结直肠癌P53蛋白定量分析及其与肿瘤生物学行为的关系

应用MD-20显微图象分析仪对30例结直肠癌的P53蛋白免疫组织化学反应产物进行定量测定,以观察P53蛋白相对含量与肿瘤生物学行为的关系.结果显示:结直肠癌P53蛋白MOD值与PCNA增殖指数呈正相关(P<0.05).P53蛋白MOD值大的肿瘤多里浸润性生长方式,且浸润至浆膜外者多(PM<0.01,P<0.05).淋巴结转移与MOD值无明显关系(P>0.05).结果提示:结直肠癌P53蛋白相对含量对肿瘤的生物学行为有重要影响.     

RT－cDNA克隆－PCR法获取犬瘟热病毒融合蛋白基因（F）

纯化鸡胚成纤维细胞培养的犬瘟热病毒（ＣａｎｉｎｅＤｉｓｔｅｍｐｅｒＶｉｒｕｓ,ＣＤＶ）,获得病毒基因组ＲＮＡ后,反转录合成双链病毒Ｆ基因ｃＤＮＡ。将此双链ｃＤＮＡ平端插入ＰＵＣ１９质粒ＳａｍⅠ位点构建重组质粒,进行ｃＤＮＡ克隆。以重组克隆质粒为模板ＰＣＲ扩增,获得ＣＤＶ全长Ｆ基因。将此Ｆ基因插入表达载体ＰＢＶ２２０,在大肠杆菌中表达,通过对表达产物的最终鉴定,可确认所获片段为ＣＤＶ全长Ｆ基因．     

天然高分子吸附剂研究进展

本文综述了蛋白质、淀粉、甲壳素、纤维素等天然高分子材料作为吸附剂使用的研究进展情况,对它们的制备方法、使用范围和应用前景作了介绍,引用文献122篇。     

胁迫处理对林生山黧豆蛋白质多肽及其含量的影响

应用二维电泳技术,分析了经水分胁迫（ＰＥＧ）、盐分胁迫（ＮａＣｌ）和热激（４０℃）处理后林生山黧豆（ＬａｔｈｙｒｕｓｓｙｌｖｅｓｔｒｉｓＬ．）体内蛋白质多肽及其含量的变化。有些蛋白质经ＰＥＧ、ＮａＣｌ和热激处理后可以产生相同的变化。两种不同的胁迫因子对某些蛋白质的影响有一定的共同性。特定的胁迫条件可以造成特定的影响。不同胁迫因子对同一蛋白质多肽可以造成不同的影响。胁迫下蛋白质的变化可能与林生山黧豆抵抗和适应胁迫条件的能力以及体内非蛋白质氨基酸的代谢有关     

人重组GM－CSF／HAS－D3嵌合蛋白（GM－HSA）在大肠杆菌中的表达及其产物稳定性的研究

将人粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人血清白蛋白第三功能区（ＨＡＳ－Ｄ３）的基因串联后,在Ｅ．ｃｏｌｉ中获高效表达,表达量占菌体蛋白的３２．６％．利用ＴＦ－１体外细胞活性测定表明,ＧＭ－ＨＳＡ的活性单位为１．０４×１０~６Ｕ／ｍｇ,虽然其比活性低于ＧＭ－ＣＳＦ,但比后者具有更高的体外热稳定性和储藏稳定性．     

Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4

Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.     

Primary Structure and Phylogenetic Relationships of Glyceraldehyde-3-Phosphate Dehydrogenase Genes of Free-Living and Parasitic Diplomonad Flagellates

ABSTRACT. Complete nucleotide sequences have been established for two genes (gap1 and gap2) coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) homologs in the diplomonad Giardia lamblia. In addition, almost complete sequences of the GAPDH open reading frames were obtained from PCR products for two free-living diplomonad species, Trepomonas agilis and Hexamita inflata, and a parasite of Atlantic salmon, an as yet unnamed species with morphological affinities to Spironucleus. Giardia lamblia gap 1 and the genes from the three other diplomonad species show high similarity to each other and to other glycolytic GAPDH genes. All amino-acyl residues known to be highly conserved in this enzyme are also conserved in these sequences. Giardia lamblia gap2 gene is more divergent and its putative translation reveals the presence of a cysteine and serine-rich insertion resembling a metal binding finger. This motif has not yet been noted in other GAPDH molecules. All sequences contain an S-loop signature with characteristics close to those of eukaryotes. In phylogenetic reconstructions based on the derived amino acid sequences with neighborjoining, parsimony and maximum-likelihood methods the four typical GAPDH sequences of diplomonads cluster into a single clade. Within this clade, G. lamblia gap1 shares a common ancestor with the rest of the genes. The latter are more closely related to each other, indicating an early separation of the lineage leading to the genus Giardia from the lineage encompassing the morphologically less differentiated genera, Trepomonas, Hexamita and that of the unnamed species. This result is discordant with the orthogonal evolution of diplomonads suggested on the basis of comparative morphology. In neighbor-joining reconstructions G. lamblia gap2 occupies a variable position, due to its great divergence. In parsimony and maximum likelihood analysis however, it shares a most recent common ancestor with the typical G. lamblia gap1 gene, suggesting that it diverged after the separation of the Giardia lineage. The position of the diplomonad clade in broader phylogenetic reconstructions is firmly within the typical cytosolic glycolytic representatives of GAPDH of eukaryotes.     

A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.     

Leishmania donovani Attachment Stimulates PKC-Mediated Oxidative Events in Bone Marrow-Derived Macrophages

We investigated activation signaling events in bone marrow-derived macrophages after infection with Leishmania donovani, an intracellular parasite of macrophages. Leishmania donovani infection caused a general suppression of activation parameters like O₂^- and NO production. However, conditions which allow parasite attachment and prevent entry resulted in triggering of O₂^- and NO production and stimulation of O₂ consumption. Optimal NO and O₂^- production occurred when bone marrow-derived macrophages and Leishmania ratio was 1:100. The activation signal for O₂^- production was initiated 15 min after parasite attachment, whereas augmentation of NO production started 6 h after attachment. Activation of O₂^- and NO generation by L. donovani attachment was inhibited by staurosporine as well as by prolonged treatment of phorbol myristate acetate suggesting a protein kinase C-dependent mechanism. Translocation studies showed that protein kinase C activity in cell membrane fraction rapidly and transiently increased following parasite attachment. No such protein kinase C translocation event occurred in L. donovani infected bone marrow-derived macrophages. Phorbol myristate acetate was found to stimulate membrane translocation of protein kinase C in parasite attached cells whereas it was impaired in infected cells. However, both attachment and infection induced a similar shift of phorbol receptors from cytosolic to membrane fraction indicating that in infected cells the translocation of protein kinase C protein was not impaired but the activity of the membrane associated enzyme was somehow inhibited. These results suggest that although internalization of intracellular parasites like L. donovani caused inhibition of nitrite and superoxide release, mere attachment on macrophage surface resulted in an activation of protein kinase C-mediated downstream oxidative events.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).