Type I iodothyronine deiodinase is a selenocysteine-containing enzyme.

Although thyroxine (3,5,3',5'-tetraiodothyronine, T4) is the principal secretory product of the vertebrate thyroid, its essential metabolic and developmental effects are all mediated by 3,5,3'-triiodothyronine (T3), which is produced from the prohormone by 5'-deiodination. The type-I iodothyronine deiodinase, a thiol-requiring propylthiouracil-sensitive oxidoreductase, is found mainly in liver and kidney and provides most of the circulating T3(1) but so far this enzyme has not been purified. Using expression cloning in the Xenopus oocyte, we have isolated a 2.1-kilobase complementary DNA for this deiodinase from a rat liver cDNA library. The kinetic properties of the protein expressed in transient assay systems, the tissue distribution of the messenger RNA, and its changes with thyroid status, all confirm its identity. We find that the mRNA for this enzyme contains a UGA codon for selenocysteine which is necessary for maximal enzyme activity. This explains why conversion of T4 to T3 is impaired in experimental selenium deficiency and identifies an essential role for this trace element in thyroid hormone action.     

优选配比条件下海藻玉壶汤加减海藻甘草反药组合对大鼠甲状腺肿大的治疗作用

 探讨海藻玉壶汤加减海藻甘草反药组合对大鼠甲状腺肿大的治疗作用。实验将Wistar 大鼠随机分为7 组,空白组灌服生理盐水,其余各组连续14 d灌服丙硫氧嘧啶复制甲状腺肿大模型,以优甲乐作为阳性对照药,各组分别给予相应治疗药物,1次/d,连续28 d。测定甲状腺系数、三碘甲状腺原氨酸(T₃)、四碘甲状腺原氨酸(T₄)、游离三碘甲状腺原氨酸(FT₃)、游离四碘甲状腺原氨酸(FT₄)、促甲状腺激素(TSH)、促甲状腺激素释放激素(TRH)水平,采用苏木精-伊红染色法(HE 染色)观察各组大鼠甲状腺组织形态。结果显示,海藻玉壶汤全方组、去海藻组、去甘草组以及去海藻甘草组的甲状腺系数较模型组明显降低,具有统计学差异(P<0.05);给药各组T₃、T₄水平较模型组有升高的趋势,但无统计学差异;给药各组对TSH 的水平有明显降低,具有统计学差异(P<0.05);全方组的TRH 水平较模型组明显降低,具有统计学差异(P<0.05),去海藻组、去甘草组和去海藻甘草组有降低的趋势,但无统计学差异。HE 染色镜下结果显示,给药各组对甲状腺肿大均有恢复作用,全方组效果最为显著。根据检测结果,优选配比条件下,含反药配伍的海藻玉壶汤全方组对甲状腺肿大的恢复作用以及甲状腺各项指标回调效果优于全方去单味海藻、甘草或同时去掉海藻甘草的配伍组合。     

Purification and cloning of amyloid precursor protein beta-secretase from human brain

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.     

Independent phosphatidylinositol synthesis in pituitary plasma membrane and endoplasmic reticulum

Phosphatidylinositol (PtdIns), the most abundant phosphoinositide, is the precursor of phosphatidylinositol 4-monophosphate which is converted to phosphatidylinositol 4,5-bisphosphate, the lipid hydrolysed as an early step in signal transduction by many stimuli. It is generally thought that a single enzyme in the endoplasmic reticulum, PtdIns synthase (CDP-diglyceride:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11), is responsible for PtdIns synthesis and that newly synthesized PtdIns is transported to the plasma membrane by exchange proteins. Several investigators have proposed that there are two functionally distinct pools of PtdIns, one responsive to stimulation and the other not, and that the stimulus-responsive pool may be synthesized at a different site within the cell, perhaps within the plasma membrane. Indeed, it was suggested that there is PtdIns synthase activity in plasma membrane isolated from rat liver. GH3 rat pituitary tumour cells are an excellent model system to study stimulation of phosphoinositide metabolism by thyrotropin-releasing hormone (TRH). Conversion of PtdIns to polyphosphoinositides and TRH (and GTP)-activated phosphoinositide hydrolysis are known to occur in plasma membrane isolated from GH3 cells. Here we report that PtdIns synthase activity in the plasma membrane of GH3 cells is distinct from that present in the endoplasmic reticulum. The plasma membrane PtdIns synthase may be responsible for a portion of PtdIns re-synthesis that occurs during cell stimulation.     

A single human gene encoding multiple tyrosine hydroxylases with different predicted functional characteristics

Catecholaminergic systems in discrete regions of the brain are thought to be important in affective psychoses, learning and memory, reinforcement and sleep-wake cycle regulation. Tyrosine hydroxylase (TH) is the first enzyme in the pathway of catecholamine synthesis. Its importance is reflected in the diversity of the mechanisms that have been described which control its activity; TH levels vary both during development and as a function of the activity of the nervous system. Recently, we deduced the complete amino-acid sequence of rat TH from a complementary DNA clone encoding a functional enzyme. Here we demonstrate that, in man, TH molecules are encoded by at least three distinct messenger RNAs. The expression of these mRNAs varies in different parts of the nervous system. The sequence differences observed are confined to the 5' termini of the messengers and involve alternative splicing events. This variation has clear functional consequences for each putative form of the enzyme and could represent a novel means of regulating catecholamine levels in normal and pathological neurons.     

高碘对小鼠脑发育及血清游离甲状腺激素水平的影响

】为了解高碘对机体脑发育及代谢的影响，本实验用高碘水喂养小鼠复制出高碘甲状腺肿动物模型。结果发现，用高碘水喂养小鼠２１０天后，高碘组小鼠体重减轻，甲状腺重量明显增加，血清ＦＴ３及ＦＴ４水平均显著高于适碘组。两组比较脑重、脑ＤＮＡ、蛋白质含量均无显著差异。揭示高碘不影响脑发育但可使代谢率增加。     

Expression and antigen activity determination of human thyroid peroxidase in silkworm and yeast

In this study, we report the expression of human thyroid peroxidase (TPO) in silkworm larvae and Pichia pastoris GS115. Recombinant TPO is sequentially purified from the hemolymph of infected silkworm larvae and yeast using a Ni-NTA resin kit. The concentration of yield of recombinant TPO is 4.87 mg per thousand larvae and 40.83 mg per liter yeast culture. However, the recombinant TPO produced in silkworm show similar binding ability with the specific anti-TPO serum to standard human TPO purified from insect cells. The lower antigen activity indicates the TPO expressed in yeast is not suitable to be used as the coating antigen in enzyme linked immunosorbent assay (ELISA). The cost of TPO expressed in B. mori is about 1/4 that of in insect cells, and the cost of TPO purified from silkworm for ELISA is only 1/8 that of TPO produced from Sf9 cells. It indicates the BmNPV-silkworm expression system is a cost-effective platform for producing TPO with high antigen activity.     

Inhibition by dopamine of (Na(+)+K+)ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor synergism

The (Na(+)+K+)ATPase, an integral membrane protein located in virtually all animal cells, couples the hydrolysis of ATP to the countertransport of Na+ and K+ ions across the plasma membrane. In neurons, a large portion of cellular energy is expended by this enzyme to maintain the ionic gradients that underlie resting and action potentials. Although neurotransmitter regulation of the enzyme in brain has been reported, such regulation has been characterized either as a nonspecific phenomenon or as an indirect effect of neurotransmitter-induced changes in ionic gradients. We report here that the neurotransmitter dopamine, through a synergistic effect on D1 and D2 receptors, inhibits the (Na(+)+K+)ATPase activity of isolated striatal neurons. Our data provide unequivocal evidence for regulation by a neurotransmitter of a neuronal ion pump. They also demonstrate that synergism between D1 and D2 receptors, which underlies many of the electrophysical and behavioural effects of dopamine in the mammalian brain, can occur on the same neuron. In addition, the results support the possibility that dopamine and other neurotransmitters can regulate neuronal excitability through the novel mechanism of pump inhibition.     

NOS在甲状腺功能低下小鼠脑中含量变化的研究

本文研究了甲状腺激素减少时,小鼠的大脑、小脑、海马、嗅脑等脑组织中一氧化氮合酶（NOS）含量的变化.采用丙硫氧嘧啶（PTU）持续灌注20日龄左右雌性昆明小鼠造成甲减模型.采用NOS的生化测定法检测甲减小鼠的大脑、小脑、嗅脑、海马等中的NOS含量;检测指标包括组织中总一氧化氮合酶（TNOS）活性,以及诱导型一氧化氮合酶（iNOS）及结构型一氧化氮合酶（cNOS）两种分型酶的活性.生化测定法显示,甲状腺功能低下小鼠NOS含量与对照组比较在大脑、小脑和嗅脑中均出现了下降,cNOS在大脑、小脑、海马、嗅脑部位也均出现了下调.而iNOS含量在甲状腺功能低下小鼠的大脑、小脑、海马、嗅脑中却出现了上调.由此可得出,甲状腺激素的分泌异常会造成NOS含量的异常,NO信号系统可能参与甲状腺激素缺乏所致的脑损害过程.     

Neuroendocrine regulation of structure and function in the endostyle of amphioxus, Branchiostoma belcheri

Using histological and histochemical methods the structure of the endostyle in amphioxus at different gonadal developmental stages is studied, and the immunocytochemical localization of thyroglobulin (Tg) and thyrotropin (TSH) in the endostyle, Hatschek's pit and brain vesicle is investigated. The results not only confirm the previous report that the endostyle is composed of 6 zones and the cells of zone 5 are thyroid hormone synthesizing cells, but also find the thyroxine synthesis in and secretion from zone 3. In addition, the epithelial cells in Hatschek's pit and the neural cells in brain vesicle are immunopositive for TSH, and the immunoactivity is correlated with gonadal cycle. The present study may provide morphological proof for the hypothesis that the secretory activity of thyroid cells is regulated by TSH from Hatschek's pit and brain vesicle.     

猪脑心肌炎病毒VR-129 B株VP2重组蛋白的构建表达及其免疫活性分析

为获得大量猪脑心肌炎VP2基因及蛋白研究的细胞模型,构建pDC315-EMCV-VP2真核表达载体,且将其转染到293T细胞,筛选出阳性质粒进行克隆。EMCV VR-129B株VP2基因序列参照GenBank(登录号:X74312),利用RT—PCR方法扩增VP2的全基因序列,将其与质粒pDC315经NheI和XhoI双酶切后连接,构建pDC315-EMCV-VP2重组表达质粒,并将其转染至293T细胞。使用荧光定量PCR方法观察pDC315-EMCV-VP2真核表达载体在细胞中的表达情况。双酶切PCR结果获得大小为780 bp的基因片段,测序结果显示与GenBank上已经公布的同名基因(登录号:X74312)序列片段同源性为100%,重组质粒构建成功。实时荧光定量PCR(QPCR)结果显示,重组质粒转染组与空质粒组之间相比,EMCV-VP2基因的表达量显著上调(P0.001);在荧光显微镜下观察转染细胞,转染成功部分出现较亮绿色荧光,EMCV-VP2基因表达稳定。从转染细胞中提取重组蛋白与EMCV阳性血清和阴性血清进行ELisa反应,具有较好免疫活性。     

脲变性兔脑肌酸激酶在复性过程中活力与构象变化的比较

研究了脲变性兔脑肌酸激酶（ＡＴＰ：Ｃｒｅａｔｉｎｅ Ｎ－ｐｈｏｓｐｈｏｔｒａｎｓｆｅｒａｓｅ ＥＣ２．７．３．２）在复性时的构象变化与活力变化的过程。在适当条件下，酶的活力恢复可达９０％以上。与兔肌肌酸激酶相似，该酶的复活过程也呈现含有三相组成的一级反应过程。在一定的酶浓度范围内，酶活力恢复过程与酶浓度无关。将用荧光、ＣＤ和紫外差吸收监测的构象变化与活力变化的速度常数比较，表明酶活力恢复前期的速度与构象变化速度相当，但在酶分子的总体构象变化完成之后，活力恢复尚未完全。     

Toxicity of the brominated flame retardant tris-（2,3-dibromopropyl） isocyanurate in zebrafish （Danio rerio）

Tris-(2,3-dibromopropyl) isocyanurate (TBC) is a heterocyclic brominated flame retardant that was recently detected in the environment in China. TBC is semi-volatile and can accumulate in the lipid of some species, but little is known about its effect on aquatic organisms. We exposed adult zebrafish to 0, 0.25, 1 and 4 mg/L TBC for 28 d and measured the effect on survival, growth, histopathology, hormone levels, enzyme activity, and gene expression. TBC exposure had no effect on survival or growth. We observed significant damage to the liver and gill, including hepatocellular swelling and fatty degeneration in the liver as well as proliferation and edema of epithelial cells in the gills. In addition, exposure to 4 mg/L TBC induced proliferation of goblet cells in the intestine of both sexes, acellular areas in the testis, and thinly scattered vitellogenic granules in vitellogenic oocytes. TBC exposure had no effect on the levels of thyroid hormones, testosterone, estradiol, liver superoxide dismutase activity, malondialdehyde content, and brain cholinesterase activity. By contrast, hepatic vitellogenin and cytochrome P4501A gene expression was significantly down-regulated in both male and female zebrafish in response to TBC exposure. Our results suggest that exposure to TBC causes a variety of potential reproductive and endocrine toxic effects.     

Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src.

The protein-tyrosine kinase activity of the proto-oncogene product p60c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527. The phosphorylation might be catalysed by a so-far-unidentified tyrosine kinase, distinct from p60c-src. Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60c-src from neonatal rat brain. We have now confirmed the specificity of this enzyme by using a mutant p60c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60c-src, represents a new type of tyrosine kinase.     

Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein

Dynein, an ATPase, is the force-generating protein in cilia and flagella. It has long been speculated that cytoplasmic microtubules contain a related enzyme involved in cell division or in intracellular organelle transport. A 'cytoplasmic dynein' has been described in sea urchin eggs, but because the egg stockpiles precursors for both cytoplasmic and ciliary microtubules, the role of this enzyme in the cell has remained unresolved. We recently found that the microtubule-associated protein (MAP) 1C (ref. 6) from brain is a microtubule-activated ATPase that produces force in the direction corresponding to retrograde organelle transport in the cell. MAP 1C has several similar properties to ciliary and flagellar dynein. Here we show directly, using scanning transmission electron microscopy, that MAP 1C is structurally equivalent to the ciliary and flagellar enzyme and is the long-sought cytoplasmic analogue of this enzyme.     

甲状腺99mTc摄取率与血清甲状腺激素的相关研究

目的：探讨甲状腺99mTc摄取率（CTU）与甲状腺功能的关系,方法：对524例不同甲状功能状态的受试者进行甲状腺99mTc过锝酸盐显像和全自动测量其CTU,并与其血清甲状腺激素水平进行了要相关性研究,结果：甲状腺瘤组（n=64)和单纯性甲状腺肿组（n=56)的CTU与健康对照组（n=89)之间差异无显著性意义,甲亢（n=270)和亚急性非化脓性甲状腺炎（亚甲炎）甲低期组（n=22)的CTU明显高于健康对照组,亚甲炎甲 亢期组（n=23)的CTU明显低于健康对照组,479例甲腺CT和血清甲状腺激素水平的直线相关分析结果显示两者呈显著正相关,45例亚甲炎CTU和血清甲腺激素水平的直线相关分析结果显示两者呈显著负相关,结论：甲状腺CTU和血清甲状腺激素水平密切相关,提示全自动定量测定CTU能反映甲状腺功能状态和严重程度,有较大的临床应用价值。     

活性炭吸附法固定猪胰脂酶的初步研究

初步研究了以活性炭为载体的猪胰脂酶吸附固定，吸附pH和吸附量对吸附固定有重要影响，吸附的最适pH在8左右，酶的比活随吸附量的增加而减小，同时固定化酶有比自由酶更好的热稳定性。     

应用比浊法测定凝血酶样酶活性

传统常用目测法观察血浆凝固时间来测定凝血酶样酶的活性,因无法微观地检测凝固过程的起始而导致灵敏度低．应用比浊法检测凝血酶样酶活性,以东菱克栓酶为标准凝血酶样酶,在血浆凝固过程中,浊度随时间基本呈直线变化直至浊度变化最终趋于恒定,表明此时血浆已完全凝固．酶活与浊度一时间变化曲线的直线斜率之间存在严格的线性关系．线性范围为０．０５～０．６５ｕ／ｍＬ．结果表明,比浊法是一种改进的凝血酶样酶测活方法．     

Ca2+ is essential cofactor for trypanocidal activity of normal human serum.

Normal human serum has been known to exert a cytotoxic effect on Trypanosoma brucei subspecies for nearly 80 yr. But in spite of many attempts, no trypanocidal factor was found in human or baboon serum, until Rifkin demostrated a high density lipoprotein (HDL) in normal human serum with trypanocidal activity. The conclusion that this was the trypanocidal factor was supported by the report that serum from patients with Tangier disease, characterised by a severe deficiency of HDL, lacked trypanocidal activity. We report here that Ca2+ is an essential cofactor for the trypanocidal activity of normal human serum, in which alpha2 macroglobulin (alpha 2) might function as a Ca2+-carrier. We further show that D-glucose, D-fructose and D-mannose can suppress the trypanocidal action of normal human serum, whereas glycerol has the opposite effect.     

几丁质·几丁聚糖对运动训练小鼠肝组织自由基代谢及血清GPT活性的影响

通过建立灌服几丁质·几丁聚糖小鼠的力竭游泳训练实验模型 ,测定了小鼠肝组织的超氧化物歧化酶 (SOD)的活性、丙二醛 (MDA)的水平以及血清谷丙转氨酶(GPT)的活性 .结果表明 ,灌服几丁质·几丁聚糖后 ,小鼠游泳运动力能明显提高 ;肝组织SOD活性显著高于各自对照组 ;MDA含量显著低于各自对照组 .说明几丁质·几丁聚糖具有较强的抗自由基损伤和抗脂质过氧化损伤的作用 ,服用几丁质·几丁聚糖可减轻运动所产生的内源性自由基对小鼠的伤害 .另外 ,几丁质·几丁聚糖对小鼠肝细胞膜有保护作用 ,可减少组织酶的外泄 .