NMR法研究天花粉蛋白TDK肽片段的溶液构象

用ＨＮＭＲ法测定ＴＤＫ肽在Ｈ２Ｏ（ＨＯＤＫ）,５０％六氟丙醇（ＦＰＤＫ）和２ｍｏｌ／ＬＧｕ．ＨＣｌ（ＧＵＤＫ）溶液构象。在ＨＯＤＫ和ＦＰＤＫ中,ＴＤＫ肽的两段序列Ａｓｐ０－Ｉｌｅ４,Ｓｅｒ９－Ｉｌｉ１７分别具有较稳定的α－螺旋含量;而ＧＵＤＫ的ＳＡＬＳ序列仍能检测到有序残存结构。并假设ＳＡＬＳ序列是肽链形成二级结构的原始核心。     

炎症介质对离体大鼠肠系膜动脉降钙素基因相关肽释放的影响

本工作目的是在离体大鼠肠系膜动脉床灌流模型上,观察几种常见炎症介质：前列腺素Ｅ２（ＰＧＥ２）、缓激肽（ＢＫ）、组胺（ＨＩＳ）、血小板活化因子（ＰＡＦ）及５－羟色胺（５－ＨＴ）对血管周围感觉神经介质ＣＧＲＰ释放的直接影响。结果显示：ＰＧＥ２（１－１００μｍｏｌ／Ｌ）和ＢＫ（５－１０μｍｏｌ／Ｌ）能引起大鼠肠系膜动脉床时间和浓度依赖性地释放ＣＧＲＰ。ＨＩＳ,ＰＡＦ和５－ＨＴ则未见明显作用。结果提示,ＰＧＥ２与ＢＫ可能是引起血管周围感觉神经兴奋和ＣＧＲＰ释放的主要炎症介质。     

下丘脑室旁核β－内啡肽在大鼠烫伤休克中的作用

以１００℃沸水接触雄性大鼠去毛的背部２０ｓ,形成占体表面积２０％的三度烫伤。收集下丘脑室旁核推挽灌流液测定β－内啡肽免疫活性物质的含量;向下丘脑室旁核微量注射β－内啡肽或其抗血清,观察烫后心血管功能指标的改变及存活时间。结果表明,烫后下丘脑室旁核灌流液中β－内啡肽免疫活性物质含量显著升高,有两个峰值。烫后给予β－内啡肽抗血清,可显著改善心血管功能指标（ＭＡＰ,ｄＰ／ｄｔｍａｘ,Ｌｖｓｐ和ＨＲ）,延长存活时间;给予β－内啡肽则作用相反。上述观察表明下丘脑β－内啡肽的过量增多是促使休克加重和加快死亡的重要原因之一。     

HMR法研究天花粉蛋白TDK肽片段的溶液构象

用１ＨＮＭＲ法测定ＴＤＫ肽在Ｈ２Ｏ（ＨＯＤＫ）,５０％六氟丙醇（ＦＰＤＫ）和２ｍｏｌ／ＬＧｕ·ＨＣｌ（ＧＵＤＫ）中的溶液构象。在ＨＯＤＫ和ＦＰＤＫ中,ＴＤＫ肽的两段序列Ａｓｐ０～Ｉｌｅ４,Ｓｅｒ９～Ｉｌｅ１７分别具有较稳定的α－螺旋含量;而ＧＵＤＫ的ＳＡＬＳ序列仍能检测到有序残存结构。并假设ＳＡＬＳ序列是肽链形成二级结构的原始核心。     

u-PA/t-PA嵌合型纤溶酶原激活剂(ut-PA)的构建、表达、纯化和性质研究

将尿激酶原（ｐｒｏ－ＵＫ）ｃＤＮＡ和组织型纤溶酶原激活剂（ｔ－ＰＡ）Ａ链ｃＤＮＡ克隆到Ｍ１３ｍｐ１８中,经过二次寡核甘酸诱导的大片段定点删除和一次寡核苷酸诱导的多位点突变,得到ｕ－ＰＡ（Ｌｅｕ１４４－Ｇｌｙ４０８）／ｔ－ＰＡ（Ｓｅｒ１－Ｔｈｒ２６３）（ｕｔ－ＰＡ）融合基因．将ｕｔ－ＰＡ融合基因克隆到表达载体ｐＣＭ－βｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞,筛选稳定表达株．收集无血清表达上清,经苯甲脒柱纯化得到ｕｔ－ＰＡ纯品,ＳＤＳ－ＰＡＧＥ和纤维蛋白自显影显示ｕｔ－ＰＡ有两种分子量形式,分子量分别为６８ｋＤ和６１ｋＤ．纤维蛋白亲和性试验表明,ＬＵＫ（低分子量尿激酶）对纤维蛋白没有亲和性,而含有ＬＵＫ的ｕｔ－ＰＡ则对纤维蛋白表现出很强的亲和性,但ｕｔ－ＰＡ的亲和性略低于亲本ｔ－ＰＡ．     

南方鲇碱性磷酸酶的分离纯化及部分性质的研究

用正丁醇抽提,硫酸铵分级沉淀,ＤＥＡＥ－纤维素和ＳｅｐｈａｃｒｙｌＳ－２００柱层析,从南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ　Ｃｈｅｎ）肠粘膜中提取出碱性磷酸酶（ＡＫＰ）。提纯倍数为３９．５０倍,比活为６８．３５μ／ｍｇ蛋白,提取酶液经ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ只呈现一条区带。该酶的分子量为１３２１４０,Ｎ末端氨基酸为门冬氨酸,最适ｐＨ为１０．１０,７．５＞ｐＨ＞１１．５时不稳定,最适温度为４０℃左右,对热不很稳定,以磷酸苯二钠为底物其Ｋ_ｍ值为１．７２×１０~（－３）ｍｏｌ／Ｌ。Ｍｇ~（２＋）、Ｍｎ~（２＋）为该酶的激活剂,ＫＨ_２ＰＯ_４、Ｌ－ＣｙＳ、ＭＥ、ＤＦＰ、ＥＤＴＡ－Ｎａ_２为抑制剂。选用ＫＨ_２ＰＯ_４和ＤＦＰ作抑制类型的判断,结果表明,ＫＨ_２ＰＯ_４属竞争性掏剂,其抑制常数为２．３ｍｍｏｌ／Ｌ;ＤＦＰ为非竞争性抑制剂,抑制常数为１．０５ｍｍｏｌ／Ｌ。     

点状产气单胞菌脯氨酰内肽酶在大肠杆菌中的高效表达

采用ＰＣＲ分段克隆法将点状产气单胞菌点状亚种（Ａｅｒｏｍｏｎａｓ ｐｕｃｔａｔａ ｓｕｂｓｐ．ｊｐｕｃｔａｔａ）脯氨酰内肽酶（ｐｒｏｌｙｌ ｅｎｄｏｐｅｐｔｉｄａｓｅ,ａｐＰＥＰ）的编码区基因分成３段扩增并拼接成编码６９０个氨基酸的完整基因ａｐＰＥＰ,将其克隆在表达载体ｐＢＬ和ｐＫＫＨ上,构建成温度和ＩＰＴＧ诱导型高效表达ａｐＰＥＰ的重组大肠杆菌ＢＬ２１／ｐＢＬ－ＰＥＰ和ＢＬ２１／ｐＫＫＨ－ＰＥＰ     

刺激大鼠视上核后电针“足三里”对蓝斑灌流液中去甲肾上腺素含量的影响

运用行为测痛结合蓝斑（ＬＣ）灌流液去甲肾上腺素（ＮＥ）的高压液相（ＨＰＬＣ）测定；观察 大鼠痛阈（ＰＴ）与ＬＣ灌流液中去甲肾上腺素含量变化间的相互关系，结果表明：（ｌ）视上核 （ＳＯＮ）内注射 １０μｍＬ－谷氨酸（Ｌ－ｇｌｕｔａｍｉｃａｃｉｄ， Ｌ－Ｇｌｕ）后 ３０分钟，大鼠ＰＴ较注射前增加１３３． ２± ２１．４％，此时ＬＣ 灌流液中ＮＥ含量从注射前的４３７．３±２０．４ｎｇ／ｍｌ降到２２９．２±１１．９ｎｇ／ｍｌ，注射 后６０分钟ＰＴ仍比注射前高８３．９±１４．７％，而灌流液中ＮＥ的含量为３２８．６±２８．０ｎｇ／ｍｌ，与人工 脑脊液（ＡＣＳＦ）对照组相比有非常明显的差别（Ｐ＜０．０５～０．００１）。（２） ＳＯＮ注射Ｌ－Ｇｌｕ后，电 针足三里３０分钟（Ｌ－ＧＩＵ＋ＥＡ组）增加到注射前的１８８．２±２３．９％，同ＡＣＳＦ电针组（ＡＣＳＦ＋ ＥＡ）的 ９４．９±７．１％相比有明显差异（Ｐ＜０．０１）。此时ＬＣ灌流液中ＮＥ的含量虽较注射前都明显 降低、分别为１３７．６±７．５ｎｇ／ｍｌ和 １５１，１±１１．５ｎｇ／ｍｌ，但两者相比无明显差异。停针后３０分钟Ｌ－ Ｇｌｕ＋ＥＡ组的ＰＴ仍比注射前高１３３．８±２７．９％，明显高于     

固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌Ａ１５０１菌体经超声破碎后,无细胞粗提液以ＰＥＧ－６０００分级沉淀,丙酮沉淀,再经蓝球脂糖亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ＫＤ和６４５ｋＤ,亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值。在以Ｇｌｕ为源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ）,５０ｍｍｏｌ／Ｌ（ＡＴＰ     

人Mn—SOD cDNA的克隆及高效表达

用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）,以人肝细胞株（Ｌ０２）总ＲＮＡ为模板,扩增了人锰超氧化物歧化酶（ｈＭｎ－ＳＯＤ）的ｃＤＮＡ。重组到Ｔ７启动子控制下的表达载体ｐＥＴ－２４ａ（＋）中,构建表爱质粒ｐＥＴ－ＭｎＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳ－ＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代－β－Ｄ－半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为２２ｋＤ的蛋白质,与抗人     

褪黑素减弱大鼠下丘脑弓状核β-内啡肽免疫反应的强度

为探讨褪黑素（ＭＥＬ）镇痛作用的机制,本文采用免疫组化方法结合计算机图像处理技术,观察了注射ＭＥＬ对大鼠下丘脑弓状核内神经细胞的β－内啡肽免疫反应的影响。实验大鼠分约药组及对照组,分别腹腔注射ＭＥＬ１１０ｍｇ／ｋｇ或配药液,１ｈ后灌注取脑、冰冻切片,进行免疫组化染色,计算机图像处理技术测定染色脑片积分光密度（ＩＯＤ）和平均光密度（ＯＤ）。结果显示,给药组大鼠弓状核内β－内啡肽免疫反应明显减弱,ＩＯ     

Arginine vasopressin induces periaqueductal gray release of enkephalin and endorphin relating to pain modulation in the rat

Previous study has proven that microinjection of arginine vasopressin (AVP) into periaqueductal gray (PAG) raises the pain threshold, in which the antinociceptive effect of AVP can be reversed by PAG pretreatment with V2 rather than V1 or opiate receptor antagonist. The present work investigated the AVP effect on endogenous opiate peptides, oxytocin (OXT) and classical neurotransmitters in the rat PAG. The results showed that AVP elevated the concentrations of leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek) and beta-endorphin (beta-Ep), but did not change the concentrations of dynorphinA(1-13) (DynA(1-13)), OXT, classical neurotransmitters including achetylcholine (Ach), choline (Ch), serotonin (5-HT), gamma-aminobutyric acid (GABA), glutamate (Glu), dopamine (DA), norepinephrine (NE) and epinephrine (E), and their metabolic products in PAG perfusion liquid. Pain stimulation increased the concentrations of AVP, L-EK, M-Ek, beta-Ep, 5-HT and 5-HIAA (5-HT metabolic product), but did not influence the concentrations of DynA(1-13), OXT, the other classical neurotransmitters and their metabolic products. PAG pretreatment with naloxone - an opiate receptor antagonist completely attenuated the pain threshold increase induced by PAG administration of AVP, but local pretreatment of OXT or classical neurotransmitter receptor antagonist did not influence the pain threshold increase induced by PAG administration of AVP. The data suggested that AVP in PAG could induce the local release of enkephalin and endorphin rather than dynophin, OXT and classical neurotransmitters to participate in pain modulation.     

The effect of altered 5-hydroxytryptamine levels on beta-endorphin content in rat brain

The purpose of the present study was to examine the effect of altering the concentration of 5-hydroxytryptamine (5-HT) on beta-endorphin (beta-Ep) content in the hypothalamus, thalamus, and periaqueductal gray (PAG)-rostral pons regions of the rat brain. The selective 5-HT reuptake inhibitor, fluoxetine (10 mg/kg), significantly lowered beta-Ep content in the hypothalamus and the PAG. Parachlorophenylalanine, which inhibits 5-HT synthesis, significantly elevated beta-Ep in all brain parts studied. Intracisternal injections of the neurotoxin, 5',7'-dihydroxytryptamine, with desmethylimipramine pretreatment, significantly increased beta-Ep content in the hypothalamus and the PAG. In adrenalectomized rats, fluoxetine significantly decreased beta-Ep levels in the hypothalamus and increased the levels in the PAG. The results indicate that 5-HT may modulate the levels of brain beta-Ep.     

Induction of lipid peroxidation in hamster organs by the carcinogen cadmium: melioration by melatonin

Cadmium is a well-known human carcinogen. Lipid peroxidation is involved in cadmium-related toxicity and carcinogenesis. Melatonin is an effective antioxidant and free radical scavenger. The potential protective effects of melatonin against cadmium-induced lipid peroxidation in hamster brain, heart, kidney, testes, lung, and liver were examined. Lipid peroxidation was induced by intraperitoneal injection of cadmium chloride [single dose of 1 mg/kg body weight (bw)]. To test whether melatonin would protect against the toxicity of the carcinogen, the melatonin was injected peritoneally at a dose of either 15 mg/kg bw or 5 mg/kg bw, 0.5 h before cadmium treatment and thereafter at 8 h intervals during the day in the 48 h interval following the cadmium injection. One group of hamsters received only a single melatonin injection (a dose of 15 mg/kg bw, 30 min prior to cadmium). Forty-eight hours after cadmium injection, lipid peroxidation increased in brain, heart, kidney, testes, and lung. Either multiple injections of melatonin at both the 5 and 15 mg/kg bw doses, or a single injection of 15 mg/kg bw, prevented the cadmium-related increases in lipid peroxidation in brain, heart and lung. Cadmium-induced lipid peroxidation in kidney was prevented by melatonin when it was given as a single dose of 15 mg/kg bw. Melatonin slightly, but not significantly, reduced cadmium-induced lipid peroxidation in testes. It is concluded that cadmium toxicity, at least with regard to the resulting lipid peroxidation, is reduced by administering melatonin. This revised version was published online in July 2006 with corrections to the Cover Date.     

家兔中脑导水管周围灰质中注射八肽胆囊收缩素（CCK—8）拮抗吗啡镇痛和...

We have reported that intracerebroventricular (i. c. v.) injection of 1-4 ng of CCK-8 to the rat produced a remarkable antagonistic effect on morphine analgesia. In order to study the species specificity and the site of action, CCK-8 was microinjected into the PAG of the rabbit, and its influence on morphine analgesia and electroacupuncture analgesia was observed. The latency of the escape response (ERL) to radiant heat focused on the snout was measured as an index of the pain threshold. Microinjections were made via cannulae chronically implanted into the PAG. The drug solutions were delivered in a volume of 1 microliter, at a speed of 0.125 microliter/min. The ERL was measured for a period of 60 or 70 minutes at 10 min intervals. 1. CCK-8 administered unilaterally to the PAG of the rabbit at a dose of 3 ng antagonized the analgesia induced by morphine (4 mg/kg, i. v.) by 73% (P less than 0.001), and reduced the analgesic effect of electroacupuncture by 67% (P less than 0.001). These effects were dose-dependent within the range from 1.5 ng to 6.0 ng. The effect of CCK-8 was reversed by CCK receptor blocker proglumide (4 microliters, intra-PAG injection). Unsulfated CCK-8 (CCK-us) had no effect in this regard. These results indicate that in the PAG of the rabbit, exogenously administered CCK-8 was capable of antagonizing opioid analgesia by the activation of CCK receptors. 2. Two groups of rabbits were given with morphine (2 mg/kg, i. v.) and simultaneous injection of CCK-8 antiserum (CCK-AS, 1 microliter) or normal rabbit serum (NRS) into the PAG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noradrenergic Inhibition and α2-Adrenergic Stimulation of Melatonin Secretion in the Pigeon

Adrenergic regulatory mechanisms of melatonin synthesis and secretion were studied in the pigeon in vivo. Late-afternoon intraperitoneal injection of noradrenaline (NA; 1 mg/kg) resulted in a significant decrease in plasma melatonin levels in 3 h. The same effect was seen after phenylephrine treatment (1 mg/kg i.p.), indicating that an alpha 1-adrenergic mechanism may mediate the inhibition. Propranolol treatment had no effect on plasma melatonin levels, supporting this concept. Detomidine (1 mg/kg i.p.), an alpha 2-adrenergic agonist, increased melatonin levels. This stimulatory effect was blocked by yohimbine, an alpha 2-adrenergic antagonist. However, yohimbine alone had no effect on the plasma melatonin levels, suggesting that alpha 2-adrenergic transmission is not primarily responsible for the nocturnal stimulation of melatonin synthesis and secretion in the pigeon.     

慢性吗啡耐受大鼠脑内孤啡肽生成与释放增加

本文彩放射免疫分析法测定了慢性吗啡耐受过程中大鼠脑室灌流液、中脑导水管周围灰质（ＰＡＧ）及杏仁核中孤啡肽（ＯＦＱ）免疫活性的动态变化。结果观察到：（１）大鼠连续５ｄ皮下注射递增剂量的盐酸吗中民慢性吗啡耐受,其脑室灌流中ＯＦＱ－ｉｒ随吗啡注射剂量和注射次数的增加逐渐上升,第５ｄ注射后较对照组升高了５２％;（２）皮下注射吗啡１ｄ、３ｄ、５ｄ的大鼠ＰＡＧ中ＯＦＱ－ｉｒ比对照组分别升高了１７％、４８％和８     

Effect of pineal indoles on activities of the antioxidant defense enzymes superoxide dismutase, catalase, and glutathione reductase, and levels of reduced and oxidized glutathione in rat tissues.

Male Sprague-Dawley rats were randomly divided into four groups. Two of the groups received a single intraperitoneal injection of melatonin and 5-methoxytryptamine (5 mg/kg body weight), respectively, at 9 PM. One group received an intraperitoneal injection of 5-methoxytryptophol (5 mg/kg body weight) at 9 AM. The remaining group received alcoholic saline (vehicle) and served as the control. All rats were sacrificed 90 min after injection and the livers, kidneys, and brains were dissected. The activities of superoxide dismutase, catalase, and glutathione reductase in the organs were measured. It was found that both melatonin and 5-methoxytryptamine were approximately equipotent in enhancing the activities of superoxide dismutase and glutathione reductase in the kidney and liver, while 5-methoxytryptophol displayed a weaker effect. Both melatonin and 5-methoxytryptamine augmented the level of reduced glutathione in the kidney and liver, while 5-methoxytryptophol did so only in the kidney. All three pineal indoles increased the activity of superoxide dismutase and lowered the ratio of oxidized to reduced glutathione in the brain.     

Through V2, not V1 receptor relating to endogenous opiate peptides, arginine vasopressin in periaqueductal gray regulates antinociception in the rat

Our previous study has proven that central arginine vasopressin (AVP) plays an important role in antinociception, and pain stimulation raises AVP concentration in the periaqueductal gray (PAG). The nociceptive effect of AVP in PAG was investigated in the rat. The results showed that microinjection of AVP into PAG increased pain threshold, whereas microinjection of V2 receptor antagonist-d(CH2)5[d-Ile2, Ile4, Ala9-NH2]AVP into PAG decreased pain threshold in a dose-dependent manner, but local administration of V1 receptor antagonist-d(CH2)5Tyr(Me)AVP did not change pain threshold; Pain stimulation elevated AVP, Leucine-enkephalin (L-Ek), Methionine-enkephalin (M-Ek) and beta-endorphin (beta-Ep), not dynorphinA(1-13) (DynA(1-13)) concentrations in PAG perfuse liquid; PAG pre-treatment with naloxone, an opiate receptor antagonist or V2 receptor antagonist completely reversed AVP-induced increase in pain threshold, however, PAG pre-treatment with V1 receptor antagonist did not influence this effect of AVP administration. The data suggest that AVP in the PAG, through V2 rather than V1 receptor, regulates antinociception, which progress relates to enkephalin and endorphin.     

In vivo and in vitro effects of beta-endorphin on glucose metabolism in the rat

The effects of beta-endorphin (beta-Ep) on plasma glucose levels in rats and on glucose metabolism in isolated rat liver cells were examined. Intravenous injection of beta-Ep (5 micrograms/100 g BW) into ether-anaesthetized rats resulted in prompt and sustained hyperglycaemia with increases in the plasma glucagon and somatostatin levels and decrease in the plasma insulin level. When liver cells isolated from fed rats were incubated in the presence of beta-Ep at concentrations of 6 X 10(-8) M to 6 X 10(-7) M, glucose release into the medium increased within 15 min in a dose-related manner. Time course experiments showed that beta-Ep increased the level of cyclic AMP within 3 min. Significant increase in gluconeogenesis in liver cells isolated from fasted rats was also observed on addition of 10(-7) M beta-Ep in the presence of 10 mM L-lactate. These results suggest that the hyperglycaemia induced by beta-Ep may be caused, at least in part, by the effects of beta-Ep on releases of pancreatic hormones and glucose production in liver cells.