Effects of tributyltin on barrier functions in human intestinal Caco-2 cells

The effect of tributyltin (TBT) on human intestinal epithelial cell functions was investigated by using human intestinal Caco-2 cell monolayers. We paid particular attention to the effect of TBT on two barrier functions: the tight junction as a physical barrier and MDR1/P-glycoprotein as a biological barrier. A loss of monolayer integrity was apparent from the TBT treatment and the paracellular permeability was increased by TBT. On the other hand, the activity of P-glycoprotein, which was examined by measuring the accumulation of Rhodamine-123 and daunomycin, was increased by prolonged TBT treatment in a concentration-dependent manner (1-100 nM). Furthermore, it was clarified by Western and Northern blots that this increase was accompanied by the increased expression of MDR1 mRNA and protein. The activation of a multidrug resistance transporter P-glycoprotein by TBT would cause a disorder of the human intestines by changing the drug pharmacokinetics.     

Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5-aminolevulinic acid

4,5-Dioxovaleric acid (DOVA) was synthesized from 5-bromolevulinic acid via formation of the pyridinium bromide of 5-bromolevulinic acid, followed by nitrone formation with p-nitrosodimethylaniline, and hydrolysis of the nitrone to yield DOVA. Partial purification of DOVA was obtained by passage of the reaction mixture through a cation exchange column. DOVA was identified by paper electrophoresis and by a specific fluorometric assay. DOVA was nonenzymatically transaminated to 5-aminolevulinic acid (ALA) with glycine serving as the amino donor. Other compounds tested were less effective amino donors. Glyoxylic acid was identified as a reaction product by paper electrophoresis and a specific calorimetric test. ALA was identified by paper electrophoresis, paper chromatography of a pyrrole derivative, reaction with Ehrlich reagent, and by its enzymatic conversion by a barley extract to porphobilinogen and uroporphyrin. The nonenzymatic transamination was inhibited by Tris and was stimulated by high pH. The existence of this nonenzymatic activity is discussed in relation to previous reports of dova transaminase activity in cell extracts.     

人FOXO3a基因真核表达载体的构建及其功能验证

目的:构建带myc标签的人FOXO3a基因真核表达载体,并对其功能进行初步检测。方法:采用PCR技术,从乳腺文库中扩增人FOXO3a基因,并将其正确插入pXJ-40-myc载体;将重组质粒与空载体分别转染人乳腺癌细胞系ZR75-1、MCF-7后,通过Western印迹检测其表达情况,并用CCK8法测定细胞生长曲线。结果:双酶切和测序鉴定表明myc-FOXO3a真核表达质粒构建成功,转染乳腺癌ZR75-1、MCF-7细胞后目的基因成功表达;细胞生长曲线结果显示,转染myc-FOXO3a的乳腺癌细胞较空载体细胞生长较慢。结论:构建了带myc标签的人FOXO3a基因真核表达载体,为进一步研究FOXO3a在乳腺癌中的功能奠定了基础。     

Release of3H-dopamine and Analogous monoamines from rat striatal tissue

1. The release of previously accumulated 3H-dopamine (DA) from minces of striatal tissue prepared from the brains of pargyline-pretreated rats was evaluated by superfusion with a physiological buffer solution in a six-chamber apparatus with silver toroid electrodes to provide electrical field stimuli. The identity of released tritium as 3H-DA was demonstrated chromatographically and 3H-DA taken up was found in a synaptosomal subcellular fraction. 2. Release of 3H-DA previously accumulated at 0.3 microM was found to be linearly dependent on stimulus intensity between 1 and 10 V (for 60 sec); 5 V was selected as a standard stimulus. 3. Release of 3H-DA did not occur from minces of rat liver, nor was there release of previously accumulated labeled urea or leucine from striatal tissue by electrical stimulation, 50 mM KCL, or 0.1 mM (+)-amphetamine. When 3H-DA was taken up in the presence of cocaine (1 mM) or benztropine (100 microM), electrically induced release of 3H-DA was markedly reduced, while spontaneous efflux was much less altered. 4. Release of 3H-DA was also induced by depolarizing concentrations of K+, as well as by Rb+ or NH4+, and by veratridine. Electrical release and that induced by 50 mM K+ or 100 microM veratridine was blocked by the omission of Ca2+ (with EDTA added) and that induced by veratridine was blocked by tetrodotoxin (30 microM).     

15-Hydroxyprostaglandin dehydrogenase can be induced by dexamethasone and other glucocorticoids at the therapeutic level in A549 human lung adenocarcinoma cells

Dexamethasone induced the expression of 15-PGDH in a time- and concentration-dependent manner in A549 human lung adenocarcinoma cells. Maximal induction was observed at 10nM. Induction of 15-PGDH expression was also achieved by other synthetic glucocorticoids. Induction was inhibited by the addition of pro-inflammatory cytokines and phorbol ester. These pro-inflammatory agents were also shown to induce COX-2 expression. PMA was found to be the most effective stimulator of COX-2 expression and the most potent inhibitor of dexamethasone-induced 15-PGDH expression. Attenuation of dexamethasone-induced 15-PGDH expression by PMA was, in part, due to a protein kinase C-mediated mechanism. The induction of 15-PGDH expression by dexamethasone was blocked by a glucocorticoid receptor antagonist RU 486 and by a nuclear translocation inhibitor geldanamycin, indicating that the induction is a genetic mechanism. The induction of 15-PGDH expression by dexamethasone and other glucocorticoids at the therapeutic level provides an additional biochemical mechanism for the anti-inflammatory action of these glucocorticoids.     

眼镜蛇毒中降压因子的提取及其降血压作用的研究

目的：从广西眼镜蛇蛇毒中分离纯化血管紧张素转换酶抑制剂(Angiotensin Converting Enzyme Inhibitor,ACEI),命名为降压因子（Hypotensive Factor,HF）,并测定其生物活性。方法：采用Sephacryl S-100凝胶过滤,CM Sepharose F.F.离子交换层析分离纯化HF,高效液相鉴定纯度,SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定其分子量,紫外分光光度法测定HF对血管紧张素转换酶（ACE）的抑制活性。用离体兔子十二指肠平滑肌测定HF增强缓激肽(Bradykinin,BK)的效应。结果：纯化的广西眼镜蛇蛇毒HF经SDS-PAGE检测显示单一条带,测得其相对分子量约为8.2kD,由十二种氨基酸组成,蛋白回收率为5.70%。HF对ACE有明显的抑制作用,其抑制作用与剂量呈正相关。IC50为1.02?g/ml。HF能增强BK对离体兔子十二指肠平滑肌的收缩效应。结论：本方法成功地从广西眼镜蛇蛇毒中纯化出降血压成分。该成分与血管紧张素转换酶抑制剂作用相似,对血管紧张素转换酶有明显的抑制作用。     

The mechanisms of reduction of protein mixed disulfides (dethiolation) in cardiac tissue

Dethiolation of proteins (reduction of protein mixed disulfides) by NADPH-dependent and glutathione (GSH)-dependent enzymes, and by nonenzymatic reaction with GSH, was studied by electrofocusing methodology with glycogen phosphorylase b and creatine kinase as substrates. Phosphorylase b was not rapidly dethiolated by reduced glutathione alone, but a cardiac extract catalyzed rapid dethiolation by both an NADPH-dependent and a GSH-dependent process. In contrast, creatine kinase was actively dethiolated by GSH. This GSH-dependent dethiolation was not enhanced by a soluble extract of bovine heart. Creatine kinase was also not dethiolated by an NADPH-dependent process. Partial purification of the phosphorylase dethiolases showed that the NADPH-dependent dethiolase had both a high-molecular-weight and a low-molecular-weight component The properties of these components were similar to those of thioredoxin and thioredoxin reductase. These two components were sensitive to inhibition by phenylarsine oxide and inhibition was reversed by addition of a dithiol. In contrast, GSH-dependent dethiolation required a single component of low molecular weight. This process was less sensitive to phenylarsine oxide inhibition. These studies show that two cytosolic proteins, phosphorylase b and creatine kinase, were dethiolated by different mechanisms. Phosphorylase b was dethiolated by both NADPH-dependent and GSH-dependent enzymes found in a soluble extract of bovine heart. In contrast, creatine kinase was rapidly dethiolated nonenzymatically by GSH alone.     

科尔沁固定沙地植被特征对降雨变化的响应

选择科尔沁固定沙地利用一种野外增减雨试验装置研究了沙地植被生长特征对降雨增减变化的响应。结果表明:(1)在6月,降雨增减变化对植物群落高度有显著影响(P0.05)。减雨60%和30%时,植物群落平均高度比对照分别降低8.8%和2.3%,增雨60%和30%时,则分别增加6.8%和1.4%;相比增雨60%,增雨30%更能促进群落盖度的增大;降雨量变化影响群落植株密度。(2)1a的降雨增减变化对沙地植被的多样性和均匀度均没有显著影响,但减雨可显著增加7月物种的丰富度(P0.05)。(3)随着降雨量的增加,地上生物量逐渐增大,在增雨30%时达到最大值;而地下生物量会随着降雨量增加而显著增大,同时,减雨60%也使地下生物量增加。此外,降雨量的增加和减少都会使地下与地上生物量的比值增加。(4)固定沙地地下生物量主要分布在0—20 cm之间,占总地下生物量的52.7%;降雨量的增加显著增加20—40 cm土壤中根系的分布,当降雨量减少60%时,20—40 cm土壤中根系的分布也略有增加,增雨60%和减雨60%对地下生物量在40—60 cm土层的分布具有明显的促进作用。     

MicroRNA-146a抑制胶质瘤细胞增殖的研究

目的：检测胶质瘤中miR-146a的表达水平,并研究miR-146a对胶质瘤细胞增殖的影响。方法：应用实时定量PCR的方法检测胶质瘤组织和癌旁组织中miR-146a的表达水平,采用脂质体细胞转染miRNA模拟物的方式过表达miR-146a,MTT法检测转染后细胞的增殖率,利用在线软件targetScan预测miRNA可能的靶基因。结果：miR-146a在胶质瘤组织中表达明显降低（P〈0.01）,相对表达水平为癌旁组织的35%,细胞转染miR-146a模拟物后,miR-146a表达明显增加,癌细胞增殖率明显降低（P〈0.01）,仅为原细胞的47%。Notch1基因是miR-146a影响胶质瘤细胞增殖活力的可能靶基因。结论：miR-146a可能通过抑制Notch1基因的表达调控胶质瘤细胞的增殖。     

miR-29a通过调控PTEN表达对脂多糖诱导人肺微血管内皮细胞损伤的保护作用研究*

目的:探讨miR-29a在脂多糖(LPS)诱导人肺微血管内皮细胞(HPMVECs)损伤中的作用及机制。方法:构建LPS损伤HPMVECs模型。RT-qPCR检测miR-29a表达变化;试剂盒测乳酸脱氢酶(LDH)释放量;MTT和流式细胞术分别检测细胞存活率及凋亡率;Western blot测蛋白质表达水平;Microcosm、starBase、Pictar、TargetScan软件预测 miR-29a的可能靶基因,双萤光素酶实验验证miR-29a和PTEN的靶向关系。结果:使用LPS处理HPMVECs,显著降低细胞中miR-29a的表达和细胞存活率,诱导LDH释放量和HPMVECs凋亡率增加,上调细胞中PTEN、Bim蛋白表达,下调p-Akt/Akt、p-FOXO3a/FOXO3a表达 (P<0.05);过表达miR-29a逆转LPS对HPMVECs的损伤作用。萤光素酶报告基因实验证实miR-29a 靶向PTEN,转染miR-29a mimics显著下调PTEN蛋白表达,转染miR-29a inhibitors明显上调PTEN蛋白表达 (P<0.05),但PTEN mRNA表达水平差异均无统计学意义(P>0.05)。结论:过表达miR-29a可能通过抑制PTEN蛋白的表达水平、激活Akt/FOXO3a/Bim信号通路对LPS致HUVECs的损伤发挥保护作用。     

The metabolic activation of cyanamide to an inhibitor of aldehyde dehydrogenase is catalyzed by catalase

The inhibition of aldehyde dehydrogenase by cyanamide is dependent on an enzyme catalyzed conversion of the latter to an active metabolite. The following results suggest that catalase is the enzyme responsible for this bioactivation. The elevation of blood acetaldehyde elicited by cyanamide after ethanol administration to rats was attenuated more than 90 percent by pretreatment with the catalase inhibitor, 3-amino-1,2,4-triazole. This attenuation was dose dependent and was accompanied by a reduction in total hepatic catalase activity. Although hepatic catalase was also inhibited by cyanamide, a positive correlation between blood acetaldehyde and hepatic catalase activity was observed. In vitro, the activation inhibitor, 3-amino-1,2,4-triazole. This attenuation was dose dependent and was accompanied by a reduction in total hepatic catalase activity. Although hepatic catalase was also inhibited by cyanamide, a positive correlation between blood acetaldehyde and hepatic catalase activity was observed. In vitro, the activation of cyanamide was catalyzed by a) the rat liver mitochondrial subcellular fraction, b) the 50-65% ammonium sulfate mitochondrial fraction and c) purified bovine liver catalase. Cyanamide activation was inhibited by sodium azide. Since much of the hepatic catalase is localized in the peroxisomes and since peroxisomes and mitochondria cosediment, the cyanamide activating enzyme, catalase, is likely of peroxisomal and mitochondrial origin.     

抗菌肽Cec4a的重组表达和抗菌活性研究*

目的:构建Cec4a的原核重组表达体系,通过诱导表达、酶切纯化获得重组蛋白,并检测产物的抗菌活性。方法:基于Cec4a的序列设计引物,克隆Cec4a基因的DNA片段。利用原核表达载体(pCold-SUMO)构建重组原核表达质粒,并将其转化到大肠杆菌C41(DE3)等感受态细胞,使用IPTG进行诱导表达。通过Ni-NTA亲和层析柱纯化,获得含有His-SUMO标签的重组Cec4a融合蛋白。在SUMO蛋白酶酶切后,再次使用Ni-NTA亲和层析纯化,得到目的蛋白,最后用鲍曼不动杆菌(ATCC19606)作为指示菌检测表达产物的抗菌活性。结果:成功构建pCold-SUMO-Cec4a原核表达质粒,测序分析其序列与预期结果一致。Cec4a融合蛋白表达量为42.8mg/L,纯化后的Cec4a重组蛋白对鲍曼不动杆菌的MIC为4 μg/mL。结论:通过原核表达,并经Ni-NTA亲和层析纯化,获得了具有抗菌活性的重组蛋白Cec4a,为研究Cec4a的生物活性、抗菌机制及应用奠定了基础。     

Factors influencing swimming in bay scallops, Argopectenirradians (Lamarck, 1819)

Factors influencing the probability, distance, and direction of swimming in bay scallops (Argopectenirradions Lamarck, 1819) were studied through a series of experimental releases in the field and in a 3-m tank. The probability of a scallop swimming was significantly influenced by the type of substratum on which it was released (sand vs. grassbed), by contact with two natural gastropod predators (Murex, Fasciolaria), and by the amount of rest allowed after a previous swim. The horizontal distance traveled by a swimming scallop was significantly influenced by artificial weight of a magnitude equivalent to a normal load of shell-encrusting organisms, by the amount of rest allowed after a previous swim, by the height attained in the water column, and by the scallop's size. The direction of scallop swimming was significantly influenced by the location along the mantle edge where a predator was contacted, and by factors probably related to the asymmetrical water flow pattern through the mantle cavity. Swimming in bay scallops apparently serves to maintain position in grassbeds and to avoid predators.     

人RHD基因的克隆和鉴定

目的克隆人RHD基因,并对其进行鉴定。方法以RhD阳性志愿者骨髓为材料,用TRIzol试剂提取总RNA;设计、合成人RHD基因扩增引物,RT-PCR方法扩增RHD基因片段;T／A克隆后将其亚克隆人pET28a（＋）载体中,经酶切、PCR和测序对重组质粒进行鉴定。结果骨髓总RNA被成功提取;RT-PCR成功扩增出RHD基因片段,其大小与预期约509bp基本一致;T／A克隆后再将其亚克隆,通过酶切和PCR证明RHD基因成功亚克隆入pET28a（＋）载体中;基因测序结果比对显示,与已公布的RHD基因（GenBank登录号为NM016124）序列基本一致,同源性为98％。结论成功克隆了RHD基因,这将为进一步研究奠定基础。     

Saccharomyces cerevisiae DNA-dependent RNA polymerase III: a zinc metalloenzyme.

Yeast nuclear RNA polymerase III was purified by batch adsorption to phosphocellulose, followed by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on DNA-Sepharose. Polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band which contained polymerase activity. The molecular weight estimated by sedimentation velocity centrifugation in a glycerol gradient was 380 000. Enzyme activity was inhibited 50% at 0.1 mM 1,10-phenanthroline and 100% of 1.0 mM, but was restored when 1,10-phenanthroline was removed by dialysis. Enzyme activity was not inhibited by 7,8-benzoquinoline, a nonchelating structural analogue of 1,10-phenanthroline. These results strongly suggest that inhibition of enzyme activity occurs by the formation of a reversible enzyme-zinc-phenanthroline ternary complex. The zinc content, measured by atomic absorption spectroscopy, was 2 g-atoms per mol of enzyme. Zinc was not removed from the enzyme by gel filtration on Sephadex G-25, by passage through Chelex-100 resin, or by dialysis against buffer containing 1,10-phenanthroline. Enzyme-bound zinc was removed by dialysis after denaturation of the enzyme with heat and sodium dodecyl sulfate. Enzyme-bound zinc did not exchange with free zinc. These results establish yeast nuclear RNA polymerase III as a zinc metalloenzyme.     

Glutamine synthetase and glutamate synthase activities during growth and sporulation in Bacillus subtilis.

Glutamine synthetase in Bacillus subtilis 168 was repressed to a greater extent by L-glutamine or L-arginine than by ammonia when each was used as sole nitrogen source. It was derepressed when either L-glutamate or nitrate was used as nitrogen source. Glutamate synthase was repressed by L-glutamate or L-arginine and, to a lesser extent, by L-glutamine but was derepressed during growth with ammonia or nitrate. Glutamine synthetase activity was unaltered during the onset of sporulation. Glutamate synthase activity, however, underwent a small and apparently transient increase in bacteria induced to sporulate by nitrogen limitation.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

Responses of the rat cardiovascular system to substance P,neurotensin and bombesin

The carotid arterial blood pressure and heart rate responses to intravenous injections of substance P, neurotensin and bombesin were compared in anaesthetized rats. In rats anaesthetized with urethane neurotensin produced only a fall in blood pressure but in rats anaesthetized with sodium thiobutabarbitone, the fall was preceded by a transient rise in blood pressure. The reason for the different responses to neurotensin with the two anaesthetics was not investigated. The hypotensive effect of neurotensin observed with both anaesthetics was abolished by mepyramine and therefore appeared to be mediated by action on H₁ receptors either of neurotensin directly or of histamine released. On the other hand, catecholamines might be implicated in the pressor response to neurotensin observed in rats anaesthetized with sodium thiobutabarbitone since it was reduced by phentolamine and hexamethonium. Low doses of substance P produced a depressor response which was not inhibited by the antagonists tested. At higher doses marked tachycardia occurred and the depressor response was less and was often followed by a pressor response. The tachycardia was abolished by propranolol but not by cervical cord section or by hexamethonium. Bombesin produced a pressor response which was unaffected by hexamethonium but was reversed to depressor by phentolamine. This depressor response to bombesin was abolished by propranolol. It was concluded that substance P produced a depressor response by action on its own specific receptors and tachycardia by catecholamine release whereas neurotensin and bombesin produced cardiovascular actions which were mediated entirely by amine release.     

HBsAg/GM-CSF融合蛋白在毕赤酵母中的表达及其免疫原性研究

为探索一种提高乙肝病毒表面抗原免疫原性的新方法,用PCR和基因重组技术构建HBsAg与GM-CSF的融合基因,并在毕赤酵母中分泌表达HBsAg/GM-CSF(S-GM)融合蛋白。表达产物用SDS-PAGE检测,W estern b lot分析,离子交换柱纯化后免疫昆明鼠,ELISA检测免疫小鼠血清中抗HBsAg的抗体水平。结果显示S-GM融合蛋白在毕赤酵母中获得了表达,离子交换柱一步纯化即可得到纯度达90%以上的S-GM。W estern b lot分析S-GM可分别与抗HBsAg及抗GM-CSF的抗体特异结合。ELISA检测发现第一次免疫后4w出现抗HBsAg的抗体,加强免疫后融合蛋白组几乎全部阳转,且抗体水平较HBsAg组(P=0.009<0.05)及HBsAg和GM-CSF的混合物组(P=0.032<0.05)高。HBsAg/GM-CSF融合蛋白能够在毕赤酵母中表达,且可增强HBsAg的免疫原性,为提高乙肝疫苗的免疫效果提供了新的思路与方法。     

Influence of Thickeners on the Fragmentation of Fish Meat Sausage by Mastication

The influence of food thickeners (potato starch, guar gum, and xanthan gum and deionized water) on the breakdown of solid food was numerically analyzed, and an investigation was made into the cumulative size distribution of food fragments, textural properties, sensory evaluation and maximum transit velocity of a bolus in the pharynx.

The results suggest that evaluating the breakability into small pieces was easily influenced by the addition ratio of the dispersion medium. However, in respect of the destruction process for the solid body, each sample was more strongly affected by the type of the dispersion medium than by the addition ratio of this medium.

The destruction process was strongly influenced by the history of the breakdown caused by mastication when a liquid dispersion medium was added to the solid. However, when a high-viscosity sol was added to the solid, the destruction process was random and not affected by any history.