甜菜碱对大鼠肝细胞膜表皮生长因子受体及其酪氨酸蛋白激酶的作用

目的：研究甜菜碱对大鼠肝细胞膜表皮生长因子（ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＥＧＦ）受体及其酪氨酸蛋白激酶（ｔｙｒｏｓｉｎｅｐｒｏｔｅｉｎｋｉｎａｓｅ，ＴＰＫ）活性的影响。方法：应用受体的放射配基结合分析法（ｒａｄｉｏｌｉｇａｎｄｂｉｎｄｉｎｇａｓａｙｏｆｒｅｃｅｐｔｏｒｓ，ＲＢＡ），比较实验组和对照组之间１２５ＩＥＧＦ与其受体的结合情况；受体酪氨酸蛋白激酶活性测定法，以大鼠肝细胞膜可溶性蛋白为酪氨酸蛋白激酶来源，比较实验组和阴性组之间受体蛋白自身磷酸化作用的差异。结果：２６ｎｍｏｌ·Ｌ－１～５．２ｍｍｏｌ·Ｌ－１的甜菜碱均能抑制正常大鼠肝细胞膜ＥＧＦ受体与其配基的结合，这种抑制作用为非竞争性抑制；１０与１００μｍｏｌ·Ｌ－１的甜菜碱可激活酪氨酸蛋白激酶活性，而１０ｍｍｏｌ·Ｌ－１的甜菜碱则抑制酪氨酸蛋白激酶活性。结论：甜菜碱可以抑制ＥＧＦ受体与其配基的结合并影响ＥＧＦ受体的酪氨酸蛋白激酶活性。     

Blockade of the spinal excitatory effect of cAMP on the startle reflex by intrathecal administration of the isoquinoline sulfonamide H-8: comparison to the protein kinase C inhibitor H-7

Drugs thought to inhibit the actions of protein kinase C (PKC) and cAMP dependent protein kinase (A-kinase) were infused intrathecally into the subarachnoid space of the lumbar region of the spinal cord, and the effects on acoustic startle were measured. Previous work has shown that intrathecal infusion of drugs thought to increase cAMP increase the startle response. The present experiment evaluated whether inhibition of A-kinase would prevent this effect. Rats were infused with the isoquinoline sulfonamide, H-8 (360 nmol) or vehicle (50% dimethyl sulfoxide), 30 min prior to infusion of 100 nmol of dibutyryl cAMP. By itself, H-8 had little effect on startle, but completely blocked the normal excitatory effect of dibutyryl cAMP on startle. In contrast, the isoquinoline sulfonamide, H-7, which is less active in blocking A-kinase, but more active in blocking PKC, did not block dibutyryl cAMP. Moreover, H-8 did not block the excitatory effect of intrathecal infusion of the 5-HT1A receptor agonist, 8-OH-dipropylaminotetraline (8-OH-DPAT). Thus, the blockade of dibutyryl cAMP by H-8 appears somewhat specific and suggests an involvement of A-kinase in the excitatory effects of dibutyryl cAMP on the acoustic startle response. In a second experiment, it was found that administration of the isoquinoline sulfonamide H-7 caused a marked, dose-dependent (150-800 nmol) facilitation of the startle reflex in comparison with its vehicle. Tris buffer (0.1 M). Like H-7, another PKC inhibitor, GT1b (20 nmol) produced a marked increase in the startle reflex versus its vehicle, 0.01 M phosphate buffer.(ABSTRACT TRUNCATED AT 250 WORDS)     

中国KM小鼠亚群遗传变异的研究

应用电泳方法和Festing下颌骨形态分析法对北京、上海、长春三大地区的四个中国KM小鼠的主要群体展开调查,并与近年从美国引进Swiss来源的NIH小鼠群体进行比较.结果显示:1.中国KM与NIH小鼠群体在Es-3,Es-10,Got-2,Glo-1,Gpt-1,和Mpl-1座位的等位基因组成存在显著差异;两者间遗传距离为0.131±0.011,证实中国KM小鼠为非Swiss来源的另一亚种。2.KM小鼠各亚群间等位基因分布无明显差异,亚群间遗传距离为0.008～0.027,与群体封闭时间成正相关.3.对群体生化和下颌骨形态学数据聚类分析表明S:KM在四个KM亚群中为特殊的一类,提示不同地区KM亚群存在的遗传差异可能对实验重复性产生影响.     

Protein kinase C is localized in focal contacts of normal but not transformed fibroblasts

Transformed cells differ from normal cells in that they fail to respond to normal signals for regulation of growth and differentiation. This disordered signal transduction probably contributes to maintenance of the transformed phenotype. Several lines of evidence suggest that changes in the Ca2(+)- and phospholipid-dependent protein kinase, protein kinase C (PKC), may be important for transformation. To determine the role of PKC in transformation, we compared the levels and subcellular distribution of total phorbol ester receptors and PKC in normal and SV40-transformed rat embryo fibroblasts (REF52 cells). We also used our alpha-PKC (Type 3)-specific monoclonal antibodies to compare alpha-PKC content and regulation. We found no differences in quantity or subcellular distribution of PKC in 100,000 x g soluble and pelletable fractions. Downmodulation, which represents a feedback loop for limiting PKC activity, occurs to the same extent in both cell types. A major difference between the normal and transformed cells was revealed by immunofluorescence of alpha-PKC. In normal cels, alpha-PKC is tightly associated with the cytoskeleton and appears to be organized into focal contacts because it colocalizes with talin. In contrast, in SV40-REF52 cells, alpha-PKC is not tightly associated with the cytoskeleton and does not colocalize with talin. The difference in subcellular localizations correlates with a loss of two alpha-PKC-binding proteins in the transformed cells. These results indicate that inappropriate subcellular location of alpha-PKC may contribute to maintenance of the transformed phenotype.     

PCNA、P21、P53在胃癌中的表达及其意义

目的 :研究PCNA、P2 1、P5 3在胃癌组织中的表达及其与临床病理因素的关系。方法 :应用免疫组化S P法检测 4 6例胃癌根治术标本中PCNA、P2 1、P5 3的表达情况 ,并与临床病理资料对比研究。结果 :4 6例胃癌PCNA、P2 1、P5 3表达阳性率分别是 80 .4 %、5 6 .5 %、4 5 .7% ,其中进展期胃癌P2 1的表达率 (6 2 .9% )明显高于早期胃癌 (36 .4 % ) ,三者与胃癌的组织学类型及分级无关。PCNA的表达与胃癌浸润深度和转移有关 ;P2 1表达与胃癌淋巴结和远处转移有关 ,而与浸润深度无关 ;P5 3的阳性表达与临床病理指标无明显相关 ;PCNA与P2 1、P5 3的表达有明显的协同性。结论 :PCNA与P2 1、P5 3表达可能与胃癌的发生、发展有关 ,是判断胃癌生物学行为的良好指标     

Effect of a Korean Traditional Formulation, Hwaotang, on Superoxide Generation in Human Neutrophils, Platelet Aggregation in Human Blood, and Nitric Oxide, Prostaglandin E2 Production and Paw Oedema Induced by Carrageenan in Mice

Hwaotang, a traditional Korean medicinal formulation, is a dried decoctum of a mixture of 7 herbal medicines, consisting of Angelica gigantis Radix, Rehmanniae radix, Paeoniae radix, Ciniamomi cortex, Cnidii rhizoma, Persicae semen and Carthami flos. We have investigated that Hwaotang water extract (HOT) has various effects on stimulus-induced superoxide generation in human neutrophils. The effects of HOT on superoxide generation in human neutrophils were investigated. HOT significantly inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid (AA). On the other hand, HOT enhanced superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) in a concentration-dependent manner. The superoxide generation induced by PMA with HOT was suppressed by staurosporine, an inhibitor of protein kinase C, but was not suppressed by genistein, an inhibitor of protein tyrosine kinase. Tyrosyl phosphorylation of a 58 kDa protein, which was increased by fMLP, was inhibited by HOT. HOT also inhibited the generation of a 47 kDa protein and platelet aggregation in human blood. The results suggest that protein tyrosine kinase participates in fMLP-mediated superoxide generation by HOT-treated human neutrophils. HOT inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. HOT reduced nitric oxide (NO) and prostaglandin E2 production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of iNOS, COX-2 or COX-1 was observed. HOT significantly reduced mouse paw oedema induced by carrageenan. Western blot analysis showed that HOT reduced the expression of iNOS and COX-2. The results indicate that HOT exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and prostaglandin E2 production, which could be due to a decreased expression of iNOS and COX-2.     

Oxidative stress in chemical toxicity

The toxic effects of compounds which undergo redox cycling via enzymatic one-electron reduction are reviewed. First of all, the enzymatic reduction of these compounds leads to reactive intermediates, mainly radicals which react with oxygen, whereby superoxide anion radicals are formed. Further oxygen metabolites are hydrogen peroxide, singlet oxygen and hydroxyl radicals. The role of these oxygen metabolites in toxicity is discussed.The occurrence of lipid peroxidation during redox cycling of quinonoide compounds, e.g., adriamycin, and the possible relationship to their toxicity is critically evaluated. It is shown that iron ions play a crucial role in lipid peroxidation induced by redox cycling compounds.DNA damage by metal chelates, e.g., bleomycin, is discussed on the basis of findings that enzymatic redox cycling of a bleomycin-iron complex has been observed. The involvement of hydroxyl radicals in bleomycin-induced DNA damage occurring during redox cycling in cell nuclei is claimed. Redox cycling of other substances, e.g., aromatic amines, is discussed in relation to carcinogenesis.Other chemical groups, e.g., nitroaromatic compounds, hydroxylamines and azo compounds are included. Other targets for oxygen radical attack, e.g., proteins, are also dealt with.It is concluded that oxygen radical formation by redox cycling may be a critical event in toxic effects of several compounds if the protective mechanisms of cells are overwhelmed.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Platelet-activating factor stimulation of p125 and p130 tyrosine phosphorylation in brain

The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat brain slices. PAF induced a time- and concentration-dependent increase in tyrosine phosphorylation of a doublet of approximately 125 kDa. These proteins were identified by immunoprecipitation as p125^FAK and p130^Cas, using monoclonal antibodies. This effect was mediated by PAF receptors, as shown by its inhibition by the action of a PAF antagonist. The tyrosine phosphorylation evoked by PAF was dependent, at least in part, on external calcium. The involvement of protein kinase C was demonstrated by the synergistic effect of TPA on PAF-stimulated tyrosine phosphorylation. The finding that PAF stimulates tyrosine phosphorylation of both focal adhesion protein p125^FAK and p130^Cas suggests that PAF might modulate the integrin mediated signal transduction in the brain.     

丝裂素活化蛋白激酶在大鼠心脏缺血预处理中的作用

目的 探讨丝裂素活化蛋白激酶家系（ＭＡＰＫｓ）在大鼠心脏缺血预处理（ＩＰＣ）保护中的作用。方法 在离体灌注的ＳＤ大鼠心脏缺血／再灌注（Ｉ／Ｒ）模型上，观察ＩＣＰ对于Ｉ／Ｒ后损伤的影响，并观察ＭＡＰＫｓ中三种激酶（细胞外信号调节激酶（ＥＲＫｓ）、蛋白激酶Ｐ３８（Ｐ３８）、应激活化蛋白激酶（ＳＡＰＫ））活性的变化及其佤保护作用的关系。结果 ＩＰＣ明显改善大鼠Ｉ／Ｒ后的心脏功能，减少Ｉ／Ｒ造成的心肌肌酸