Separation of Monomerizing and Lysozyme Activities of Destabilase from Medicinal Leech Salivary Gland Secretion

Destabilase, endo--(-Glu)-Lys-isopeptidase, was prepared from the salivary gland secretion of the medicinal leech (Hirudo medicinalis). The secretion prepared by the known method of Rigbi et al. (1987) (secretion-K) lacks the destabilase-characteristic highly specific isopeptidase activity (the D-dimer-monomerizing activity) because of its degradation by proteolytic activity (the substrate of Glp-Ala-Ala-Leu-pNA) due to contamination with leech intestinal channel contents. Therefore, we have elaborated a new technique for preparation of a true leech secretion (secretion-I). This secretion is characterized by the complete absence of the leech intestinal channel contents and has no proteolytic activity. For the first time the destabilase-specific D-dimer-monomerizing and lysozyme activities were separated by fractionation of secretion-I by HPLC gel filtration through Superose S-12. For the purified destabilase preparation, these activities were separated by reversed-phase chromatography in an acetonitrile gradient (0-60%) in the presence of 0.1% trifluoroacetic acid. The monomerizing activity of destabilase is responsible for the ability of secretion-I to dissolve stabilized fibrin via isopeptidolysis of - and - fibrin chains bound by -(-Glu)-Lys-isopeptide bonds.     

The internal pH of synaptic-like microvesicles in rat pinealocytes in culture

Synaptic-like microvesicles (SLMVs) are morphological and functional equivalents of neuronal synaptic vesicles, and are responsible for the storage and secretion of classical neurotransmitters in various endocrine cells. Vacuolar H+-ATPase acidifies the internal space of these organelles and provides a driving force for the uptake of neurotransmitters. Thus, the luminal pH is an important determinant of the function of SLMVs, although its value in living cells is unknown. Here, we determined the luminal pH of SLMVs in living rat pinealocytes by means of an immunoelectronmicroscopic procedure basedon the distribution of an amphipathic amine, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP). Use of double-labeling techniques with antibodies against 2,4-dinitrophenol for DAMP and synaptophysin for SLMVs, and of frozen ultrathin sections enabled us to determine the number of immunogold particles for DAMP per microm2 of SLMVs. Using the density of gold particles, the luminal pH of SLMVs was calculated to be 5.11 +/- 0.01. Treatment with either 1 microm bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, or 50 mm ammonium chloride, a dissipater of the transmembrane pH gradient, increased the luminal pH to 6.04 +/- 0.07 and 6.05 +/- 0.11, respectively. Simultaneously, the lysosomal pH was found to be 5.14 +/- 0.07, which increased to 5.77 +/- 0.09 and 5.93 +/- 0.13 with bafilomycin A1 and ammonium chloride, respectively. It is concluded that the luminal pH of SLMVs is comparable to that of lysosomes in vivo.     

Casein-derived phosphopeptides disrupt tight junction integrity,and precipitously dry up milk secretion in goats

Mammary involution is triggered by local stimuli, but the precise mechanism has not been defined. Milk stasis accumulate local signals, which makes the tight junctions (TJ) leaky. The aim of the study was to check the hypothesis that casein hydrolyzates (CNH) compromise TJ integrity and dry up milk secretion. A single dose of CNH transiently (12 to 24 h) compromised TJ integrity in the treatedudder. This was associated by a transient (12 to 96 h) decline in milk secretion. No such changes were recorded in the contralateral gland that served as a control. Four repeated doses of CNH after each milking caused drastic changes in mammary secretion and composition, which were associated with irreversible cessation of milk secretion within 96 h. No such changes were recorded in goats treated with de-phosphorylated casein (control). We conclude that CNH are the milk-borne factors that cause the disruption of TJ integrity and induction of involution, and that the serine-Ps in the CNHs are essential for the excretion of biological activity.     

Cytoplasmic TSC-22 (transforming growth factor-beta-stimulated clone-22) markedly enhances the radiation sensitivity of salivary gland cancer cells

We transfected a salivary gland cancer cell line, TYS, with three different forms of TSC-22 (transforming growth factor-beta-stimulated clone-22) gene: full-length TSC-22 (TSC-22FL) containing nuclear export signal, TSC-box and leucine zipper, truncated TSC-22 (TSC-22LZ) containing only TSC-box and leucine zipper, and truncated TSC-22 with nuclear localization signal (NLS-TSC-22LZ). High expression of TSC-22FL in the cytoplasm markedly enhanced the radiation-sensitivity of TYS cells, while, moderate expression of TSC-22FL marginally affected the radiation-sensitivity. TSC-22LZ, which was expressed in the cytoplasm and the nucleus, enhanced the radiation-sensitivity of TYS cells irrespective to its expression level. NLS-TSC-22LZ, which was expressed only in the nucleus, marginally affected the radiation-sensitivity of the cells even at high expression level. Interestingly, cytoplasmic TSC-22 translocates to nucleus concomitant with radiation-induced apoptosis. These results suggest that cytoplasmic localization of TSC-22 and translocation of TSC-22 from cytoplasm to nucleus is important for regulating the cell death signal after irradiation-induced DNA damage.     

大鼠泪腺睾酮受体直空负压ABC法光镜观察与免疫金探针超微结构定位

动物实验发现睾酮能改善干眼病动物模型干眼症状,促进泪腺分泌,但作用机理不明,本研究探讨泪腺细胞中是否存在睾酮受体。雌雄各8只大鼠的泪腺分别制成石蜡切片和超薄切片,睾酮分别采用ABC法及免疫金标记,通过真空负压ABC法光镜观察及免疫金探针超微结构定位进行细胞睾酮受体检查。结果：光镜下泪腺导管上皮细胞的胞浆及胞核中出现免疫染色阳性,泪腺上皮细胞则很少见到;电镜下泪腺细胞胞浆及核中可见金颗粒,对照组则染色阴性。结论 泪腺细胞存在着睾酮受体,睾酮通过受体对泪腺发生作用。     

Comparative image analysis of EGF immunoreaction in rat submandibular gland using 3,3'-diaminobenzidine with metal enhancer substrate

We have shown the efficacy of image analysis using 3,3'-diaminobenzidine (DAB) with a metal enhancer substrate for demonstrating quantitative differences in the amount of epidermal growth factor (EGF) in the submandibular gland from normal, castrated, and testosterone propionate (TP) treated castrated rats. Immunohistochemical determination of EGF visualized by DAB-nickel reagent was performed with image analysis using a computed image analyzer system (ACAS 570). Immunohistochemistry for EGF disclosed positive staining in granular convoluted tubule cells in the tissue sections from each experimental group. Using the tools of a competent data program installed in the ACAS 570 software, we measured quantitative differences among the experimental glands examined. Castration was shown to elicit a significant reduction in the EGF-positive area and staining intensity, and administration of TP to the castrated animals restored these parameters to levels greater than those of normal rats. Our study demonstrates that a simple, inexpensive, commercially available metal enhancer substrate can be applied accurately to the computer assisted quantification of histochemical hormone-induction studies.     

山羊乳腺生物反应器表达人促红细胞生成素的分离纯化

以离子交换、疏水作用、HPLC和凝胶过滤层析等方法对山羊乳腺生物反应器表达的人促红细胞生成素 (erythropoietin ,EPO)进行了分离纯化 ,初步探讨了其分离工艺 ,结果得到了纯度达到 96%以上的纯品 ,所纯化的EPO分子质量为 32kDa ,糖基化程度根据理论分子质量测算为40 %。Western blot证明所得纯化产品具有天然EPO免疫原性 ,N端氨基酸序列测定与文献报道一致 ,ELISA检测免疫活性为 2 4 0 0 0 0IU mg,表明通过 4步纯化步骤可以得到高纯度的产品 ,为今后工艺放大提供了有力的实验依据。     

整合人lactoferrin基因的山羊体细胞支持核移植克隆胚的体外发育

克隆了人lactoferrin基因和山羊[[beta]]-casein基因5′端调控区, 构建了人lactoferrin的乳腺表达载体, 并将该载体利用脂质体介导转染了奶山羊胎儿成纤维细胞, 获得了稳定整合人lactoferrin基因的转基因体细胞克隆17个, 其中PCR和Southern Blot检测阳性的细胞克隆14个, 阳性率82.4%。以转基因体细胞为供体细胞进行了核移植, 获得了能够体外发育的山羊转基因克隆胚胎, 体内成熟卵母细胞来源的核移植囊胚率为64.8%, 体外成熟卵母细胞来源的核移植囊胚率为51.7%, 证明了山羊转基因体细胞能够支持克隆胚的进一步发育。     

乌龟脑垂体显微及其腺垂体超微结构的研究

乌龟脑垂体由柄形神经垂体和椭圆形腺垂体两部分组成,神经垂体位于腺垂体后部上方呈背腹型排列。神经垂体中神经叶不发达,腺垂体分为远侧部和中间部,特殊空泡结构成为垂体门脉系统的特征。远侧部细胞分为嗜酸性细胞、嗜碱性细胞和嫌色细胞3种。通过透射电镜观察,腺垂体远侧部主要有5种分泌激素细胞:即生长激素(GH)分泌细胞、催乳激素(PRL)分泌细胞、促甲状腺激素(TSH)分泌细胞、促肾上腺皮质激素(ACTH)分泌细胞、促性腺激素(GTH)分泌细胞和非分泌类型滤泡-星形细胞(FS)。生长激素分泌细胞核大、分泌颗粒少的特征成为乌龟与其他动物最大的区别,可能与乌龟具有生长慢、寿命长的生物学特性有关。