Novel Form of Phosphate Activated Glutaminase in Cultured Astrocytes and Human Neuroblastoma Cells,PAG in Brain Pathology and Localization in the Mitochondria

A novel form of phosphate activated glutaminase (PAG), catalyzing the synthesis of glutamate from glutamine, has been detected in cultured astrocytes and SH-SY5Y neuroblastoma cells. This enzyme form is different from that of the kidney and liver isozymes. In these cells we found high enzyme activity, but no or very weak immunoreactivity against the kidney type of PAG, and no immunoreactivity against the liver type. PAG was also investigated in brain under pathological conditions. In patients with Down's syndrome the immunoreactivity in the frontoparietal cortex was significantly reduced. The findings leading to our conclusion of a functionally active PAG on the outer face of the inner mitochondrial membrane are discussed, and a model is presented.     

Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα-, αγ-, and γγ-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα-enolase was the predominant isoenzyme, and αγ-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα-enolase and a lower proportion of γγ-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα-enolase and lowest proportion of γγ-enolase.     

一种新的抑癌候选基因——NDR2在人类正常组织及相应肿瘤中的表达

为观察NDR2基因在人类正常组织及相应肿瘤组织中的表达分布特点,收集人脑与胶质瘤、肺与肺癌、胃与胃癌、结肠与结肠癌组织标本,分别进行组织石蜡切片和总RNA提取,应用免疫组化方法和RT-PCR技术检测NDR2蛋白质及mRNA表达水平,并通过DNA测序验证PCR产物的正确性.免疫组化结果表明,在上述各组织均有NDR2蛋白不同程度的表达.RT-PCR结果显示,在人脑和胶质瘤组织、肺与肺癌组织、胃与胃癌组织、结肠与结肠癌组织中均有NDR2 mRNA表达,其表达水平以脑组织为最高.NDR2 mRNA在正常脑和肺组织的表达水平分别显著高于胶质瘤与肺癌组织,而结肠与结肠癌组织,胃与胃癌组织NDR2 mRNA表达水平则无显著差别.以上结果表明,NDR2基因可能广泛表达于人体正常组织内,而在胶质瘤与肺癌中的表达较相应正常组织减低,提示该基因可能与神经系统及呼吸系统肿瘤的发生发展有关,为进一步探讨该基因功能提供了线索.     

Expression of the mRNA for τ Proteins During Brain Development and in Cultured Neurons and Astroglial Cells

Two tau cDNA probes of 1.6 and 0.3 kilobases (kb) have been used to study the expression of the tau mRNAs during mouse brain development and in highly homogeneous primary cultures of neurons and astrocytes. (1) Whatever the stage, a 6-kb mRNA was detected with the two probes. In the astrocytes a 6-kb mRNA hybridized clearly only with the 1.6-kb probe. (2) During brain development the abundance of tau mRNA increases from a late fetal stage (-4 days) until birth, remains high until 6 days postnatal, and then markedly decreases to reach very low values in adulthood. Such a marked decrease in the abundance of tau mRNA parallels that of alpha-tubulin mRNA. These data suggest that: (1) depending on the stage of development and on the cell type (neurons or astrocytes) tau mRNAs of the same size encode several tau proteins differing in molecular weight: several tau proteins are expressed either during early stages of development (juvenile tau proteins of 48 kilodaltons) or in adulthood (mature tau proteins of 50-70 kilodaltons) or are specific of the astrocyte (83 kilodaltons). (2) The expression of the two major components of axonal microtubules, tubulin and tau proteins, seems to be developmentally coordinated.     

非小细胞肺癌脑部转移瘤适形与调强放疗的剂量研究

为探讨NSCLC脑部转移瘤调强放疗与适形放疗的剂量特点,本研究选取57例非小细胞肺癌脑转移瘤患者,其中单个脑转移灶患者5例,多个脑转移灶患者52例,分别设计全脑放疗+适形放疗与调强放疗计划,用均匀指数(HI)和适形指数(CI)评价靶区剂量,危及器官(OAR)剂量用近似最大剂量D_2%(串联)和中位剂量D_50%(并联)进行评价。研究发现,单个脑转移灶IMRT与WBRT+CRT比较中,CI为(PTV,(0.80±0.15) cGy,(0.34±0.19) cGy, p=0.00),HI为(PTV,(0.52±0.03) c Gy,(0.71±0.12) cGy, p=0.24),两者OARs剂量比较:脑干为((4 348±236) cGy,(4 593±149) cGy, p=0.01),脑垂体为((4 258±166) cGy,(4 581±123) cGy, p=0.02);在多个脑转移灶中,IMRT与WBRT+CRT比,较CI为(PTV,(0.59±0.33) cGy,(0.49±0.27) cGy, p=0.03),HI为(PTV,(0.93±0.01) cGy,(0.58±0.03) cGy, p=0.19),两者OARs剂量比较:脑干为((4 946±132) cGy,(4 843±196) cGy, p=0.51),脑垂体为((4 597±180) cGy,(4 705±149) cGy, p=0.70)。本研究的结果说明,单个脑转移灶患者,IMRT较WBRT+CRT有更好的靶区适形性、稍差的靶区异质性,脑干和垂体的IMRT剂量低于WBRT+CRT,而眼球、晶体的剂量两者差别不明显。多个脑转移灶患者,IMRT较WBRT+CRT有更好的靶区适形性、稍差的靶区异质性,而OARs剂量,IMRT较WBRT+CRT差异不明显。在临床实践过程中,应当根据患者不同的病灶情况选择合适的放疗方案,以获取更优的临床治疗效果。     

CD117、CD34、DOG-1、WT-1 在胃肠道间质瘤中的表达及意义

目的:探讨DOG-1、CD117、CD34、WT-1在胃肠道间质瘤(GIST)中的表达及临床意义。方法:应用免疫组织化学SP法检测DOG-1、CD117、CD34、WT-1在39例GIST患者肿瘤组织中的表达。结果:GIST光镜下主要由梭形细胞和(或)上皮样细胞或多形性细胞混合或单一性组成。DOG-1、CD117、CD34和WT-1在GIST肿瘤组织中阳性表达率分别为92.3%(36/39)、71.7%(28/39)、64.1%(25/39)、23.1%(9/39),四者的阳性表达率在各风险程度组(极低及低度危险性GIST、中度危险性GIST、高度危险性GIST)两两比较中差异均无统计学意义(P>0.05)。DOG-1与CD117相比,在极低及低度危险组中表达有显著差异(P<0.05),DOG-1与CD34相比,在极低及低度危险组、高度危险组中表达有显著差异(P<0.05);WT-1与CD117相比,在中度及高度危险组中表达有显著差异(P<0.05),WT-1与CD34相比,在中度危险组中表达有显著差异(P<0.05)。对照组平滑肌瘤、纤维瘤、神经鞘瘤中CD117、DOG-1在GIST中的表达明显高于其他多种梭形细胞的表达(P<0.05)。结论:DOG1是GIST较为敏感和特异的标记物,WT-1在极低及低度危险性GIST中有一定的诊断价值,在GIST的诊断中加入两者将使患者更加受益。     

The Roles of IAPs with Ubiquitin Ligase Activity in the Occurrence and Development of Malignant Tumors

The inhibitors of apoptosis proteins (IAPs) are a family of highly conserved proteins involved in apoptosis. Recent studies indicate that IAPs with RING domains act as ubiquitin E3 ligases and play an important role in the occurrence and development of malignant tumors through inhibiting the caspases and regulating MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor kappa-B) signaling. The mechanisms of IAPs in malignant tumors are complex and diverse, including resistance to cell death, inflammatory response, invasion and metastasis. IAPs inhibit apoptosis through both intrinsic and extrinsic pathways. They promote inflammatory response and regulate immune response. Besides, they both promote and inhibit tumor cell migration. Recent studies indicated that IAPs are positively correlated with poor prognosis in most malignant tumors, and negatively correlated with poor prognosis in some other few malignant tumors. The conclusions above show that it will be particularly necessary to further explore the relationship among IAPs, the occurrence and development of malignant tumors and the prognosis of patients. This review summarizes the latest research of IAPs that serve as E3s, in particular XIAP (X-chromosome linked IAP), c-IAP1 (cellular IAP1), c-IAP2 (cellular IAP2) and ML-IAP (melanoma IAP), covering the structures, functions in the malignant tumors, the signaling pathways and their correlation with the development and prognosis of malignant tumors, as well as the progress of anti-tumor drugs and therapies for IAPs. Furthermore, this review explores the problems and challenges in the current studies, which may provide new directions and strategies for future research.     

6天龄与10天龄大鼠的睾丸组织的基因差异表达

通过基因芯片技术,利用Roche-NimbleGen公司制作的大鼠12×135K全基因组表达谱芯片,对日龄为6d和10d的大鼠睾丸组织进行全基因组表达差异分析。结果显示:具有2倍以上的差异表达基因有4298个,其中表达上调的基因共1878个,表达下调的基因共2420个。这些差异表达的基因中有3154个基因具有基因本体注释,参与了154个生物学通路。进一步分析表明具有8倍以上差异表达的基因有13个,这些基因参与了生物学过程、细胞组分和分子功能等基因本体分类,进一步选择3个差异表达的基因,LOC686076、Cxcl6和Trib3,做了实时定量RT-PCR检测。其结果趋势与芯片数据一致。因此,我们初步认为精原干细胞的发生与增殖在大鼠早期的发育过程中已经有大量的基因参与,是一个多基因协调表达的过程。     

生长抑素受体在人神经内分泌癌组织中的表达及受体激活对细胞生长的抑制

生长抑素（somatostatin,SST）通过与细胞膜上的G蛋白偶联的生长抑素受体（somatostatin receptors,SSTRs）结合而发挥其抑制细胞增殖的作用,因而生长抑素类似物（somatostatin analogue, SSA）常被用于肿瘤辅助治疗。然而,治疗效果存在相当大的个体差异,推测生长抑素类似物治疗效果不佳,与内源性生长抑素受体表达缺失或者表达量和亚型组合有关。为此,检测各亚型SSTR在几例罕见的神经内分泌肿瘤中的表达,并检测过表达SSTR2和SSTR5以及受体激活对细胞增殖的抑制效果,分析受体激活的可能机制,有助于临床筛选适合SSA肿瘤辅助治疗的病例,预估SSA的治疗效果。免疫组化检测肿瘤组织SSTR1-5的表达。在培养的293T细胞中过表达SSTR2和SSTR5,免疫共沉淀检测受体相互作用,免疫荧光和共聚焦显微镜检测受体细胞内定位。用MTT法检测受体过表达及激活对培养的人肺癌细胞NCI-H460细胞增殖的影响,用流式细胞技术检测细胞周期分布。SSTR1-5在10例神经内分泌肿瘤组织中均有不同程度的表达,表达亚型及表达量与肿瘤类型和年龄无关,SSTR5在所有肿瘤组织中均表达。SSTR2与SSTR5可形成受体相互作用。SSTR2与SSTR5活化后相互作用增加并定位于细胞质。共表达SSTR2和SSTR5显著抑制细胞增殖,并与受体激活剂呈现剂量相关性。SSTR2/SSTR5的共表达及激活显著减少S期的细胞而滞留于G₁期。     

Inhibition of Hyaluronan Synthesis Protects against Central Nervous System (CNS) Autoimmunity and Increases CXCL12 Expression in the Inflamed CNS

Hyaluronan (HA) may have proinflammatory roles in the context of CNS autoimmunity. It accumulates in demyelinated multiple sclerosis (MS) lesions, promotes antigen presentation, and enhances T-cell activation and proliferation. HA facilitates lymphocyte binding to vessels and CNS infiltration at the CNS vascular endothelium. Furthermore, HA signals through Toll-like receptors 2 and 4 to stimulate inflammatory gene expression. We assessed the role of HA in experimental autoimmune encephalomyelitis (EAE), an animal model of MS by administration of 4-methylumbelliferone (4MU), a well established inhibitor of HA synthesis. 4MU decreased hyaluronan synthesis in vitro and in vivo. It was protective in active EAE of C57Bl/6 mice, decreased spinal inflammatory infiltrates and spinal infiltration of Th1 cells, and increased differentiation of regulatory T-cells. In adoptive transfer EAE, feeding of 4MU to donor mice significantly decreased the encephalitogenicity of lymph node cells. The transfer of proteolipid protein (PLP)-stimulated lymph node cells to 4MU-fed mice resulted in a delayed EAE onset and delayed spinal T-cell infiltration. Expression of CXCL12, an anti-inflammatory chemokine, is reduced in MS patients in CSF cells and in spinal cord tissue during EAE. Hyaluronan suppressed production of CXCL12, whereas 4MU increased spinal CXCL12 in naive animals and during neuroinflammation. Neutralization of CXCR4, the most prominent receptor of CXCL12, by administration of AMD3100 diminished the protective impact of 4MU in adoptive transfer EAE. In conclusion, hyaluronan exacerbates CNS autoimmunity, enhances encephalitogenic T-cell responses, and suppresses the protective chemokine CXCL12 in CNS tissue. Inhibition of hyaluronan synthesis with 4MU protects against an animal model of MS and may represent an important therapeutic option in MS and other neuroinflammatory diseases.     

Piperlongumine Inhibits Zika Virus Replication In vitro and Promotes Up-Regulation of HO-1 Expression,Suggesting An Implication of Oxidative Stress

Virologica Sinica - Owing to the widespread distribution of mosquitoes capable of transmitting Zika virus, lack of clinical vaccines and treatments, and poor immunity of populations to new...     

Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue

The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr³⁰⁸ and Ser⁴⁷³ upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser⁴⁷³ and Thr³⁰⁸ phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser⁴⁷³ and Thr³⁰⁸ phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser⁴⁷³-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.     

牦牛LIFR 基因的克隆及其在繁殖周期卵巢中的表达

白血病抑制因子受体(LIFR)与白血病抑制因子(LIF)结合,在排卵、胚胎发育及胚胎附植等过程中起重要的调节作用,与哺乳动物生殖过程密切相关。为进一步研究白血病抑制因子受体(LIFR)基因在卵巢中的表达,本研究对牦牛LIFR 基因进行克隆和序列分析,并利用RT-PCR 技术研究其在繁殖周期卵巢中的表达情况。本研究克隆得到牦牛3 329 bp 的LIFR 基因cDNA 序列,内含3 288 bp 开放阅读框序列。与家牛的相应序列具有很高的同源性(99 5% ),表明LIFR 基因在进化过程中较为保守。实时定量RT-PCR 检测LIFR 基因在繁殖周期卵巢中的表达,结果显示卵巢中LIFR 基因在妊娠期表达最强。表明LIFR 基因在牦牛繁殖周期中具有不可忽视的作用。     

葡萄扇叶病毒外壳蛋白基因的克隆,序列分析及其在大肠杆菌中的表达

通过RT-PCR方法把葡萄扇叶病毒(GFLV)外壳蛋白基因(CP gene)分成两部分扩增,扩增产物克隆入pGEM-5Zf(+)载体,并通过BglⅢ位点连接成一完整的外壳蛋白基因,通过序列分析测得全长外壳蛋白基因为1512bp,编码504个AA＇s,与国外株系GFLV-F13相比,核苷酸同源性为88.4％,氨基酸同源性为95.8％。并且这一外壳蛋白基因在大肠杆菌E.coli DH-5α中得到了表达。     

用套式RT-PCR检测兰州地区肺炎患儿下呼吸道分泌物中的RSV和HPIV-3

为了调查兰州地区肺炎患儿中RSV和HPIV-3的感染状况,分别在RSV的G蛋白基因的保守序列和HPIV-3的HN基因的保守序列处,设计了套式引物,采用套式RT-PCR的方法检测了60份兰州地区肺炎患儿下呼吸道分泌物样本,结果显示,60份样本中RSV阳性为14例(23%),HPIV-3阳性为12例(20%)。并分别随机对其中的5份样本的扩增产物进行了基因序列测定,结果5份RSV阳性样本与参考株序列的同源性均大于97%,5份HPIV-3阳性样本与参考株序列的同源性大于97%。表明兰州地区下呼吸道感染患儿中,RSV和HPIV-3是常见的感染病原体。     

人脑髓鞘碱性蛋白cDNA体外扩增、克隆和鉴定

采用聚合酶链反应(PCR)从人脑cDNA文库中扩增出600bp的髓鞘碱性蛋白(MBP)cDNA片段,与载体pGEM-3Zf(+)平端连接.重组质粒DNA转化宿主菌JM109,在含X-gal和IPTG的平板上直接筛选阳性克隆.限制性内切酶分析和成套引物扩增鉴定证明,该克隆含有7个外显子的21.5kD人脑MBP全长编码序列.     

人巨细胞病毒磷蛋白65及糖蛋白B在大肠杆菌中的高效表达、免疫原性研究及初步应用

实验成功的构建了含有pp65与gB主要抗原决定簇基因的表达质粒pET-pp65、pET-gB;表达产物通过SDS-聚丙烯酰胺凝胶电泳、免疫印迹、间接酶联免疫等一系列鉴定试验进行了分析。表达蛋白经初步纯化后免疫BALB/c小鼠,经0、2、4周免疫三次,免疫后2、4、6周眼眶采血收集抗血清,进行中和试验和ELISA检测。动物试验表明两种表达蛋白具有免疫原性,并可在小鼠体内诱导产生特异性抗体,其中用重组gB蛋白免疫小鼠后得到的抗血清在体外试验中能抑制天然病毒感染细胞。同时将这两种表达蛋白作为诊断用抗原,对临床94份血清进行检测,证实pp65具有较好的特异性,为今后研制HCMV诊断试剂提供了科学依据。