱醛脱氢酶基因的功能分化

甜菜碱醛脱氢酶(BADH)在植物抗逆反应中发挥着重要作用。文中从胡杨cDNA克隆到2个甜菜碱醛脱氢酶基因,分别命名为PeBADH1和PeBADH2。PeBADH1和PeBADH2均编码503个氨基酸的蛋白质,预测分子量分别是54.93 kDa和54.90 kDa。组织表达模式分析发现这2个基因在正常生长、盐和H2O2胁迫下,在不同组织中的表达模式有较大差异。在大肠杆菌中表达并纯化了2个基因的重组蛋白。酶活性分析显示PeBADH1和PeBADH2蛋白对底物的活性分别是0.073μmol/(min.mg)和0.107μmol/(min.mg)。热力学稳定性分析显示这2个蛋白的热力学稳定性具有明显差异。因此,基因表达模式差异与蛋白质酶学性质的不同预示着这2个基因可能存在功能上的分化。     

Different protective roles in vitro of alpha- and beta-domains of growth inhibitory factor (GIF) on neuron injuries caused by oxygen free radicals.

It was well known that beta-amyloid (Abeta) and tau protein play an important role in pathological procedure of Alzheimer's disease (AD), a senile dementia. The growth inhibitory factor (GIF, also named metallothionein-3, MT-3) had been demonstrated to inhibit the outgrowth of cortex neurons in the medium with extract of the AD patient brain. In our experiments, it was found that the neurons of cortex and the PC12 (pheochromocytoma) cells could be protected from the cytotoxicity of beta-amyloid 25-35 in presence of GIF and its domains. Additionally, GIF can scavenge the hydroxyl radical efficiently in CytC-VitC radical producing system and its alpha-domain shown more effective potentials than its beta-domain. The electron paramagnetic resonance spectra also show that the alpha-domain has more potential ability for eliminating reactive oxygen free radicals than its beta-domain. The results suggest that GIF could act as an efficient scavenger against free radicals in vitro and the alpha-domain in GIF molecule shows more potential in protecting against reactive oxygen species injury than the beta-domain.     

Clinical manifestation, imaging, and genotype analysis of two pedigrees with spinocerebellar ataxia

The objective of this study was to analyze the clinical manifestation, imaging characteristics, genotype, and the relationship between the three aforementioned parameters in two pedigrees suffering from spinocerebellar ataxia. To evaluate the clinical manifestation of the two pedigrees and to compare the characteristics, we performed the MRI analysis of some patients from both pedigrees, while 2 ml of the peripheral blood sample was collected for gene analysis. The gene analysis data showed that pedigree 1 was certified spinocerebellar ataxia type-2 (SCA2); the CAG repeats in the proband, proband’s mother, and proband’s brother were 44, 36, and 38, respectively. The MRI revealed brainstem cerebellar atrophy and “cross sign” and “ordinate sign” of pons. Pedigree 2 was certified SCA1; the CAG repeats of the proband, proband’s aunt, and proband’s asymptomatic cousin were 60, 51, and 52, respectively. The MRI revealed cerebellar atrophy in these individuals. We, therefore, concluded that it was difficult to diagnose the SCA subset solely through the clinical manifestation. The imaging characteristics analysis and final diagnosis depended basically on gene analysis data.     

Contraction of fibroblast-containing collagen gels: Initial collagen concentration regulates the degree of contraction and cell survival

Remodeling of extracellular matrix involves a number of steps including the recruitment, accumulation, and eventual apoptosis of parenchymal cells as well as the production, organization, and rearrangement of extracellular matrix produced by these cells. The culture of fibroblasts in three-dimensional gels made of type I collagen has been used as a model of tissue contraction which characterizes both wound repair and fibrosis. The current study was designed to determine the effect of initial collagen concentration on the ability of fibroblasts to contract collagen gels and on cell survival. Native type I collagen was extracted from rat tail tendons and used to prepare collagen gels with varying collagen concentrations (0.75-2.0 mg/ml). Human lung fibroblasts (HFL-1) were cast into the gels and cultured in Dulbecco modified Eagle medium with 0.1% fetal calf serum for 2 wk. The gel size, collagen content, and deoxyribonucleic acid (DNA) content were determined. Gels prepared with an initial concentration of 0.75 mg/ml contracted more rapidly and to a smaller final size than gels prepared from 2 mg/ml initial collagen concentration (final size 7.1 versus 36.4% of initial size, P < 0.01). There was no significant degradation of the collagen in the gels under either condition. Hence, the dramatically increased contraction of the lower density gels resulted in a higher final density (P < 0.01). Cell density was estimated from DNA content. In low initial density gels, the final DNA content was significantly less than that in higher initial density gels (0.73 versus 1.88 microg/gel, P < 0.05). This was accompanied by an increased percentage of apoptotic cells at day 14 (43.3 versus 34.1%, P < 0.05). If the gels were maintained in the attached state which largely prevents contraction, apoptosis was significantly reduced, suggesting that contraction rather than matrix composition was a requirement for the increased apoptosis. In summary, these findings indicate that the initial matrix composition can lead to differing outcomes during fibroblast-mediated wound contraction.     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.     

Optimization of a series of quinazolinone-derived antagonists of CXCR3

The evaluation of the CXCR3 antagonist AMG 487 in clinic trials was complicated due to the formation of an active metabolite. In this Letter, we will discuss the further optimization of the quinazolinone series that led to the discovery of compounds devoid of the formation of the active metabolite that was seen with AMG 487. In addition, these compounds also feature increased potency and good pharmacokinetic properties. We will also discuss the efficacy of the lead compound 34 in a mouse model of cellular recruitment induced by bleomycin.     

Homology modeling threedimensional structure of AnxB1 and reducing its immunogenicity by sequence-deleted mutagenesis

AnxB1,a novel annexin previously isolated from Cysticercus cellulose,shows high thrombi affinity and anticoagulant activity in vivo.In order to investigate the relationship between structure and biological function,a predicted three-dimensional(3D)model of AnxB1 was generated by homology modeling.This model contains four homologous internal-domains and the Cα trace of domain Ⅰ,Ⅱ and IV shows high similarity.Based on the structure characterization,four sequence-deleted mutants were constructed and expressed as GST fusion proteins in E.coli.Two of the mutants,GST-M3 and GST-M4 reserved high anticoagulant activity(p＜0.01 vs.GST).Furthermore,compared with the wild type GST-AnxB1,the immunogenicity of GST-M3 and GST-M4 was reduced significantly(p＜0.01)and the molecular weight was lowered to 27 kD and 34 kD,respectively.These observations laid a solid foundation for further study on developing new thrombolytic agents with higher efficiency and lower side effect.