Fractionation of the rat ventral prostate with respect to isolation and exocytosis of the prostatic secretion protein

The ventral prostrate was fractionated into one mitochondrial and three microsomal fractions. The different fractions were characterized morphologically and chemically. An interesting finding was that upon homogenization the endoplasmic reticulum membranes often turned ‘inside-out’ giving rise to microsomes with ribosomes attached to the inside of the vesicles. The secretion of the protatic secretion was studied by means of isotopic pulse labeling using radioactive leucine. Peak radioactivity in the secretory fluid was obtained at 2 h after injection with a relativity rapid fall. The radioactivity in the secretory fluid displayed a continuous increase up to 8 h followed by a plateau. When prostatic secretion was purified from secretory fluid and microsomes using a Con A-Sepharose column it showed a typical precursor-product relationship with an early peak at 60 min in microsomal prosatatic secretion protein followed by a peak in secretory fluid at 4 h. Vinblastine blocked the release of labeled secretion protein into the secretory fluid, a phenomenon characteristic for secretory proteins which are exocytosed by means of fusion between secretory granules and the plasma membrane. Following intravenous injection of [³H]estramustine, accumulation was seen in the secretory fluid. Some estramustine probably binds to newly synthesized protatic secretion protein and follows the same route of intracellular transport and extracellular discharge as does prostatic secretion protein.     

Some features of secretory systems in plants

Synopsis Recent work on secretion in plants is reviewed, with emphasis on the anatomy and physiology of root cap cells in higher plants, the stalked glands ofDrosera capensis, and the secretory mechanism ofDionaea muscipula. Cells of the root cap of higher plants switch from a geo-perceptive role to one of mucilage secretion at maturation. Features of this process, the role of the Golgi and the pathway for mucilage distribution are reviewed. In contrast, the stalked glands of the leaves ofDrosera capensis are much longer lived and have a complex anatomy. The mechanisms for mucilage secretion, protein absorption and the role of the cell membranes in the internal secretion of the protein are described, using data from X-ray microscopv. The secretion of fluid and protein byDionaea is stimulated by various nitrogen-containing compounds. Uric acid, often excreted by captured insects, is particularly effective in this respect.     

Localization of the type I ribosome inactivating protein, luffin, in adult and embryonic tissues of Luffa cylindrica L. Roem

The subcellular localization of the type I ribosome-inactivating protein, luffin, has been investigated by means of immunofluorescence light microscopy. A different pattern of protein distribution has been observed in embryonic and somatic tissues. In mature seeds luffin is accumulated within protein bodies in the storage tissue; vacuolar compartmentation in cells of the cotyledonary leaves is maintained during germination of the seedlings. In adult tissues, such as mature leaves and stems, the targeting of the protein is different, since luffin is found in the extracellular spaces. This localization outside the plasma membrane has been confirmed by enzymatic activity determination on the intercellular fluid present in the apoplastic space. Results on luffin localization are discussed with respect to the putative function(s) of this enzyme.Keywords: Luffa cylindrica L. Roem., luffin, ribosome-inactivating proteins (RIPs), secretory proteins, subcellular compartmentation.      

The Infundibular Organ of Branchiostoma lanceolatum

The infundibular organ of adult and larval Branchiostoma was studied by means of the electron microscope. The secretion produced by the infundibular cells is released into the ventricular fluid from secretory vacuoles, fusing with the apical plasmalemma and forming a Reissner's fiber in the ventricle. The basal cell processes reach the external limiting membrane but no basal release of secretory material has been observed. No synapses are in contact with the infundibular cells. The organ seems to function autonomously with neither nervous control from the brain tissue nor chemical regulation from the ventricular fluid.     

毕赤酵母外源蛋白分泌途径的研究进展

毕赤酵母作为一种重要的表达外源蛋白的宿主,提高其外源蛋白的分泌量非常有必要。近年来很多学者报道了与毕赤酵母外源蛋白分泌相关的基因、蛋白质,同时毕赤酵母基因组的公布加快了这方面的研究进展。文章根据外源蛋白分泌的途径,分步骤地总结了涉及的基因和蛋白,有利于分析控制蛋白分泌效率的具体步骤,为构建更加高效的毕赤酵母表达系统提供参考。     

芽胞杆菌中重组蛋白分泌表达的优化策略及研究进展

芽胞杆菌属具有良好的蛋白表达和分泌能力,在工业酶的生产中被广泛应用,是理想的工业宿主菌,但实现蛋白分泌表达的普遍高效性还存在许多瓶颈。本文综述了芽胞杆菌的蛋白分泌表达策略,从启动子、信号肽、分泌途径、宿主和培养条件这5个方面总结了提高芽胞杆菌中分泌表达重组蛋白的方法,对芽胞杆菌高效生产工业酶有一定的参考价值,最后展望了优化芽胞杆菌分泌表达的研究方向,各种新型生物技术的发展必将推进芽胞杆菌在分泌表达领域有更深入的应用。     

What's special about secretory lysosomes?

Lysosomes are organelles specialised for their role in intracellular protein degradation. A small number of cell types also use their lysosomes as regulated secretory organelles. These secretory lysosomes package additional secretory products, respond to extracellular stimuli and fuse with the plasma membrane to release their contents. Recent research has identified unique components of the secretory machinery in these cells. However, studies on conventional lysosomes in non-secretory cells reveal that even their lysosomes can fuse with the plasma membrane in response to membrane damage. What then is special about secretory lysosomes?     

Pancreatic islet cell membranes: Extraction of a possible glucoreceptor

Solubilized cell membranes of dog pancreatic islets, in contrast to membranes from acinar cells, include a component which forms complexes with glucose, mannose, and fructose, each of which is a stimulus to insulin secretion. Galactose and 3-O-methylglucose, which are not secretory stimuli, are not complexed. Thus, this component has the specificity expected of the receptor site through which these cells are stimulated to secrete insulin.     

The human FK506-binding proteins: characterization of human FKBP19

Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in the acinar cells of mouse pancreas.     

Adaptations for matrotrophy in the female reproductive system in the pseudoscorpion Chelifer cancroides (Chelicerata: Pseudoscorpiones,Cheliferidae)

Pseudoscorpiones (pseudoscorpions, false scorpions) is an order of small terrestrial chelicerates. While most chelicerates are lecithotrophic, that is, embryos develop due to nutrients (mostly yolk) deposited in the oocyte cytoplasm, pseudoscorpions are matrotrophic, that is, embryos are nourished by the female. Pseudoscorpion oocytes contain only a small amount of yolk. The embryos develop within a brood sac carried on the abdominal site of the female and absorb nutrients by a pumping organ. It is believed that in pseudoscorpions nutrients for developing embryos are produced in the ovary during a postovulatory (secretory) phase of the ovarian cycle. The goal of our study was to analyze the structure of the female reproductive system during the secretory phase in the pseudoscorpion Chelifer cancroides, a representative of the family Cheliferidae, considered to be one of the most advanced pseudoscorpion taxa. We use diverse microscopic techniques to document that the nutritive fluid is produced not only in the ovaries but also by the epithelial cells in the oviducts. The secretory active epithelial cells are hypertrophic and polyploid and release their content by fragmentation of apical parts. Our observations also indicate that fertilization occurs in the oviducts. Moreover, in contrast to previous findings, we show that secretion of the nutritive material starts when the fertilized oocytes reach the brood sac and thus precedes formation of the pumping organ. Summing up, we show that C. cancroides exhibits traits of advanced adaptations for matrotrophy due to coordinated secretion of the nutritive fluid by the ovarian and oviductal epithelial cells, which substantially increases the efficiency of nutritive fluid formation. Since the secretion of nutrients starts before formation of the pumping organ, we suggest that the embryos are able to absorb the nutritive fluid also in the early embryonic stages.     

Secretion by ependymal cells of the neurohypophysis and saccus vasculosus of Polypterus ornatipinnis (Osteichthyes)

Electron microscopic studies of the neurohypophysis and saccus vasculosus of the bichir (Polypterus ornatipinnis) reveal an apocrine release of secretory material by ependymal cells (e.g., crown cells). The secretory material appears to migrate along the microtubular apparatus and the ciliary filaments to the cell buds. It is postulated that the formation of the buds and their release is mediated by cilial action. Secretion buds are noted in the cerebrospinal fluid and vascular sinusoids. Bulbous projections of neurohypophyseal secretory tracts extend into the ependymal lumen which also contains elementary secretion granules. Specialized “liquorkontaktneurone” are interspersed with ependymal cells. An interrelation between the hypothalamus, neurohypophysis, saccus vasculosus, and the meta-adenohypophysis is postulated.     

In vitro translocation of bacterial secretory proteins and energy requirements

The recent establishment ofin vitro assay systems has made biochemical studies on the process of membrane translocation of secretory proteins possible. This review summarizes what we have learned, using thesein vitro systems, concerning the biochemical process of protein translocation, with special reference to energy requirements. Both ATP and the protonmotive force participate in the translocation reaction. The requirement of ATP is obligatory, whereas that of the protonmotive force differs, in terms of its level, with the secretory protein species. The possible roles of ATP and the protonmotive force in protein translocation are discussed with special reference to the function of SecA, an essential component of the secretory machinery. The effect of positive charges, which precede or follow the hydrophobic domain of signal peptides, on translocation is also discussed.     

The effects of phorbol esters on fluid transport and blood flow in the small intestine

Studies were designed to examine the effects of phorbol esters on intestinal fluid transport and blood flow in the anesthetized cat and enteropooling in the conscious rat. Intraluminal administration of phorbol ester into a segment of isolated small bowel produced a copious intestinal secretion and a concomitant mesenteric hyperemia in the cat. Net fluid movement in the intestine was converted from absorption in the control state to secretion following phorbol ester administration. Intravenous atropine reduced the phorbol ester-induced secretion by 56%; clonidine abolished the remaining secretory response. In the rat, intragastric administration of phorbol ester produced enteropooling comparable to that of other potent intestinal secretagogues. Since phorbol esters are known to activate protein kinase C, these studies suggest that activation of protein kinase C in the small intestine may lead to a full secretory response. The evidence suggests that this secretion is accompanied by a metabolic hyperemia. These results suggest that protein kinase C plays an important role in the regulation of intestinal fluid transport.     

Effects of cholinergic and adrenergic agonists on the secretion of fluid and protein by submandibular glands of the guinea-pig and the mouse

Significant differences were observed between the guinea-pig and the mouse in terms of the secretion of fluid, protein and secretory granules from submandibular glands in response to pilocarpine, phenylephrine and isoproterenol. In both the guinea-pig and the mouse, the secretory responses induced by pilocarpine, phenylephrine and isoproterenol were inhibited by pretreatment with 4-DAMP, phentolamine and propranolol, respectively. The results suggest that the submandibular glands of the guinea-pig and the mouse have M₃-cholinoreceptors, as well as α- and β-adrenoceptors, and that these receptors play different roles in the secretion of fluid, protein and secretory granules from guinea-pig and mouse submandibular glands.     

Ultrastructural observations on paracnids. I: Coelogynopora axi Sopott (Turbellaria,Proseriata)

The Schlauchdrüsen or paracnids of Coelogynopora axi Sopott, 1972 consist of two components: a muscle cell and a secretory cell.The secretory cell is provided with a tube, which bears a border of microvilli. In the normal position the tube is situated in the interior of the secretory cell, and the microvilli stand at the inner side of the tube. After expulsion of the tube the microvilli are situated at its free surface.The evagination takes place in response to chemical stimuli and is effected by the contraction of the myofibrils of the muscle cell.The paracnids are supposed to be mechanisms of defense.However, conformities with nematocysts and spirocysts of the cnidarians do not exist.The paracnids in other species of the Coelogynoporidae, for example in Invenusta paracnida (Karling, 1966) and Carenscoilia bidentata Sopott, 1972 differ from those of C. axi in many details.Abbreviations bl- basement lamina - ep- epidermis - hd- hemidesmosomes - mc- muscle cell - mt- microtubules - mv- microvilli - nsc- nucleus of the secretory cell - sb- bowl containing secretion granules - sc- secretory cell - sd- septate desmosome-like structures - sg- secretion granules - t- tube - tf- tonofilaments     

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes,is essential for the biogenesis of secretory organelles

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome‐like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin‐like receptor and syntaxin‐6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab‐GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps‐C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal‐related compartments, the vacuole and immature apical secretory organelles. Conditional knock‐down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock‐down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite‐specific functions in the biogenesis of their unique secretory organelles.     

Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-³H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.     

Ultrastructure and histochemistry of the male reproductive system of the genus Callinectes Stimpson, 1860 (Brachyura: Portunidae)

We described the ultrastructure and histochemistry of the reproductive system of five Callinectes species, and evaluate the seasonal variation in weight of the reproductive system and hepatopancreas by comparing annual changes of somatic indices. The somatic indices changed little throughout the year. In Callinectes, spermatogenesis occurs inside the lobular testes and, within each lobule, the cells are at the same developmental stage. Spermatogenesis and spermiogenesis follow the same development pattern in all Callinectes studied. Mature spermatozoa are released into the seminiferous ducts through the collecting ducts. Cells of the vas deferens are secretory as evidenced by rough endoplasmic reticulum, Golgi complex, and secretory vesicles that produce the seminal fluid. The anterior vas deferens shows two portions: proximal and distal. In proximal portion (AVDp), spermatozoa are clustered and embedded in an electron-dense, basophilic glycoproteinaceous secretion Type I. In the distal portion (AVDd), the spermatophore wall is formed by incorporation of a less electron-dense glycoproteinaceous secretion Type II. The secretion Type I change to an acid polysaccharide-rich matrix that separates the spermatophores from each other. The median vas deferens (MVD) stores the spermatophores and produces the granular glycoproteinaceous seminal fluid. The posterior vas deferens (PVD) has few spermatophores. Its epithelium has many mitochondria and the PVD seminal fluid changes into a liquid and homogeneous glycoprotein. Many outpocketings in the PVD and MVD help to increase the fluid production. Overall, the reproductive pattern of Callinectes is similar to other species that produce sperm plugs. The secretions of AVD, MVD, and PVD are responsible for the polymerization that forms the solid, waxy plug in the seminal receptacle. The traits identified here are common to all Portunidae species studied so far.     

Direct hypertonic stimulation of the rat supraoptic nucleus increases c-fos expressionin glial cells rather than magnocellular neurones

We investigated whether hypertonicity acts directly on supraoptic neurones to activate c-fos expression. Hypertonic artificial cerebrospinal fluid was infused into the supraoptic nucleus (SON) via a microdialysis probe implanted 24 h previously. The rats were decapitated after 90 min for immunohistochemistry with a Fos protein antibody. Direct hypertonic stimulation increased Fos protein expression in glial cells, identified by glial fibrillary acidic protein immunoreactivity, but not in magnocellular neurones. Similarly, with in situ hybridisation c-fos mRNA expression was predominantly seen in glial cells. Fos expression in SON neurones was stimulated by systemic hypertonicity even with a microdialysis probe in the SON, and magnocellular neurones expressed Fos after direct microinjection of cholecystokinin-8S into the SON. Thus, while direct hypertonic stimulation of SON neurones activates secretion of vasopressin and oxytocin, the c-fos gene is not activated, unlike following systemic hypertonic stimulation. This indicates that excitation of neuronal electrical and secretory activity does not necessarily lead to activation of the c-fos gene. Activation of c-fos expression in glial cells by direct hypertonic stimulation may reflect their role in regulating brain extracellular fluid composition. Received: 11 March 1996 / Accepted: 24 July 1996