用2DNMR鉴定倍半萜多元酯的结构

利用2DNMR结合其他波谱方法从苦皮藤种子油中鉴定了2个新倍半萜多元酯,它们分别是1α-苯甲酰氧-8β-烟酰氧-9α、11β-2乙酰氧-β-二氢沉香呋喃,1β-苯甲酰氧-2α、8β、9α、11β-4乙酰氧-β-二氢沉香呋喃,命名为苦皮藤酯2和3。     

毛枝南蛇藤根皮中南蛇藤素的分离鉴定及含量测定

从卫矛科南蛇藤属植物毛枝南蛇藤(Celastrus hookeri Prain)根皮中首次分离出一个红色立方体形结晶,经紫外、红外、高分辨质谱和~(13)C核磁共振谱分析,并与有关文献数据对照,确认是南蛇藤素。作者用薄层扫描和高效液相两种方法初步测定了毛枝南蛇藤根皮和根的木栓化表皮的南蛇藤素含量,结果表明南蛇藤素主要集中在根的木栓化表皮,含量在12%以上,而在整个根皮中的含量大约为1%。     

活性氧对巨噬细胞呼吸爆发影响及云芝多糖的保护作用

用化学发光法观察到叔丁基氢过氧化物对培养的小鼠腹腔巨噬细胞呼吸爆发有强烈的抑制作用。云芝多糖经腹腔注射后,能增强巨噬细胞呼吸爆发功能对叔丁基氢过氧化物损伤的抵抗力。云芝多糖处理的巨噬细胞谷胱甘肽过氧化物酶基础活力显著提高,在叔丁基氢过氧化物作用下,云芝多糖处理的巨噬细胞仍有较高的谷胱甘肽过氧化物酶活力。说明巨噬细胞的免疫功能与谷胱甘肽过氧化物酶活力有关,非特异性免疫多糖可提高细胞抗氧化能力,减轻活性氧损伤作用。     

人α1/158Ⅴ型(α1c型)干扰素基因在大肠杆菌中的表达及其产物的初步纯化

采用DNA聚合酶链反应(PCR)技术,将本实验室从中国人胎肝细胞染色体DNA中发现和分离的IFN-α1/158V基因的原始克隆,改造成适于进行非融合蛋白原核表达的结构形式,并在大肠杆菌中获得高效表达。测得重组IFN-α1/158V的抗病毒活性为1.9×10~7单位/升菌液。随后又采用以单克隆抗体亲和层析为主的纯化流程对表达产物进行初步纯化,获得了在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带的纯化产物。     

改变乙型肝炎病毒核心抗原基因3′端序列对其在原核细胞中表达的影响

乙型肝炎病毒(HBV)的核心抗原基因(C基因)编码185个氨基酸残基,在原核细胞或痘苗病毒系统中能表达并装配成27nm大小的核心抗原(HBcAg)多聚体颗粒。已证实HBV C基因3′端编码近40个氨基酸的碱基序列,不是表达形成HBcAg颗粒所必需的。用外源基因替换这部分序列,已表达出表面带有外源基因产物的杂合颗粒,它具有很好的免疫原性,成为新型的基因工程多决定簇颗粒载体疫苗。但我们的实验中发现,用另外的外源基因替换3′端序列能显著影响HBV C基因在大肠杆菌中的表达,不同组成的外源基因其影响程度有所不同。     

Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

Summary C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.     

氟中毒抑制大鼠泌鼠的实验研究

The effect of fluorosis on lactation, lactotroph function and ultrastructure were studied in lactating rats. The results were as follows: 1) Inhibition of lactation in lactating rats with chronic fluorosis was assessed by stunting growth of pups and decrease in the amount of milk suckled by pups in 30 min. Metoclopramide, a blocker of dopamine receptor, could improve lactation in these rats. 2) During chronic fluorosis serum PRL level was decreased, however, PRL content in pituitary was increased. Electronmicroscopic examination showed accumulation of large mature secretory granules and appearance of extremely large abnormal secretory granules in lactotroph cytoplasma. These findings indicate that hormone release of pituitary lactotrophs is obstructed in lactating rats with fluorosis, and the toxic effect of fluoride is mediated by an enhanced function of dopaminergic system in hypothalamus.     

Ligand-triggered conformational perturbations elicit changes at the single cysteinyl residue of spinach calmodulin

Following application of stoichiometric amounts of Ca2+ or specific partner peptides to spinach calmodulin, dynamic changes in the nanosecond range could be monitored at a strategically anchored fluorescence or spin probe. For these studies the single cysteinyl residue 26 of spinach calmodulin was labelled with a thiol-specific proxyl (i.e. 2,2,5,5-tetramethyl-1-pyrrolidinyl-oxyl) spin probe or with a bimane fluorescence probe. With Ca2+ and a specific ligand (mastoparan) present, fluorescence studies (anisotropy, lifetime) indicated that the rotational motion of the protein complex becomes slower relative to the motion of calmodulin in the absence of the specific ligand. The probe's attachment site 26 appears to reside in a fairly polar microenvironment as reported by a series of proxyl spin probes varying in label length. The rotational correlation time of the shortest spin probe markedly changed upon binding of a specific peptide to a calmodulin region distant from that of the monitoring spin probe. We interpret these observations as indicating that ligand-triggered conformational perturbations are eliciting specific responses at the cysteinyl residue 26 of spinach calmodulin.     

Reaction of pyruvate kinase with the new nucleotide affinity labels 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Two new reactive nucleotides have been synthesized and characterized: 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate (8-BDB-TADP and 8-BDB-TATP). ADP or ATP was converted to 8-thio-ADP (-ATP) via 8-bromo-ADP (-ATP), followed by condensation with 1,4-dibromobutanedione. Rabbit muscle pyruvate kinase is inactivated by both reagents in a biphasic manner with an initial rapid loss of 75% activity, followed by a slow total inactivation. The initial fast reaction with both compounds exhibits nonlinear dependence on reagent concentration, indicating formation of a reversible enzyme-reagent complex prior to covalent attachment. The presence of the gamma-phosphoryl group improves the performance of the affinity label: KI values for the fast phase are similar (about 100 microM), whereas kmax for 8-BDB-TATP is about three times greater than that of 8-BDB-TADP (0.286 min-1 vs 0.0835 min-1). After an 80-min incubation with 175 microM of either reagent, about 2 mol/mol of subunit is incorporated with 76% inactivation caused by 8-BDB-TADP and 97% inactivation by 8-BDB-TATP. Loss of activity is prevented by substrates, with the best protection afforded by a combination of ATP, Mn2+, K+, and phosphoenolpyruvate. Reaction of pyruvate kinase with either compound in the presence of protecting ligands leads to incorporation of about 1 mol of reagent/mol of subunit with only about 15% loss of activity. These results suggest that 8-BDB-TADP and 8-BDB-TATP react with two groups on the enzyme, one of which is at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.