INTRACELLULAR LOCALIZATION OF KYNURENINE IN THE FATBODY OF DROSOPHILA

Light blue fluorescent globules accumulate in the cells of the anterior region of the fatbody of Drosophila larvae near the time of pupation. This fluorescent material appears in the Ore-R wild type strain as well as mutant strains in which the synthesis of both the red and brown eye pigments is affected. The vermilion mutant, which is characterized by the absence of the brown pigment component in the eye, was the only strain among those examined which did not develop the light blue fluorescent globules. Utilizing chromatographic techniques together with the information gained by examination of the mutant strains, the fluorescent material has been identified as kynurenine. Of particular interest is the manner of appearance of the fluorescent material in the vicinity of the nuclear membrane of the fat cells.     

INTRACELLULAR LOCALIZATION OF SAXITOXINS IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The potent neurotoxin saxitoxin, and possibly several of its derivatives, are localized in two types of sites within the marine dinoflagellate Gonyaulax tamarensis Lebour. Immunocytochemical techniques using a polyclonal antibody and epifluorescence microscopy demonstrate toxin localization within the nucleus as well as on the periphery of small granules thought to be starch grains. In the nuclear region, the labelling occurred on or close to the permanently-condensed chromosomes as well as in an area within the two arms of the nucleus in the vicinity of the nucleous. No binding was observed in a closely-related, non-toxic dinoflagellate. Different binding affinities were observed between the nucleus and the grains at high and low antibody dilutions. This may relate to the polyclonal nature of the antiserum and to the presence of multiple toxins within the G. tamarensis isolate studied. Mechanistic interpretations of these labelling patterns remain speculative, especially the localization of the antigen at the outer edge of starch grains, but the distinct labelling in the nuclear region suggests that saxitoxin, with its two positively charged guanidinium groups, may bind to nucleic acids or nuclear proteins in a manner analogous to the polyamines and other cations. The labelling patterns reported here suggest that the saxitoxins may not simply be secondary metabolites but instead could be important compounds involved in the structure and function of the G. tamarensis genome.     

GnRH相关肽在大鼠垂体前叶的细胞学定位

本研究应用特异性抗GnRH相关肽(GAP)N端11个氨基酸的抗血清和六种垂体前叶激素的抗血清,通过免疫组织化学双重染色技术观察GAP在大鼠垂体前叶细胞的定位。结果发现,GAP样免疫反应性物质存在于LH细胞和FSH细胞,而未见于GH、PRL、TSH和ACTH细胞。本文首次证明GAP存在于正常大鼠垂体促性腺激素细胞,为GAP调节LH和FSH的分泌提供了形态学证据;也支持GAP的功能序列在其分子的N端,或GAP进一步裂解出N端片段而发挥作用。     

人胎垂体生长激素样免疫反应细胞的发育

为探讨人胎垂体生长激素细胞发育规律,本文用免疫细胞化学结合图像分析,研究１９例不同胎龄人胎垂体生长激素细胞的形态、大小、光密度变化。结果如下：①胎１６周已存在生长激素免疫反应细胞,随胎龄增加,细胞多排列成团或索,并由周边部向远侧部的中央延伸。②随胎龄增加,生长激素样免疫反应细胞数量逐渐增多,细胞逐渐增大,胞浆逐渐增多,核浆比值逐渐变小。至胎龄３２周,生长激素样免疫反应细胞以大型细胞为主,多为圆形或卵圆形。③生长激素样免疫反应细胞的光密度随胎龄增加而逐渐增高,２６周达高峰,以后呈下降趋势,但仍高于２４周的水平,结果提示：垂体生长激素细胞的发育是从幼稚到成熟的过程,生长激素细胞的功能在胎２４－２６周最旺盛。     

INTRACELLULAR LOCALIZATION OF PHENOL SULPHOTRANSFERASE IN RAT BRAIN

—The intracellular localization of phenol sulphotransferase in rat brain was studied The distribution pattern found after differential centrifugation closely resembles that of lactate dehydrogenase and does not change during postnatal development. The distribution of the enzyme in discontinuous and continuous sucrose gradients, however, shows a deviation from the lactate dehydrogenase pattern and a shift towards a higher sucrose concentration during development. In the adult the phenol sulphotransferase coincides with monoamine oxidase, succinate dehydrogenase and β-glucuronidase. Disruption experiments, purification of mitochondria and electron microscopy exclude localization of phenol sulphotransferase in mitochondria. These studies support the idea of phenol sulphotransferase as a cytoplasmic enzyme with a preferential binding to or localization in oligodendroglial cells or, more probably, a specific type of synaptosomes.     

INTRACELLULAR LOCALIZATION OF NATIVE AUXIN IN AVENA COLEOPTILE

Intracellular localization of the native auxin in the Avenacole-optile tip was investigated by separating cellular fractionsby differential centrifugation. Each fraction was extractedwith ether and the auxin activity was measured by the sensitizedAvena curvature test. After the removal of the native free auxin,each fraction was alkaline-hydrolyied, and from these hydrolyzatesthe bound auxin was extracted with ether and its activity wasmeasured. Both the native free auxin and the native bound auxinin these extracts were identified as IAA by paper chromatography.The results show that the native free auxin occurs only in thesupernatant soluble cytoplasm, and that the native bound auxinlocalizes also in the supernatant. The distribution of the externallyapplied IAA was also investigated. (Received February 27, 1962; )     

LOCALIZATION AND DISTRIBUTION OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE IN THE RAT EPIDIDYMIS

Previous investigation has provided evidence for the control of electrogenic chloride secretion by pituitary adenylate cyclase-activating polypeptide (PACAP) across the rat epididymal epithelium using electrophysiological measurement of transepithelial transport in cultured epididymal system. Hence, it suggests that epididymal and sperm functions are subject to control by a local PACAP system in the rat epididymis. In the present study, localization and distribution of PACAP in the rat epididymal duct was studied by an indirect immunofluorescence technique in conjunction with confocal laser scanning microscopy. Immunoreactivity for PACAP was found in all regions of the epididymal duct. However, the intensity of immunoreactivity for PACAP was stronger in the caput and corpus regions when compared to that of the cauda epididymidis. Much weaker immunostaining for PACAP, as compared to those found in other regions, was observed in the cauda epididymidal tubules which are in close proximity to the vas deferens. No immunoreactivity for PACAP was found in epididymal spermatozoa. Together with the previous finding, the present results suggest that PACAP may exhibit a regional difference in its expression along the epididymal duct and it may act in a paracrine or autocrine fashion in the regulation of epididymal chloride secretion and hence fluid secretion, thus regulating epididymal and sperm functions along the epididymal duct.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

花臭蛙消化道6种激素阳性细胞的免疫组织化学定位

目的应用6种胃肠激素抗血清对花臭蛙（Rana schmackeri）消化道激素阳性细胞进行了免疫组织化学定位。方法SP（Streptavidin peroxidase）免疫组织化学法。结果五羟色胺阳性细胞在消化道各段都有分布,以胃幽门部密度最高,胃体其次,食道和直肠较少;生长抑素阳性细胞主要分布于胃和小肠,其中幽门部较多,食管和直肠未见分布。胃泌素阳性细胞只在十二指肠和空肠两个部位检测到。而胰多肽、胰高血糖素和P-物质阳性细胞在消化道各段均未见其分布。结论花臭蛙消化道这六种内分泌细胞分布与其他两栖类动物比较,既显示了两栖类动物在生活习性及动物消化生理方面消化道激素阳性细胞分布的某些共性,又显示了不同物种在消化道的结构特点、生活环境、食性等方面存在的种间差异。     

ULTRASTRUCTURAL LOCALIZATION OF INTRACELLULAR ANTIGEN USING ENZYME-LABELED ANTIBODY FRAGMENTS

The efficiency of small enzyme-labeled tracers for the demonstration of intracellular antigen was investigated in tissues fixed with picric acid-formaldehyde. The influence of fixation on the immunological activity was tested in vitro by radial immunodiffusion. The experimental model consisted of newborn pig jejunum after absorption of ferritin from the intestinal lumen. Ferritin was located after 1 hr in vacuoles scattered in the cytoplasm of the absorptive cells and represented an easily recognizable intracellular antigen. After immunohistochemical treatments with antiferritin preparations, the distribution of labeling enzyme reaction product was examined by morphometry. The ratio of the labeled volume to the total volume of vacuoles containing ferritin indicated the degree of specific labeling of the antigen. In both direct and indirect methods, the degree of labeling was low when enzyme-labeled immunoglobulin G was the tracer. With antigen binding fragments (Fab), the labeling was significantly increased. In the indirect method, the degree of labeling was influenced by the first-step reagents. Onlywhen the serum titer was optimum was a high degree of labeling obtained. With antigen binding fragments or papain-digested serum the effect of the titer was negligible and maximum labeling was achieved. In both methods, with peroxidase as the labeling enzyme, a diffuse nonspecific deposition of reaction product was observed. This could be avoided by using cytochrome c instead.     

THE INTRACELLULAR LOCALIZATION OF INORGANIC CATIONS WITH POTASSIUM PYROANTIMONATE : Electron Microscope and Microprobe Analysis

Potassium pyroantimonate, when used as fixative (saturated or half-saturated, without addition of any conventional fixative) has been demonstrated to produce intracellular precipitates of the insoluble salts of calcium, magnesium, and sodium and to preserve the general cell morphology. In both animal and plant tissues, the electron-opaque antimonate precipitates were found deposited in the nucleus—as well as within the nucleolus—and in the cytoplasm, largely at the site of the ribonucleoprotein particles; the condensed chromatin appeared relatively free of precipitates. The inorganic cations are probably in a loosely bound state since they are not retained by conventional fixatives. The implications of this inorganic cation distribution in the intact cell are discussed in connection with their anionic counterparts, i.e., complexing of cations by fixed anionic charges and the coexistence of a large pool of inorganic orthophosphate anions in the nucleus and nucleolus.     

稀有鮈鲫表达8种脑垂体激素基因的细胞在垂体中的定位

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INTRACELLULAR LOCALIZATION OF THE TASTE/ODOR METABOLITE 2-METHYLISOBORNEOL IN OSCILLATORIA LIMOSA (CYANOPHYTA)1

The monoterpene derivative, 2-methylisoborneol (MIB), is produced by many blue-green algae and often is responsible for the “musty” taste/odor in aquaculturally raised finfish and potable water supplies. Although previous researchers have suggested that taste/odor metabolites are partitioned among cell constituents and that coregulation with pigment biosynthesis occurs, no structural evidence for these hypotheses exists. MIB was localized in cells of Oscillatoria limosa (Roth) Agardh at the ultrastructural level using standard gold-labeled antibody techniques. There was no apparent relationship between age of the cells and MIB synthesis; cells that were and were not undergoing active division had similar amounts of MIB label. There was no consistent partitioning of MIB label within cells. However, occasionally, specific label was observed along photosynthetic lamellae, suggesting a potential linkage of MIB synthesis and/or binding to the pigment systems.     

SUBCELLULAR LOCALIZATION OF PROTEIN CARBOXYL-METHYLASE AND ITS SUBSTRATES IN RAT PITUITARY LOBES

Subcellular distribution of protein carboxyl-methylase (PCM) and its endogenous substrates the methyl acceptor proteins (MAP) have been studied in the anterior, the posterior and the intermediate lobes of the rat pituitary gland. In all three lobes, PCM was found to be a cytosolic enzyme whereas the highest MAP specific capacity was observed in the lysate of the granular fractions. The methylated proteins from the lysates of the granular fractions and the cytosolic fractions were analyzed with a novel electrophoretic system which prevents the hydrolysis of the protein-methyl esters. Gel electrophoretic profiles from the lysates of the granular fractions were different from those of the cytosolic fractions. In the lysatcs of the granular fractions. the MAP had a molecular weight of less than 25,000 suggesting that the methylated polypeptides in these fractions were the pituitary peptide hormones (and their subunits) and neurophysins.     

左旋十八甲基炔诺酮对大鼠卵巢颗粒细胞和垂体细胞激素合成及分泌的影响

研究了左旋十八甲基炔诺酮对大鼠卵巢颗粒细胞和垂体细胞激素合成及分泌的直接作用。结果显示左旋十八甲基炔诺酮单独作用于无血清培养的颗粒细胞时,抑制雌二醇的合成,但刺激孕酮的分泌;在与促卵泡素合并处理时,颗粒细胞雌二醇、孕酮的分泌量随着左旋十八甲基炔诺酮浓度的增加而增加。利用促性腺激素生物测定方法证明大鼠整体用左旋十八甲基炔诺酮处理后,垂体中促卵泡素和促黄体生成素活性明显下降;同时外周血清中促卵泡素的活性亦下降。培养的垂体单纯用左旋十八甲基炔诺酮处理后,其培养液经生物测定呈现抑制颗粒细胞雌二醇和孕酮的分泌,促性腺激素释放激素可减弱左旋十八甲基炔诺酮的抑制作用。提示左旋十八甲基炔诺酮除通过垂体卵巢轴系起作用外,还能直接作用于卵巢。     

鲑鱼促性腺激素释放激素（sGnRH）调节鲤鱼脑垂体生长激素分泌的离体研究

ｓＧｎＲＨ同时刺激离体灌流孵育的鲤鱼脑垂体ＧｔＨ和ＧＨ分泌,ＧｔＨ和ＧＨ对ｓＧｎＲＨ脉冲式刺激的分泌反应的时间进程和剂量依存关系相似。生长抑素显著抑制基础的ｓＧｎＲＨ刺激的ＧＨ分泌,但对ＧｔＨ分泌没有影响。阿扑吗啡以剂量依存方式显著抑制基础的ｓＧｎＲＨ促进的ＧｔＨ分泌,并显著刺激ＧＨ分泌,但对ｓＧｎＲＨ促进的ＧＨ分泌无影响。