Localization of some enzymes in the main venom glands of viperid snakes

Several secretory and nonsecretory enzymes were localized histochemically in the main venom gland of 13 viperid snakes. All secretory cells show the intracellular oxidative enzymes succinate dehydrogenase and monoamine oxidase. The granular reactions obtained for both enzymes resemble mitochondria in distribution. Distinctive cells with a very high succinate dehydrogenase activity are dispersed among the secretory cells of all species except Atractaspis. Nonspecific acid phosphatase activity is found in the supranuclear region of the secretory cells in species that do not secrete this enzyme and throughout the cytoplasm in snakes that secrete the enzyme. Nonspecific alkaline phosphatase activity occurs in the secretory cells of those snakes whose venom shows this activity. Leucine amino peptidase (aryl amidase) activity is found in the venom and in the secretory cells of all the species. In Vipera palaestinae both the venom and the secretory cells of the main venom gland contain nonspecific esterase, L-amino acid oxidase and phosphodiesterase activities. The localization of phosphodiesterase and L-amino acid oxidase do not show major differences between glands at different intervals from an initial milking. Adenosine-monophosphate phosphatase activity is localized in the supranuclear region of the secretory cells in the glands of Vipera palaestinae and Aspis cerastes. Its activity is found in the venom of Aspis only.     

Demonstration and maintenance of mucus secretion in cultured human gallbladder epithelial cells

Summary The method of human gallbladder epithelial cell culture has been developed successfully with active mucus secretory function. Human gallbladder epithelial cells were dissociated by Dispase digestion from the specimens obtained by cholecystectomy for uncomplicated gallbladder stone cases. The dissociated cells formed a monolayer in Eagle’fs minimum essential medium supplemented with 10% fetal bovine serum within 24 h after the inoculation. These cells were maintained for at least 2 wk without fibroblastic overgrowth. Cultured cells contained periodic acid Schiff-positive material in cellular cytoplasm for 3 d. On transmission electron microscopy these materials were identified as mucous secretory granules. Mucous secretory function was determined by [³H]glucosamine incorporation. Sixty percent of the secreted glycoproteins labeled with [³H]glucosamine was eluted in excluded fractions of Sepharose 4B gel filtration, which were considered to be mucous glycoprotein, because they were found to be resistant to proteoglycan-specific enzymes such as hyaluronidase, chondroitinase ABC, heparitinase, and heparinase. The mucous glycoprotein secretion was maintained for 3 d and found to be inhibited in a dose-dependent manner by monensin (10⁻⁷ to 10⁻⁵ M) which is a known blocker of secretory function.     

Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L.

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Δ11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1α was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.     

The occurrence of some enzymatic activities in the differentiation of nerve tissue of gallus embryos in cultures in vitro

Summary The occurrence and localization of Succinicodehydrogenase, Lacticodehydrogenase, Monoaminoxidase and Acetylcholinesterase have been studied in cultures in vitro of spinal cord, dorsal root ganglia, cerebellum and optic tectum of chicken embryos. Explantations have been made before the morphological and functional differentiation of the neurons of the centres considered took place. All the neurons differentiated in vitro show a high concentration of SDH and LDH, whereas the activity of these two enzymes results to be much lower in the other cultures' elements (glial cells, fibroblasts). Research-works on the AChE and MAO distribution and concentration within the neurons differentiated in vitro show that there differentiate in culture neurons which have morphological and cytochemical characters similar to those of the corresponding neurons in vivo. An important exception is the AChE occurrence in the Purkinje neurons differentiated in vitro; the possible reasons for this behaviour are examined in the text. The data mentioned in the paper allow the conclusion that the various types of neuroblasts explanted in vitro are not only able to reach the morphological differentiation, but also to acquire specific biochemical characters, directly related to the neuron functionality.     

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation. Accepted: 10 June 1999     

Etude histologique,histochimique et ultrastructurale de la pars intercerebralis chez Locusta migratoria L. (Orthoptere)

Summary A histological, histochemical and ultrastrucutral study of the pars intercerebralis (PI) has been made in Locusta migratoria. The acellular neural lamella is made up of an elastic tissue and collagen fibrils. The cells of the perilemma contain numerous lysosome structures and lipid granules.Three different types of neurosecretory cells (NSC A, B and C) have been distinguished in the PI associated with giant neurons.The cells termed A and B seem not to have an activity cycle during the two last larval instars. At the moment of sexual maturity the NSC A show an important accumulation of neurosecretory material and their number increases at the expense of the NSC B. The NSC A, which are characterized by a highly developped endoplasmic reticulum, contain numerous secretory granules which appear to be individualized in the Golgi complex in three different ways. The NSC B, with a reduced endoplasmic reticulum and an almost quiescent Golgi complex, contain abundant lysosome structures and more seldom some neurosecretory granules. In fact, the study of the fine structure shows different intermediate types, linking in a continuous way typical A cells and typical B cells. NSC A and NSC B might correspond to two opposed stages of secretory activity of one single cell type: the A cell representing the activity stage and the B cell the quiescent stage.NSC C show an accumulation of their neurosecretory products in relation to metamorphosis and sexual maturity. Ultrastructural evidence confirms their neurosecretory activity.A mode of regulating neurosecretion in NSC A and B by internal catabolism of the secretion and formation of lysosome like structures is discussed in the present paper.The giant neurons, which are surrounded by a glial envelope (trophospongium), contain several dense granules originated from Golgi complex.     

Immunocytochemical Study of the Distribution of hcs2 Gene Products in Command Neurons of the Helix lucorum Snail

The distribution of the putative protein products of gene hcs2 in giant command neurons of the parietal ganglia of the terrestrial snail Helix lucorum has been studied using light- and electron-microscopic immunocytochemistry. The product of the hcs2 gene is a hybrid protein belonging to the EF-hand family of Ca²⁺-binding proteins and is a precursor of several neuropeptides. Polyclonal antibodies to neuropeptides CNP3 and CNP4 and the C-terminal Ca²⁺-binding domain of the precursor protein have been used to determine their intracellular localization. The targets for all three types of antibodies have been found in cytoplasmic secretory granules. The label (colloidal gold) density in the secretory granules is two times higher in the case of neuropeptides CNP3 and CNP4 than in the case of the Ca²⁺-binding domain. Thus, a specific association between the putative products of the hcs2 gene and the cell secretory apparatus has been demonstrated. This agrees with the earlier hypothesis that hcs2 products may serve as neurotransmitters or neuromodulators.     

Sex steroid effects on the development and functioning of the growth hormone axis

Summary 1. The secretory pattern of growth hormone (GH) is sexually dimorphic in the adult rat. However, this difference between the sexes does not become apparent until after the onset of puberty, suggesting that pubertal sex steroids play an important role in the manifestation of this phenomenon.2. We have addressed the question as to whether there exists a sexual dimorphism in the hypothalamic neuropeptides that regulate GH release from the anterior pituitary,i.e., somatostatin (SS) and growth hormone-releasing hormone (GHRH). In addition, we have investigated whether the developmental changes in the GH secretory pattern are correlated with changes in these neuropeptides. The effect of testosterone treatment on SS and GHRH neurons during both the neonatal period and adulthood have also been studied.3. We have found that the synthetic capacity, as reflected in relative messenger RNA (mRNA) levels, of both SS and GHRH neurons changes throughout development in both male and female rats. These mRNA levels are sexually dimorphic at certain times during maturation and can be modulated by changes in testosterone levels, suggesting that sex steroid modulation of these two neuropeptide systems could at least partially account for the sexual dimorphism seen in the adult GH secretory pattern.4. The neonatal steroid environment has also been suggested to be involved in the generation of the final adult GH secretory pattern, although the mechanisms underlying this effect are even less well understood. In support of the hypothesis that the neonatal steroid environment plays an important role in organizing the GH axis, we have found that the number of GHRH neurons in the adult brain, as well as their sensitivity to adult steroids, is modulated by neotatal testosterone treatment. The number of SS neurons in the periventricular and paraventricular nuclei were not modulated by neonatal steroids; however, the synthetic capacity of these neurons does appear to be influenced by the neonatal steroid environment.5. These studies suggest that both the neonatal and adult sex steroid environments influence the adult GH secretory pattern by modulating GHRH and SS neurons.     

Signal peptidases and signal peptide hydrolases

Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources. Seven signal peptidases have been purified, two fromE. coli, two from mammalian sources, and three from mitochondrial matrix. The mitochondrial enzymes are soluble and function as a heterogeneous dimer. The mammalian enzymes are isolated as a complex and share a common glycosylated subunit. The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes. The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the –3, –1 rule of von Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide. The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site. The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class of proteases as they are resistant to chelators and most protease inhibitors. There are no data concerning the substrate binding site of these enzymes. In vivo, the signal peptide is rapidly degraded. Three different enzymes inEscherichia coli that can degrade a signal peptidein vitro have been identified. The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degredation of an intact signal peptidein vivo. It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.     

A histochemical study of the cells of the ventral cord ganglia of the horseshoe crab,Limulus polyphemus (L.)

Summary The ventral cord ganglia of the horseshoe crab, Limulus polyphemus, contains six distinct cell types: three appear to be ordinary neurons and three exhibit the staining affinities of neurosecretory cells.The presumed neurosecretory cells have been termed neurosecretory cell I (NSC I), NSC II and NSC III. NSC I cells contain a colloid-like inclusion which may occur as a single small vacuole or occupy more than one-half of the cell volume. Colloid inclusions occur with greater frequency toward the periphery, although small cells of similar staining affinity occur in cords extending to the fibrous core. The histochemical tests suggest that the cytoplasm is positive for proteins, but contains no strong acidic groups which may have been derived from S-S or S-H groups. The presence of carbohydrate is also indicated.NSC II cells exhibit distinct secretory cycles. Early in the cycle the cytoplasm becomes phloxinophilic and progresses to a distinct fuchsinophilic stage. Small homogeneous irregular inclusions are found in the axon hillock during the latter stages of the cycle. Histochemical tests suggest the presence of a carbohydrate and strong acidic groups which may have been derived from S-S or S-H groups. There are small cells present which appear to be immature neurosecretory cells.NSC III cells are characterized by a perinuclear ring of cytoplasm which is stained by chrome alum hematoxylin but not by paraldehyde fuchsin. A secretory cycle may also be present in this cell type.The three cell types presumed to be ordinary neurons exhibit no particular staining affinity for the stains or tests used in this study.This study was supported in part by a grant from the Central Fund for Research of the Pennsylvania State University.     

Genetic Analysis of Growth Inhibition of Yeast Cells Caused by Expression of Aspergillus oryzae RNase T1

Even though most fungal hydrolytic enzymes have been successfully secreted in S. cerevisiae cells by expression of corresponding cDNA, overexpression of A. oryzae RNase T1 causes severe growth inhibition in yeast. We observed that yeast strains carrying RNase T1 cDNA under control of the GAL1 promoter with a single-copy vector were able to grow on galactose medium while those with a multi-copy vector were not. It was found that overexpression of three mutated versions of RNase T1 with low enzymatic activity did not affect the growth. We also observed that expression of RNase T1 without a signal sequence severely inhibited growth of the transformant even on the single-copy plasmid. Subcellular fractionation showed that overexpressed myc-tagged RNase T1 was localized in the membrane fraction. In the yeast secretory pathway, while the mutants defective in translocation into the ER, ER-Golgi trafficking and vacuole formation had severe growth inhibition during expression of RNase T1 from the single-copy plasmid. These results suggest that a mislocalization of active RNase T1 in cytosol by overflow from the secretory apparatus has toxic effects on the host cells.     

SECRETORY CELLS OF LILY PISTILS. I. FINE STRUCTURE AND FUNCTION

The fine structure of the cells which line the canal of Lilium longiflorum pistils confirms the secretory function which has been ascribed to them. The cells differ in structure from the secretory cells which cover the stigma surface and are therefore referred to as canal cells rather than stigmatoid cells. Their most striking feature is an elaborate wall, 8–14 μ in maximum depth, on the side facing the canal, which with associated structures, we term the secretion zone. Pollination, which triggers chemotropic activity in the style and secretory activity in the canal cells, is not correlated with marked changes in the fine structure of the canal cells. The canal cells appear to fit well into that category which Gunning and Pate have termed “transfer cells.”     

Histochemical studies on the morphology of the golgi apparatus and on the enzymes of carbohydrate metabolism in various nuclei of the rat mesencephalon

Summary Detailed histochemical studies have been conducted on the distribution of various enzymes such as thiamine pyrophosphatase, α-glucan phosphorylase, hexokinase, glucose-6-phosphate dehydrogenase, aldolase, lactate dehydrogenase and succinate dehydrogenase in various components of the nucleusEdinger-Westphali, nucleus n. oculomotorii, nucleus ruber and nucleus niger of healthy adult male Wistar strain rats. The thiamine pyrophosphatase reaction showed the morphological patterns of the Golgi apparatus characteristic for each nucleus. The Golgi apparatus was well developed in the nucleusEdinger-Westphali, composing a network of highly fenestrated plates in the nucleus n. oculomotorii and nucleus ruber, and a simple network in the nucleus niger. These results indicate that the former three nuclei need a rich energy supply and argue against the possibility that the four nuclei have a secretory role. The neurons of the nucleusEdinger-Westphali may derive their energy mainly from glucose of the circulating blood, but glial cells may serve as energy donators to the neurons in the pars compacta of the nucleus niger, and the neurons of the other nuclei may derive energy from both sources. These conclusions are consistent with the morphological patterns of the Golgi apparatus. It is suggested that the neurons of the nucleusEdinger-Westphali, nucleus n. oculomotorii, nucleus ruber and of the pars lateralis of the nucleus niger may be equipped almost equally with the Embden-Meyerhof pathway and with the hexose monophosphate shunt. But, the hexose monophosphate shunt is dominant in the pars compacta of the nucleus niger. It is also suggested that the pattern of distribution of succinate dehydrogenase may parallel that of lactate dehydrogenase. The nucleus n. oculomotorii, and nucleus ruber have a higher level of oxidative metabolism than the nucleusEdinger-Westphali and the nucleus niger. The nucleusEdinger-Westphali may be representative of autonomic nuclei with low oxidative metabolism whereas the nucleus n. oculomotorii may represent motor nuclei with high oxidative metabolism. Predominance of hexose monophosphate shunt, intense hexokinase reaction around the neurons, and weak activity of succinate dehydrogenase indicate that the pars compacta of the nucleus niger belongs to the category of “exceptional nuclei”.     

Ultrastructural Analysis of the Neuroendocrine Apparatus of Oncopeltus fasciatus (Heteroptera)

• 1 In Oncopeltus fasciatus, the A-cells of the pars intercerebralis and their tracts are stainable in situ with the performic acid-victoria blue (PAVB) method. The axons from these cells, after traversing the corpus cardiacum, terminate in the anterior part of the aorta which thus serves as the neurohemal organ.
• 2 Ultrastructurally, four types of secretory neurons are distinguishable in the pars intercerebralis region: pic-I with granules measuring 1000–3000 Å in diameter; pic-II with granules of irregular size and shape, the elongate ones showing mean dimensions of 2400 × 1400 Å; pic-III with less electron-dense granules measuring 1000–2700 Å in diameter; pic-IV, present not only in the pars intercerebralis but also in adjacent regions of the brain, with variable proportions of granules measuring 700–1800 A and dense-cored vesicles measuring 1000–2400 Å.
• 3 The nervi corporis cardiaci contain at least three types of neurosecretory axons, based on granule content, presumably representing pic-I, pic-II and pic-III neurons.
• 4 The wall of the aorta contains endings of at least three distinct types, again representing pic-I, pic-II and pic-III neurons, and thus provides the neurohemal site for these three types of protocerebral neurosecretory cells. Axons of pic-IV neurons appear to enter the cerebral neuropil.
• 5 The corpus cardiacum is composed of two types of parenchymal secretory cells, with electron-dense granules measuring 1300–3000 Å and 1000–2300 Å in diameter, respectively. The corpus cardiacum also contains interstitial cells and some axons of extrinsic origin, with and without granules.
• 6 The corpus allatum may be paired or median, and receives a small number of at least two types of axons. The corpora allata of some reproducing females show a large number of PAVB-stainable inclusions which appear to be modified cytoplasmic organelles, but are definitely not neurosecretory material.
• 7 The hypocerebral ganglion is composed of two types of secretory-appearing neurons and glial cells. The two neuronal types contain secretory granules, 1000–3000 Å and 900–2100 Å in diameter, respectively. Axons of the recurrent nerve also may contain occasional granules.
• 8 In this heteropteran insect, the two principal functions of the corpus cardiacum appear to be spatially separated: the neurohemal function is subserved by the aortic wall, which permits release of material into both the aortic lumen and the hemocoel, and the intrinsic endocrine function is possessed by the parenchymal cells.

     

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I₂ assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.     

Isolation of Functional Golgi-derived Vesicles with a Possible Role in Retrograde Transport

Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis-side and exit in transport vesicles budding from the trans-side. Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretory flow must be recycled constantly by retrograde transport in the opposite direction. In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells. We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cisternal membranes. These vesicles appeared to be depleted of secretory cargo. They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus. Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack.     

Identification of a complex peptidergic neuroendocrine network in the hard tick, <Emphasis Type="Italic">Rhipicephalus appendiculatus</Emphasis>

Neuropeptides are crucial regulators of development and various physiological functions but little is known about their identity, expression and function in vectors of pathogens causing serious diseases, such as ticks. Therefore, we have used antibodies against multiple insect and crustacean neuropeptides to reveal the presence of these bioactive molecules in peptidergic neurons and cells of the ixodid tick Rhipicephalus appendiculatus. These antibodies have detected 15 different immunoreactive compounds expressed in specific central and peripheral neurons associated with the synganglion. Most central neurons arborize in distinct areas of the neuropile or the putative neurohaemal periganglionic sheath of the synganglion. Several large identified neurons in the synganglion project multiple processes through peripheral nerves to form elaborate axonal arborizations on the surface of salivary glands or to terminate in the lateral segmental organs (LSO). Additional neuropeptide immunoreactivity has been observed in intrinsic secretory cells of the LSO. We have also identified two novel clusters of peripheral neurons embedded in the cheliceral and paraspiracular nerves. These neurons project branching axons into the synganglion and into the periphery. Our study has thus revealed a complex network of central and peripheral peptidergic neurons, putative neurohaemal and neuromodulatory structures and endocrine cells in the tick comparable with those found in insect and crustacean neuroendocrine systems. Strong specific staining with a large variety of antibodies also indicates that the tick nervous system and adjacent secretory organs are rich sources of diverse neuropeptides related to those identified in insects, crustaceans or even vertebrates. This work was supported by Slovak grant agencies: Agentúra na podporu vyskumu a vyvoja (APVV-51-039105) and Vedecká grantová agentúra (VEGA 2-6090-26 and 2/6155/26).     

The paraventricular and posterior recess organs of elasmobranchs: A system of cerebrospinal fluid-contacting neurons containing immunoreactive serotonin and somatostatin

Summary The paraventricular organ (PVO) and the posterior recess organ (PRO) of two elasmobranch species, the spiny dogfish,Squalus acanthias, and the skate,Raja radiata, were investigated by use of scanning and transmission electron microscopy and immunocytochemistry employing a series of primary antisera. The PVO and PRO contained four types of cerebrospinal fluid (CSF)-contacting neurons. One type was free of secretory granules and projected a dendrite-like process into the ventricle. The other three types were distinguished according to the size of their secretory granules. The ventricular extensions of these cells were filled with secretory granules. By means of immunocytochemistry three types of CSF-contacting neurons were observed in the PVO and PRO. Type I contained only serotonin; type 2 displayed only somatostatin; type 3 was endowed with both serotonin and somatostatin. Type I dominated in the PRO, whereas type 3 was the most frequent in the PVO. The latter cells appear to be the site of origin of a loose tract formed by serotonin- and somatostatinimmunoreactive fibers projecting from the PVO into the neuropil of the PRO. Compact bundles formed exclusively by serotonin fibers were also shown to extend between the PVO and PRO. The basal processes of the CSF-contacting neurons of the PRO penetrated into the underlying neuropil. This neuropil is rich in synapses and can be regarded as an integrative area to which the basal processes of the local CSF-contacting neurons, serotonin and somatostatin fibers from the PVO, and fibers containing immunoreactive thyrotropin-releasing hormone of unknown origin, support a conspicuous input. The present findings indicate that the PVO and PRO of elasmobranchs are functionally integrated structures.Dedicated to Professor Erik Dahl on the occasion of his 75th birthday.     

On‐site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip‐growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long‐range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three‐dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub‐apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short‐range “on‐site” secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.