HSF2 mRNA在热应激和大剂量11酸睾酮诱导恒河猴生精细胞凋亡中的表达变化

为了探讨HSF2 mRNA在热应激和超生理剂量睾酮诱导恒河猴生精细胞凋亡中的表达变化,我们建立了手术诱导单侧隐睾和注射大剂量11酸睾酮（TU）恒河猴动物模型,应用3′末端标记分析（TUNEL）和原位杂交方法,检测睾丸细胞的凋亡信号和HSF2的表达变化。TUNEL结果显示热应激和超生理剂量睾酮能够诱导生精细胞出现凋亡信号,它分别于处理后第5天和第30天达到最强,表明热应激和睾酮干扰精子发生可能是通过生精细胞凋亡的方式来实现的。HSF2 mRNA水平在生精细胞凋亡早期（凋亡信号达到最强以前）略有降低,而在凋亡高峰期之后其表达急剧下降。Hsf2基因与我们以前研究的Hsp70-2基因的表达具有时间上的相关性,表明HSF2蛋白可能调控Hsp70-2基因的表达,而且HSF2可能通过多种方式影响精子的发生以及抑制生精细胞的凋亡。     

一氧化氮在大鼠肢体缺血／再灌注后肾组织细胞凋亡中的意义

目的：探讨NO在肢体缺血／再灌注（LI／R）后肾组织细胞凋亡发生中的作用。方法：采用我室常规方法复制大鼠LI／R模型,采用生物化学方法检测血液及肾组织中一氧化氮及其相关指标,利用免疫组织化学方法及原位末端标记法（TUNEL）检测各组动物肾组织细胞凋亡情况。结果：与对照组比较,大鼠LI／R后4h,肾小管上皮细胞、血管内皮细胞呈凋亡改变;预先给予L-Arg的大鼠,凋亡细胞数明显减少;而经L-NAME处理的大鼠,凋亡细胞数明显增多。结论：大鼠LI／R后肾损伤可能与细胞凋亡增强有关;NO可通过影响凋亡基因的激活及相关蛋白的表达减轻LI／R后肾组织损伤。     

P16蛋白和生精细胞凋亡对热压和11酸睾酮诱导的恒河猴无精子症和少精子症中的作用

探讨了P16蛋白和生精细胞凋亡在热压和11酸睾酮诱导恒河猴无精子症和少精子症中作用间的关系。3′末端标记分析（TUNEL）结果显示热应激和超生理剂量睾酮能够诱导生精细胞出现凋亡信号,它分别于处理后第5天和第30天达到最强,免疫组化结果显示,热压或TU主要诱导精原细胞和其它生精细胞以及Sertoli细胞P16的表达。P16蛋白的表达在生精细胞凋亡晚期,即隐睾手术第10天或注射TU第60天后迅速升高并维持在热压或11酸睾酮诱导的早期精母细胞和精子细胞的凋亡和在晚期对精原细胞有丝分裂的抑制,二者共同作用导致热压或TU诱导的恒河猴无精子症和少精子症。     

缺血后适应对局灶性脑缺血/再灌注大鼠caspase-3表达的影响

目的：探讨缺血后适应对大鼠局灶性脑缺血/再灌注损伤后caspase-3表达的影响。方法：大脑中动脉线拴法复制大鼠局灶性脑缺血/再灌注损伤动物模型。将30只雄性SD大鼠随机分为3组（n=10）：假手术组（sham组）、缺血/再灌注（I/R）组和缺血后适应（IP）组。利用原位缺口末端标记法观察神经细胞凋亡的变化。应用Western blot检测大鼠局灶性脑缺血/再灌注损伤后caspase-3蛋白表达水平的变化。结果：大鼠脑缺血/再灌注后凋亡细胞数量和caspase-3蛋白表达水平均显著升高,而缺血后适应组凋亡细胞数量和caspase-3蛋白表达水平均显著低于缺血/再灌注组（P〈0.01）。结论：缺血后适应可抑制大鼠脑缺血/再灌注后细胞凋亡的发生,此作用可能与下调caspase-3蛋白表达有关。     

地塞米松诱导培养的大鼠大脑皮质星形胶质细胞凋亡

目的　研究地塞米松诱导纯化培养的大鼠大脑皮质星形胶质细胞凋亡的作用。方法　不同浓度的地塞米松(浓度为 10 -3 、 10 -4、 10 -5mol/L)与纯化培养的大鼠大脑皮质星形胶质细胞共同孵育 18小时后 ,吖啶橙染色荧光显微镜观察细胞凋亡形态学改变 ,流式细胞仪检测细胞凋亡和结晶紫比色法酶标仪测定活细胞数。结果　 (1)吖啶橙染色荧光显微镜观察 :10 -4组偶见细胞凋亡 ,10 -3 组可见许多细胞有典型的凋亡形态学改变核固缩 ,深染 ,或肿胀 ,碎裂 ,并可见凋亡小体。 (2 )流式细胞仪检测细胞凋亡 :10 -3 组细胞凋亡率为 15 99% ,与其它三组相比明显增高 ,有显著性差异 (P <0 0 1)。 (3)结晶紫法酶标仪测定活细胞数 :10 -3 组OD值为 0 . 185与其它三组相比明显下降 ,有显著性差异 (P <0 0 1) ,表明活细胞数明显减少。结论　大剂量地塞米松可诱导体外的星形胶质细胞凋亡。     

药物诱导的玉米根尖细胞凋亡

同时应用ＤＮＡ Ｌａｄｄｅｒｉｎｇ、ＤＮＡ Ｇｅｌ Ｂｌｏｔ以及基于染色体涂片 原位末端标记技术,从染色体、细胞核和ＤＮＡ不同水平对细胞毒素类药和的放线菌Ｄ、放线菌酮和秋水仙碱诱导的玉米（Ｚｅａ ｍａｙｓ Ｌ．）根尖分生组织细胞死亡作了检测。结果表明：同动物中一样,这些药物诱导的玉米根尖分生组织细胞死亡也具有ＤＮＡ Ｌａｄｄｅｒ、染色质和细胞核浓缩等典型的调亡特征,说明这些细胞毒素类药物能够诱导植     

鸡胚巨噬细胞AcP酶变化及AcP酶与凋亡细胞的关系

本研究采用电镜及酶细胞化学的方法观察了鸡胚脾脏不同胚龄组巨噬细胞溶酶体酸性磷酸酶（AcP酶）的变化、凋亡实验组巨噬细胞及其AcP酶与凋亡细胞的关系。取10天、13天和17天鸡胚脾脏,按Gomori法显示AcP酶,各胚龄脾脏巨噬细胞AcP酶细胞化学反应阳性,按AcP酶染色阳性做溶酶体计数,结果显示随着胚龄的增加溶酶体数随之增加,尤以第17天组溶酶体数增加最为明显,所得数据经统计分析表明各胚龄组间溶酶体数的差异有统计学意义。凋亡实验组采用放线菌酮诱导15天鸡胚脾脏细胞凋亡,结果显示凋亡细胞为各类幼稚血细胞,以幼稚淋巴细胞为主。巨噬细胞未见凋亡,而是吞噬了大量的凋亡细胞和凋亡小体,AcP酶反应颗粒不仅出现在巨噬细胞的溶酶体、吞噬体,还见于高尔基复合体、内质网等。细胞AcP酶反应强度数字化结果表明：凋亡组酶活性显著高于对照组,差别有统计学意义,提示胚胎巨噬细胞在凋亡细胞出现时AcP酶活性增强,说明巨噬细胞吞噬和消化凋亡细胞或凋亡小体是通过AcP酶等活性物质来实现的。     

大鼠脑缺血再灌注后神经元和星形胶质细胞凋亡的比较研究

目的比较研究大鼠局灶性脑缺血再灌注后神经元和星形胶质细胞的凋亡规律。方法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,在缺血再灌注后1、3、7、14d断头取脑,应用流式细胞分选技术和原位末端标记法分别检测各组MCAO后不同时期神经元和星形胶质细胞凋亡情况。结果局灶性脑缺血再灌注后,海马区星形胶质细胞凋亡数量超过神经元,其凋亡以再灌注3d最为显著,而神经元则以7d最为显著;而皮层区神经元凋亡数量超过星形胶质细胞,两种细胞凋亡均在再灌注后7d达高峰。结论脑缺血再灌注后,皮层和海马区的神经元及星形胶质细胞均可发生凋亡,海马区星形胶质细胞比皮层区更易凋亡,而皮层区神经元比海马区更易凋亡。     

NOS抑制剂对甲醛炎性痛诱导的大鼠痛反应及脊髓神经元凋亡的影响

目的：观察甲醛炎性痛是否可诱导脊髓神经元凋亡以及一氧化氮（NO）对甲醛炎性痛诱导的大鼠痛反应以及脊髓神经元凋亡的影响。方法：采用行为学方法观察大鼠自发痛反应,流式细胞术检测脊髓神经元凋亡率。结果：与正常大鼠比较,甲醛炎性痛可诱导大鼠脊髓神经元凋亡率明显增加,于注射甲醛后3d时最为明显。预先鞘内注射NOS抑制剂L-NAME可剂量依赖性抑制足底注射甲醛诱导的大鼠第一相和第二相痛反应,并可剂量依赖性抑制足底注射甲醛诱导的脊髓神经元凋亡过程;正常大鼠鞘内注射L-Arg也可诱发出伤害性反应和脊髓神经元凋亡。结论：甲醛炎性痛可诱导脊髓神经元发生凋亡,于注射甲醛后3d神经元凋亡最为明显;炎性痛诱导的NO产生增多促进了脊髓伤害性信息传递过程,并在炎性痛诱导的脊髓神经元凋亡过程中发挥促进作用。     

内毒素所致SIRS/MODS小鼠脾细胞损伤的形态学观察

目的 应用大肠杆菌内毒素脂多糖（LPS）腹腔注射制备小鼠全身炎症反应综合片/多器官功能失常综合征（SIRS/MODS）模型。方法 用免疫组化的方法检测了Bcl-2的表达。结果 随LPS作用时间的延长,脾损伤逐渐加重,HE染色中,生发中心和动脉周围淋巴鞘分别在LPS注射后9小时和18小时出现大量深浅不一的颗粒状细胞碎片。TUNEL阳性染色细胞随时间延长而增多,电镜下可见到凋亡的淋巴细胞,在18小时还见到细胞坏死现象,Bcl-2的表达随着LPS作用时间的延长而明显下降,与TUNEL染色的相关分析表明,Bcl-2的表达与细胞凋亡有显著的负相关。结论 在LPS所致的SIRS/MODS模型中,脾细胞损伤以凋亡为主,随LPS作用时间延长而加重,在晚期还会出现细胞坏死。Bcl-2参与了LPS引起的脾细胞凋亡效应。     

甲型H1N1流感病毒在季流病毒、禽流感病毒共感染时变异可能性的验证

目的 甲型H1N1流感病毒A/California/7/2009分别与A/Brisbane/10/07和A/ShenZhen/406H/06共感染小型香猪,预测甲流病毒在与季流H3N2病毒/甲流病毒与禽流感病毒共感染时是否会发生变异.方法 分别将A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)对5～6月龄小型猪共感染,小型猪经复方氯胺酮0.1 mL/kg麻醉后进行滴鼻感染,感染后第5天安乐死动物,取动物肺组织作病毒测序分析.结果 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2)共感染后,A/California/7/2009病毒PB1基因993位G→A突变,PA基因1659位G→A突变,没有氨基酸的变异.A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后A/California/7/2009病毒PB2基因1711位T→C突变.碱基的突变未引起氨基酸的变异.结论 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后在猪的体内没有发生病毒重组、变异.     

Pathogenesis of primary defects in mitochondrial ATP synthesis

Maternally inherited mutations in the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase) of the respiratory chain/oxidative phosphorylation system are responsible for a subgroup of severe and often-fatal disorders characterized predominantly by lesions in the brain, particularly in the striatum. These include NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh syndrome), and FBSN (familial bilateral striatal necrosis). Of the five known pathogenic mutations causing these disorders, four are located at two codons (156 and 217), each of which can suffer mutations converting a conserved leucine to either an arginine or a proline. Based on the accumulating data on both the structure of ATP synthase and the mechanism by which rotary catalysis couples proton flow to ATP synthesis, we propose a model that may help explain why mutations at codons 156 and 217 are pathogenic.     

Glycoprofiling with micro-arrays of glycoconjugates and lectins

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.     

Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida

Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.     

Heparin sequencing

Heparin is a highly sulfated glycosaminoglycan widely used as an anticoagulant. Modifications in its relatively uniform structure appear to be key to its recognition and modulation of serine proteases, growth factors, chemokines, and extracellular proteins, as has been most clearly demonstrated in the antithrombin binding site. We sequenced the major oligosaccharides released from mastocytoma heparin by partial nitrous acid using a highly sensitive technique tailored for sequencing of metabolically radiolabeled heparin. It utilizes partial nitrous acid cleavage to allow simultaneous sequencing of the internal components of the oligosaccharide under investigation by specific lysosomal exoenzymes. Sequencing revealed that although the majority of the heparin disaccharides are N-, 2-O-, and 6-O-sulfated, the less sulfated disaccharides (lacking 2-O- or 6-O-sulfates) seem to be spaced out along the chain. The technique may be particularly useful for characterizing heparin from novel sources, such as the glial progenitor cells and Ascidia, as well as for sequencing protein binding sites.     

A new kind of carbohydrate array,its use for profiling antiglycan antibodies,and the discovery of a novel human cellulose-binding antibody

In this study, we use a novel glycan array to analyze the glycan-binding antibody repertoire in a pool of affinity-purified IgG collected from a healthy human population. The glycan array used is based on mono- and oligosaccharides covalently linked to the surface via a long linker at their reducing ends. They are thus presented to the medium with a well-defined orientation and are accessible for specific binding by glycan-binding proteins, such as antibodies and lectins. A novel anticellulose antibody was detected that binds specifically to beta4-linked saccharides with a preference for glucopyranose over galactopyranose residues. We also found previously known antiglycan antibodies against mono- and oligosaccharides that are constituents of commonly occurring bacterial polysaccharides. We propose that this array can facilitate high-throughput screening of glycan-binding proteins and the search for biomarkers for personalized medicine.     

N-glycan branching requirement in neuronal and postnatal viability

The structural variations among extracellular N-glycans reflect the activity of glycosyltransferases and glycosidases that operate in the Golgi apparatus. More than other types of vertebrate glycans, N-glycans are highly branched oligosaccharides with multiple antennae linked to an underlying mannose core structure. The branching patterns of N-glycans consist of three types, termed high-mannose, hybrid, and complex. Though most extracellular mammalian N-glycans are of the complex type, some cells variably express hybrid and high-mannose forms. Nevertheless, a requirement for hybrid and complex N-glycan branching exists in embryonic development and postnatal function among mice and humans inheriting defective Mgat1 or Mgat2 alleles. The resulting defects in formation N-glycan branching patterns cause multiple abnormalities, including neurologic defects, and have inferred the presence of distinct functions for hybrid and complex N-glycan branches among different cell lineages. We have further explored N-glycan structure-function relationships in vivo by using Cre-loxP conditional mutagenesis to abolish hybrid and complex N-glycan branching specifically among neuronal cells. Our findings show that hybrid N-glycan branching is an essential posttranslational modification among neurons. Loss of Mgat1 resulted in a unique pattern of neuronal glycoprotein deficiency concurrent with caspase 3 activation and apoptosis. Such animals exhibited severe locomotor deficits, tremors, paralysis, and early postnatal death. Unexpectedly, neuronal Mgat2 deletion resulting in the loss of complex but not hybrid N-glycan branching was well tolerated without phenotypic markers of neuronal or locomotor dysfunction. Structural features associated with hybrid N-glycan branching comprise a requisite posttranslational modification to neuronal glycoproteins that permits normal cellular function and viability.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

Carbohydrate-recognition domains of galectin-9 are involved in intermolecular interaction with galectin-9 itself and other members of the galectin family

Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9.