适用于分析细胞内蛋白质组分研究的两向电泳法

哺乳动物体细胞内含有上万种不同蛋白质,各组分在细胞内的丰度因组织和细胞类型而异,也会在转化、分化和激素反应过程中发生变化。每个细胞内各种蛋白质含量的大致范围是10~3—10~9个分子。到目前为止细胞内所含蛋白质已被仔细研究过的还不足1000种,多数属     

羊垂体促黄体素(LH)mRNA在兔网织红细胞溶胞物系统中翻译的研究

高等哺乳动物垂体前叶合成并分泌两种与生殖有密切关系的激素,它们是促黄体素(LH)和促滤泡素(FSH)。其合成一方面受丘脑下部促黄体素释放激素(LH-RH)的控制,同时也受性甾体激素的控制。例如雌二醇主要是控制促性腺激素的β-亚单位的合成。近年来有人报道,用Sephadex G-100纯化LH,发现有分子量超过100,000的具有LH免疫结合活性的大分子,并且在血液中也有分子量大于32,000的LH分子。本研究采用正交试验法,建立了羊垂体LH mRNA体外蛋白质翻译系统,并对其翻译产物作了鉴定,实验结果表明,在翻译产物中除了分子量为32,000的LH分子外,也有分子     

FSP27基因敲除小鼠——初步揭开白色脂肪细胞单房油滴之谜

脂肪组织储存甘油三酯等中性脂质,维持能量平衡,在肥胖、胰岛素抵抗和2型糖尿病研究中备受关注,脂肪细胞内油滴也被看成是具有活跃功能的细胞器。众所周之,哺乳动物白色脂肪细胞,胞浆部分95％以上空间被一个单房的大油滴（直径10～200μm不等）所占据;棕色脂肪细胞内含丰富的线粒体和多房的小油滴;其它组织,如肝脏、心肌、肾脏等细胞胞浆内含有多房且体积很小的油滴,提示白色脂肪细胞内单房大油滴是甘油三酯储存的主要形式。     

促黄体素β基因表达中的转导通路及转录因子

促性腺激素释放激素 (GnRH)为下丘脑促垂体激素 ,其脉冲式地释放调节垂体促卵泡素(FSH)和促黄体素 (LH)的合成与释放 ,进而调节动物的生殖活动。LH是由α亚基和 β亚基组成的异二聚体糖蛋白激素 ,其中 β亚基决定激素的特异性。LHβ基因的表达是由GnRH诱发的 ,此过程主要依靠PKC和Ca2 两类信号通路 ,并调节LHβ基因的表达。目前已经发现 ,多种转录因子 ,如早期生长反应基因 (Egr 1)、核受体SF 1基因、Ptx1基因和Sp1基因等 ,通过与LHβ亚基基因的启动子区直接结合 ,而对该基因的表达进行调控。     

在分化条件下甜菊愈伤组织分生区细胞超微结构研究

对甜菊（Ｓｔｅｖｉａｒｅｂａｕｄｉａｎａ）愈伤组织中尚未发生器官分化的分生细胞团进行了超微结构研究．结果表明,在器官分化条件下,愈伤组织中形成的分生区域的细胞体积小,细胞核大,核仁明显,且具核仁泡,部分细胞核中含有核内含物．大量小液泡分布在细胞的边周或散布于整个细胞中．液泡中通常含有陷入的细胞质成分和膜状物．部分液泡的形成与内质网膨大有密切关系．同时也观察到由内质网形成的多圈膜和双层膜包围细胞质成分的同心环结构．高尔基体及其小泡丰富,有时聚集分布在细胞某一区域．核糖体密集,有的聚集成多聚核糖体．因此,愈伤组织中分生区的细胞与分生组织中的液泡化和分裂的细胞类似．分生区细胞的另一明显特征是出现质膜内陷．推测这些超微结构特征可能反映了甜菊愈伤组织器官分化前的某些形态变化。     

亚胺环己酮可模拟抑素对垂体促性腺激素的调控作用

抑素(inhibin)是由卵巢颗粒细胞或睾丸支持细胞分泌的一种糖蛋白激素,它对垂体促性腺激素具有负反馈调节作用,亚胺环己酮(cycloheximide)是一种蛋白质生物合成的强抑制剂。为了研究抑素的作用机制,作者比较了抑素(32KDa,从猪卵泡液提取)和亚胺环己酮分别对离体培养的大鼠腺垂体细胞合成与分泌促性腺激素的作用,结果表明:(1)抑素和亚胺环己酮都能抑制腺垂体卵泡刺激素(FSH)的基础分泌及细胞内 FSH 含量(ED_(50)分别为1.0ng/ml 和22.5ng/ml),但它们都不改变黄体生成素(LH)的基础分泌及细胞     

银杏营养贮藏蛋白质的亚细胞定位

在电子显微镜下,对银枵（Ginkgo biloba L.）枝条营养贮藏蛋白质的超微结构特征及在亚细胞水平的定位进行了系统研究。结果表明：银杏营养贮藏蛋白质主要存在于韧皮薄壁细胞的液泡内。银杏韧皮薄壁细胞内的营养贮藏蛋白质在细胞质内合成,由内质网膨大的槽库、质膜内折或高尔基体小泡发育形成贮藏蛋白质的液泡。液泡蛋白质主要以不定形块状、絮状或颗粒状形态存在。贮藏蛋白质在整个越冬期一直保持高含量,直到翌年春季萌芽时,贮藏蛋白质迅速转移再利用。随着新梢的生长,到了夏末秋初,又重新开始积累贮藏蛋白质。     

降香黄檀次生韧皮部薄壁组织细胞液泡蛋白质的形成和积累的超微结构研究

热带豆科树木降香黄檀（Ｄａｌｂｅｒｇｉａｏｄｏｒｉｆｅｒａ）落叶时期末端小枝次生韧皮部薄壁组织细胞的中央液泡中有大量的贮藏蛋白质。用透射电镜技术研究了这些蛋白质的形成和积累。在这些细胞的周缘细胞质中有丰富的游离核糖体和许多粗糙内质网（ＲＥＲ）。在这些核糖体之间,总是分散着纤维状物质,有时还出现一些类似液泡蛋白质的结构。常常看到与ＲＥＲ相连的含有稀疏纤维状物质的小泡,以及含有类似液泡蛋白质的形状不规则的小泡和含有更多类似液泡蛋白质的光滑小泡。从连接ＲＥＲ的小泡到形状不规则小泡,再到光滑小泡,可能存在个体发生的连续性。通过内吞作用细胞质中的纤维状物质和核糖体以及其它结构被并入上述３种小泡。光滑小泡通常与中央液泡的膜融合而释放其内含物到液泡中。     

大鼠促性腺激素细胞内蛋白激酶C的改变对黄体生成素mRNA表达的影响

目的:分析大鼠黄体生成素(LH)表达的受体后信号转导机制。方法:促性腺激素(GTH)细胞内蛋白激酶C(PKC)兴奋或抑制后,用促性腺激素释放激素(GnRH)脉冲刺激,然后用实时荧光定量PCR方法测定细胞LH的β亚基(LHβ)mRNA的表达量,并与空白组比较。结果:LHβmRNA随着PKC活性的升高而显著升高,随着PKC活性的降低而显著降低。结论:GnRH脉冲刺激引起LHβmRNA表达,其受体后的信号转导是PKC-Ca2+途径。     

甜菊叶片细胞液泡系的电镜观察

本文系应用电子显微镜技术,着重观察甜菊叶片细胞不同发育时期内,液泡系的形态及内含物的变化。观察到幼叶细胞内,液泡小而数目多,呈半透亮状的圆形或椭圆形;在生长旺盛的叶片细胞内,有许多形态特化的液泡,内含物丰富,呈大小不一的颗粒状或囊泡状;至老叶细胞内,液泡内含物又逐渐分解乃至消失。这一形态特点与我们对叶绿体亚微结构观察,PAS 反应测定以及不同叶龄“甜菊糖苷”含量相比较存在着一定的对应关系。我们推测:“甜菊糖苷”主要贮存在液泡内,并与液泡的酿造机能有关。     

Caerulein-induced acute pancreatitis in rats: changes in glycoprotein-composition of subcellular membrane systems in acinar cells

Caerulein-induced acute pancreatitis is characterized by the occurrence of two membrane-bound vacuolar systems in acinar cells. Beside digestive enzymes containing secretory vacuoles, lysosomal autophagic structures can be identified at the ultrastructural level. In the present study glycoconjugate patterns of the surrounding membranes were characterized by ultrastructural lectin-binding experiments using five colloidal-gold labeled lectins with distinct sugar specificities. Furthermore, the profile of membrane glycoproteins of isolated vacuolar fractions was studied by SDS-PAGE and lectin-blotting. In pancreatitis, membranes of secretory vacuoles showed a significant lower degree of lectin-binding compared to normal zymogen granules. In contrast, newly appearing autophagic vacuoles in pancreatitis revealed a strong membrane labelling for most lectins used. The pattern of membrane glycoproteins of secretory and autophagic vacuoles as determined by SDS-PAGE and lectin-blotting differed from those of normal zymogen granules resembling the protein profile of smooth microsomes. Since this pattern requires a previous passage through Golgi stacks, it is assumed that the two types of vacuoles derive from Golgi elements. For the pathogenesis of caerulein pancreatitis these vacuolar post-Golgi structures seem to play an important role.     

Caerulein-induced acute pancreatitis in rats: changes in glycoprotein-composition of subcellular membrane systems in acinar cells

Summary Caerulein-induced acute pancreatitis is characterized by the occurrence of two membrane-bound vacuolar systems in acinar cells. Beside digestive enzymes containing secretory vacuoles, lysosomal autophagic structures can be identified at the ultrastructural level. In the present study glycoconjugate patterns of the surrounding membranes were characterized by ultrastructural lectin-binding experiments using five colloidal-gold labeled lectins with distinct sugar specificities. Furthermore, the profile of membrane glycoproteins of isolated vacuolar fractions was studied by SDS-PAGE and lectin-blotting. In pancreatitis, membranes of secretory vacuoles showed a significant lower degree of lectin-binding compared to normal zymogen granules. In contrast, newly appearing autophagic vacuoles in pancreatitis revealed a strong membrane labelling for most lectins used. The pattern of membrane glycoproteins of secretory and autophagic vacuoles as determined by SDS-PAGE and lectin-blotting differed from those of normal zymogen granules resembling the protein profile of smooth microsomes. Since this pattern requires a previous passage through Golgi stacks, it is assumed that the two types of vacuoles derive from Golgi elements. For the pathogenesis of caerulein pancreatitis these vacuolar post-Golgi structures seem to play an important role.     

Effects of a gonadotropin-releasing hormone antagonist on gonadotropin levels in Masu salmon and Sockeye salmon

The salmon gonadotropin-releasing hormone (sGnRH) is considered to be involved in gonadal maturation via gonadotropin (GTH) secretion in salmonid fishes. However, there is no direct evidence for endogenous sGnRH-stimulated GTH secretion in salmonids. In this study, to clarify whether endogenous sGnRH stimulates GTH secretion, we examined the effects of the mammalian GnRH (mGnRH) antagonist [Ac-Delta(3)-Pro(1), 4FD-Phe(2), D-Trp(3,6)]-mGnRH on luteinizing hormone (LH) levels in 0-year-old masu salmon Oncorhynchus masou and sockeye salmon Oncorhynchus nerka. First, the effects of the GnRH antagonist on LH release were examined in 0-year-old precocious male masu salmon. GnRH antagonist treatment for 3 hr significantly inhibited an increase in plasma LH levels that was artificially induced by exogenous sGnRH administration, indicating that the GnRH antagonist is effective in inhibiting LH release from the pituitary. Subsequently, we examined the effect of the GnRH antagonist on LH synthesis in 0-year-old immature sockeye salmon that were pretreated with exogenous testosterone for 42 days to increase the pituitary LH contents; the testosterone treatment did not affect the plasma LH levels. GnRH antagonist treatment slightly but significantly inhibited an increase in the testosterone-stimulated pituitary LH content levels. However, no significant differences in the plasma LH levels were observed between the GnRH antagonist-treated and control groups. These results suggest that endogenous sGnRH is involved in LH secretion in salmonid fishes.     

Effect of estradiol-17 β on the cytology of the liver,gonads and pituitary,and on plasma electrolytes in the female freshwater eel

Summary Female freshwater eels injected with estradiol-17 (E2) for 15–78 days appear paler and secrete more mucus than controls. The resulting strongly opalescent blood plasma indicates that vitellogenin synthesis occurs in the liver, which shows a significant hypertrophy, an increased vacuolization (lipid material) and glycogen depletion. Plasma sodium is lowered, but calcium levels are considerably increased. The gonosomatic index increases (0.92±0.1 to a maximum of 2.21). Oocytes are enlarged, but the incorporation of vitellogenin remains discrete. Gonadotrophs (GTH cells), small and scarcely visible in the pituitary of control eels, are hypertrophied and contain numerous glycoprotein granules after E2-administration. E2 may act on the pituitary and/or hypothalamus via a positive feedback to induce gonadotrophin (GTH) synthesis; GTH release seems to be very limited as indicated by the ovarian response. The differentiation of GTH cells in eels treated with fish pituitary extracts is most probably due to secretion of E2 by the ovary, which reacts on the pituitary. Various hypotheses are considered to explain the low GTH release.Thyrotrophs, somatotrophs and prolactin cells of the pituitary are stimulated. In the pars intermedia, MSH and PAS-positive cells appear less active. A possible antidopaminergic effect of E2 is discussed.E2 administration constitutes a simple and economic technique to induce the synthesis of GTH and will facilitate the biochemical and biological study of the latter hormone in eels.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs

Summary Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Stimulatory effects of insulin-like growth factor 1 on expression of gonadotropin subunit genes and release of follicle-stimulating hormone and luteinizing hormone in masu salmon pituitary cells early in gametogenesis

Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadotropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGF-I on expression of genes encoding glycoprotein alpha (GPalpha), follicle-stimulating hormone (FSH) beta, and luteinizing hormone (LH) beta subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GPalpha, FSHbeta, and LHbeta mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-I increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-I + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-I itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs.

Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtiiy1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins. Received: 29 September 1999 / Accepted: 20 November 1999     

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.     

Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules.

An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules.