The exocyst complex controls multiple events in the pathway of regulated exocytosis.

Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.

Condensation of the ß-cell secretory granule luminal cargoes pro/insulin and ICA512 RESP18 homology domain.

ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of ß-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 µm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.

Evaluation of secretory activity in carp pancreatocytes (Cyprinus carpio L. ) when using lactobacillus-based chelate and probiotic supplemental feed complexes / Avaliação da atividade secretora em pancreatócitos de carpa (Cyprinus carpio L. ) ao usar quelato à base de lactobacilos e complexos suplementares de alimentação probiótica

The experimental research was carried out on the juvenile carp (Cyprinus carpio L.). The impact from supplemental feeds consisting of variable concentrations of chelate compounds, biogenic trace elements and probiotic lactobacillus-based product Bacillus subtilis VKPM B-2335 was evaluated. Optical and qualitative parameters of the lactobacillus base were studied in order to identify the major group of substances potentially able to influence the end result. The purpose of this research was to identify changes in the structure of the zymogen granules and their dimensions at which supplemental feeds produce a stimulating effect on the synthesis of zymogens in exogenous cells of the secretory part of pancreas. At the outcome of the study, for the first time, it was possible to prove that the integrated action of chelates and lactobacillus-based probiotics complemented each other. Metal chelate compounds contributed to enlargement of the zymogen granules, if compared to the control values. The bacterial products accelerated production of the zymogen granules in acinar cells diffusely located in carp hepatopancreas.(AU)

A pesquisa experimental foi realizada em carpas juvenis (Cyprinus carpio L.). O impacto de suplementos alimentares consistindo em concentrações variáveis de compostos quelatos, oligoelementos biogênicos e produto probiótico baseado em lactobacilos Bacillus subtilis VKPM B-2335 foi avaliado. Parâmetros ópticos e qualitativos da base de lactobacilos foram estudados a fim de identificar o maior grupo de substâncias potencialmente capazes de influenciar o resultado final. O objetivo dessa pesquisa foi identificar mudanças na estrutura dos grânulos de zimogênio e suas dimensões nas quais alimentos suplementares produzem um efeito estimulante na síntese de zimogênios em células exógenas da parte secretora do pâncreas. No resultado do estudo, pela primeira vez, foi possível comprovar que a ação integrada dos quelatos e dos probióticos à base de lactobacilos se complementava. Compostos quelatos metálicos contribuíram para o aumento dos grânulos de zimogênio, se comparados aos valores de controle. Os produtos bacterianos aceleraram a produção dos grânulos de zimogênio nas células acinares localizadas difusamente no hepatopâncreas da carpa.(AU)

Evaluation of secretory activity in carp pancreatocytes (Cyprinus carpio L. ) when using lactobacillus-based chelate and probiotic supplemental feed complexes / Avaliação da atividade secretora em pancreatócitos de carpa (Cyprinus carpio L. ) ao usar quelato à base de lactobacilos e complexos suplementares de alimentação probiótica

The experimental research was carried out on the juvenile carp (Cyprinus carpio L.). The impact from supplemental feeds consisting of variable concentrations of chelate compounds, biogenic trace elements and probiotic lactobacillus-based product Bacillus subtilis VKPM B-2335 was evaluated. Optical and qualitative parameters of the lactobacillus base were studied in order to identify the major group of substances potentially able to influence the end result. The purpose of this research was to identify changes in the structure of the zymogen granules and their dimensions at which supplemental feeds produce a stimulating effect on the synthesis of zymogens in exogenous cells of the secretory part of pancreas. At the outcome of the study, for the first time, it was possible to prove that the integrated action of chelates and lactobacillus-based probiotics complemented each other. Metal chelate compounds contributed to enlargement of the zymogen granules, if compared to the control values. The bacterial products accelerated production of the zymogen granules in acinar cells diffusely located in carp hepatopancreas.

A pesquisa experimental foi realizada em carpas juvenis (Cyprinus carpio L.). O impacto de suplementos alimentares consistindo em concentrações variáveis de compostos quelatos, oligoelementos biogênicos e produto probiótico baseado em lactobacilos Bacillus subtilis VKPM B-2335 foi avaliado. Parâmetros ópticos e qualitativos da base de lactobacilos foram estudados a fim de identificar o maior grupo de substâncias potencialmente capazes de influenciar o resultado final. O objetivo dessa pesquisa foi identificar mudanças na estrutura dos grânulos de zimogênio e suas dimensões nas quais alimentos suplementares produzem um efeito estimulante na síntese de zimogênios em células exógenas da parte secretora do pâncreas. No resultado do estudo, pela primeira vez, foi possível comprovar que a ação integrada dos quelatos e dos probióticos à base de lactobacilos se complementava. Compostos quelatos metálicos contribuíram para o aumento dos grânulos de zimogênio, se comparados aos valores de controle. Os produtos bacterianos aceleraram a produção dos grânulos de zimogênio nas células acinares localizadas difusamente no hepatopâncreas da carpa.

Syntaxin 12 and COMMD3 are new factors that function with VPS33B in the biogenesis of platelet α-granules.

Platelet α-granules regulate hemostasis and myriad other physiological processes, but their biogenesis is unclear. Mutations in only 3 proteins are known to cause α-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized α-granule proteins through megakaryocyte (MK) endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Syntaxin 12 (Stx12), a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12-deficient MKs display reduced α-granule numbers and overall levels of α-granule proteins, thus revealing Stx12 as a new component of the α-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B, suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in MKs contains COMMD3, one of 10 COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced α-granule numbers and overall levels of α-granule proteins, establishing the COMMD3/CCC complex as a new factor in α-granule biogenesis. Furthermore, P-selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in α-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of α-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in MKs to make α-granules.

SNAP23 is essential for platelet and mast cell development and required in connective tissue mast cells for anaphylaxis.

Degranulation, a fundamental effector response from mast cells (MCs) and platelets, is an example of regulated exocytosis. This process is mediated by SNARE proteins and their regulators. We have previously shown that several of these proteins are essential for exocytosis in MCs and platelets. Here, we assessed the role of the SNARE protein SNAP23 using conditional knockout mice, in which SNAP23 was selectively deleted from either the megakaryocyte/platelet or connective tissue MC lineages. We found that removal of SNAP23 in platelets results in severe defects in degranulation of all three platelet secretory granule types, i.e., alpha, dense, and lysosomal granules. The mutation also induces thrombocytopenia, abnormal platelet morphology and activation, and reduction in the number of alpha granules. Therefore, the degranulation defect might not be secondary to an intrinsic failure of the machinery mediating regulated exocytosis in platelets. When we removed SNAP23 expression in MCs, there was a complete developmental failure in vitro and in vivo. The developmental defects in platelets and MCs and the abnormal translocation of membrane proteins to the surface of platelets indicate that SNAP23 is also involved in constitutive exocytosis in these cells. The MC conditional deletant animals lacked connective tissue MCs, but their mucosal MCs were normal and expanded in response to an antigenic stimulus. We used this mouse to show that connective tissue MCs are required and mucosal MCs are not sufficient for an anaphylactic response.

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues.

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane.

Rapid vesicle replenishment after the immediately releasable pool exocytosis is tightly linked to fast endocytosis, and depends on basal calcium and cortical actin in chromaffin cells.

The maintenance of the secretory response requires a continuous replenishment of releasable vesicles. It was proposed that the immediately releasable pool (IRP) is important in chromaffin cell secretion during action potentials applied at basal physiological frequencies, because of the proximity of IRP vesicles to voltage-dependent Ca2+ channels. However, previous reports showed that IRP replenishment after depletion is too slow to manage such a situation. In this work, we used patch-clamp measurements of membrane capacitance, confocal imaging of F-actin distribution, and cytosolic Ca2+ measurements with Fura-2 to re-analyze this problem in primary cultures of mouse chromaffin cells. We provide evidence that IRP replenishment has one slow (time constant between 5 and 10 s) and one rapid component (time constant between 0.5 and 1.5 s) linked to a dynamin-dependent fast endocytosis. Both, the fast endocytosis and the rapid replenishment component were eliminated when 500 nM Ca2+ was added to the internal solution during patch-clamp experiments, but they became dominant and accelerated when the cytosolic Ca2+ buffer capacity was increased. In addition, both rapid replenishment and fast endocytosis were retarded when cortical F-actin cytoskeleton was disrupted with cytochalasin D. Finally, in permeabilized chromaffin cells stained with rhodamine-phalloidin, the cortical F-actin density was reduced when the Ca2+ concentration was increased in a range of 10-1000 nM. We conclude that low cytosolic Ca2+ concentrations, which favor cortical F-actin stabilization, allow the activation of a fast endocytosis mechanism linked to a rapid replenishment component of IRP.

Pancreatic ß-Cell-Specific Deletion of VPS41 Causes Diabetes Due to Defects in Insulin Secretion.

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic ß-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in ß-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.

Ceramide induces a multicomponent intracellular calcium increase triggering the acrosome secretion in human sperm.

Exocytosis of spermatozoon's secretory vesicle, named acrosome reaction (AR), is a regulated event that plays a central role in fertilization. It is coupled to a complex calcium signaling. Ceramide is a multitasking lipid involved in exocytosis. Nevertheless, its effect on secretion is controversial and the underlying cellular and molecular mechanisms remain unknown. Human spermatozoa are useful to dissect the role of ceramide in secretion given that the gamete is not capable to undergo any trafficking mechanisms other than exocytosis. We report for the first time, the presence of sphingolipid metabolism enzymes such as neutral-sphingomyelinase and ceramide synthase in sperm. Ceramidases are also present and active. Both the addition of cell-permeable ceramide and the rise of the endogenous one, increase intracellular calcium acting as potent inducers of exocytosis. Ceramide triggers AR in capacitated spermatozoa and enhances the gamete response to progesterone. The lipid induces physiological ultrastructural changes in the acrosome and triggers an exocytosis-signaling cascade involving protein tyrosine phosphatase 1B and VAMP2. Real-time imaging showed an increment of calcium in the cytosol upon ceramide treatment either in the absence or in the presence of extracellular calcium. Pharmacological experiments demonstrate that at early stages the process involves ryanodine receptors, CatSper (calcium channel of sperm), and store-operated calcium channels. We set out the signaling sequence of events that connect ceramide to internal calcium mobilization and external calcium signals during secretion. These results allow the coordination of lipids and proteins in a pathway that accomplishes secretion. Our findings contribute to the understanding of ceramide's role in regulated exocytosis and fertilization.

Galectin-10, the protein that forms Charcot-Leyden crystals, is not stored in granules but resides in the peripheral cytoplasm of human eosinophils.

A predominant protein of human eosinophils is galectin-10 (Gal-10), also known as Charcot-Leyden crystal protein (CLC-P) because of its remarkable ability to form Charcot-Leyden crystals (CLCs), which are frequently found in tissues from patients with eosinophilic disorders. CLC-P/Gal-10 is highly expressed in human eosinophils and considered a biomarker of eosinophil involvement in inflammation. However, the intracellular sites where large pools of CLC-P/Gal-10 constitutively reside are still unclear, and whether this protein is derived or not from eosinophil granules remains to be established. Here, we applied pre-embedding immunonanogold transmission electron microscopy combined with strategies for optimal antigen and cell preservation and quantitative imaging analysis to investigate, for the first time, the intracellular localization of CLC-P/Gal-10 at high resolution in resting and activated human eosinophils. We demonstrated that CLC-P/Gal-10 is mostly stored in the peripheral cytoplasm of human eosinophils, being accumulated within an area of ∼250 nm wide underneath the plasma membrane and not within specific (secretory) granules, a pattern also observed by immunofluorescence. High-resolution analysis of single cells revealed that CLC-P/Gal-10 interacts with the plasma membrane with immunoreactive microdomains of high CLC-P/Gal-10 density being found in ∼60% of the membrane area. Eosinophil stimulation with CCL11 or TNF-α, which are known inducers of eosinophil secretion, did not change the peripheral localization of CLC-P/Gal-10 as observed by both immunofluorescence and immuno-EM (electron microscopy). Thus, in contrast to other preformed eosinophil proteins, CLC-P/Gal-10 neither is stored within secretory granules nor exported through classical degranulation mechanisms (piecemeal degranulation and compound exocytosis).

The absence of interleukin 10 affects the morphology, differentiation, granule content and the production of cryptidin-4 in Paneth cells in mice.

Paneth cells (PCs) are specialized epithelial cells of the small bowel that contain multiple secretory granules filled with antimicrobial peptides and trophic factors, which are essential for the control of the microorganisms growth and maintaining intestinal integrity. Alterations in their function are associated with an imbalance of the normal microbiota, gastrointestinal infections and inflammatory processes, such as Crohn's disease (CD). One of the most common murine models for studying CD is IL-10-/- mouse. IL-10-/- mice when housed in conventional conditions and take contact with commensal microorganisms develop an acute enterocolitis mediated by a Th1 immune response. Even though, alterations in PCs function are related to CD, they had not been characterized yet in this mouse model. Here we show that in specific pathogen free conditions IL-10-/- mice have aberrant granules and a large number of immature PCs at the bottom of the crypt in the ileum of IL-10-/- mice before developing intestinal inflammation, along with a reduced expression of Indian Hedgehog. In addition, IL-10-/- Paneth cells presented a reduced expression of cryptidin-4, and a heterogeneous distribution of lysozyme+ granules. The alterations in the maturation of the PCs at the bottom of the crypt were not modified after the colonization by the conventional microbiota. On the other hand, depletion of microbiota altered the phenotype, but did not normalize PCs. Our results suggest that IL-10 could be necessary for the integrity of PCs. Moreover, our results help to explain why IL-10-/- mice develop enterocolitis in response to microorganisms.

Proteomic and structural characterization of self-assembled vesicles from excretion/secretion products of Toxoplasma gondii.

After the cell invasion, the parasite Toxoplasma gondii locates within a parasitophorous vacuole to proliferate. It continuously modifies the composition of the parasitophorous vacuole by the secretion of GRA and ROP proteins, some of which become inserted into the vacuole membrane, remain as soluble proteins or involved in the intravacuolar network. In this report, we analyze the excretion/secretion products and the vesicles released by extracellular tachyzoites, this structures were morphologically analyzed by electron microscopy and characterized by mass spectrometry. The structural analysis showed parasites secreting in vitro individual vesicles with similarities to ectosomes and exosomes and which characterized to self-assembly in vitro forming vesicle-tubular structures morphologically similar to the intravacuolar network from infected cells. The vesicle-tubular structures were recognized with antibodies against ROP2 and GRA2. In addition, analysis by Western blot evidenced proteins from the secretory organelles. A detailed proteomic analysis of exosomes, ectosomes and soluble proteins released in vitro is here reported. Presence of GRA proteins in secretions from resting extracellular parasites indicates that these molecules are not exclusively secreted within the parasitophorous vacuole of the infected cell as reported but they are constitutively excreted/secreted even in an extracellular condition. Data are available via ProteomeXchange with identifier PXD013767. SIGNIFICANCE: Extracellular tachyzoites constitutively secrete components that previously were considered be secreted only within the parasitophorous vacuole, suggesting that in the infected host these molecules are in direct interaction with cells and molecules of the host cell including those of the immune response.

The dense granule protein 8 (GRA8) is a component of the sub-pellicular cytoskeleton in Toxoplasma gondii.

After host cell invasion, Toxoplasma secretes a variety of dense granule proteins (GRA proteins) from its secretory dense granules, which are involved in the biogenesis of the parasitophorous vacuole (PV). TgGRA8I is predicted to contain proline-rich domains, which are structural features of some cytoskeleton-related proteins. In agreement with this observation, previous proteomic analyses revealed the presence of TgGRA8I in the Toxoplasma sub-pellicular cytoskeleton. In the present study, we show (1) by docking analyses that TgGRA8I may interact with both Toxoplasma ß-tubulin and actin; (2) by immunoelectron microscopy, proteomic, biochemical, and cellular approaches that TgGRA8I associates with sub-pellicular microtubules and actin at the parasite sub-pellicular cytoskeleton; (3) that type I parasites (RH strain) lacking the GRA8 gene (RHΔku80Δgra8) exhibit loss of conoid extrusion, diminished cell infection, and egress capabilities, and that these motility impairments were likely due to important alterations in their sub-pellicular cytoskeleton, in particular their sub-pellicular microtubules and meshwork. Parasites lacking the GRA4 gene (RHΔku80Δgra4) did not show modifications in the organization of the sub-pellicular cytoskeleton. Collectively, these results demonstrated that TgGRA8I is a dense granule protein that, besides its role in the formation of the PV, contributes to the organization of the parasite sub-pellicular cytoskeleton and motility. This is the first proline-rich protein described in the Toxoplasma cytoskeleton, which is a key organelle for both the parasite motility and the invasion process. Knowledge about the function of cytoskeleton components in Toxoplasma is fundamental to understand the motility process and the host cell invasion mechanism. Refining this knowledge should lead to the design of novel pharmacological strategies for the treatment against toxoplasmosis.

Biogenesis of the Insulin Secretory Granule in Health and Disease.

The secretory granules of pancreatic beta cells are specialized organelles responsible for the packaging, storage and secretion of the vital hormone insulin. The insulin secretory granules also contain more than 100 other proteins including the proteases involved in proinsulin-to insulin conversion, other precursor proteins, minor co-secreted peptides, membrane proteins involved in cell trafficking and ion translocation proteins essential for regulation of the intragranular environment. The synthesis, transport and packaging of these proteins into nascent granules must be carried out in a co-ordinated manner to ensure correct functioning of the granule. The process is regulated by many circulating nutrients such as glucose and can change under different physiological states. This chapter discusses the various processes involved in insulin granule biogenesis with a focus on the granule composition in health and disease.

Measuring Autophagy in Pancreatitis.

Acute pancreatitis is one of the first pathological processes where autophagy has been described in a human tissue. Autophagy, autodigestion, and cell death are early cellular events in acute pancreatitis. Recent advances in understanding autophagy highlight its importance in pathological conditions. However, methods for monitoring autophagic activity during complex diseases, involving highly differentiated secretory cells, are complicated, and the results are sometimes misinterpreted. Here, we describe methods used to identify autophagic structures and to measure autophagic flux in cultured cell models and animal models of pancreatitis. We also briefly describe the pancreas specific autophagy mouse model that was useful to understand the actual role of autophagy in pancreatitis and to identify a novel selective autophagy pathway named zymophagy. Lastly, we describe the immunomagnetic isolation of autophagosomes and the detection of autophagy in pancreatic tissue samples derived from humans.

The exocrine pancreas is an extracardiac source of atrial natriuretic peptide.

Previous studies have shown that atrial natriuretic peptide (ANP) regulates exocrine pancreatic function in health and disease. As extracardiac sources of ANP have been identified and ANP-like immunoreactivity has been reported in the exocrine pancreas, in the present work we sought to establish whether ANP was produced in the rat exocrine pancreas and if conditions like fasting/feeding or acute pancreatitis were reflected on ANP expression. By using RT-PCR, immunoblotting, and immunofluorescence microscopy assays, it was found that both mRNA and protein ANP were present in the acinar cells of the exocrine pancreas. The amount of ANP in the pancreas was lower in than the atrium but similar to other tissues like the kidney and liver. Immunogold labeling electron microscopy studies revealed that ANP was localized in zymogen granules and the endoplasmic reticulum suggesting local synthesis and package into granules. ANP protein expression was significantly increased not only in fasting but also in acute pancreatitis, the latter probably related to impaired secretion. Natriuretic peptide receptor type C which mediates ANP biological effects in the exocrine pancreas was also present in acinar cells and its expression did not change with either fasting or acute pancreatitis. Present findings show that the exocrine pancreas is a relatively important extracardiac source of ANP and further support previous studies strongly suggesting the active role of the peptide in pancreatic physiology and pathophysiology.

Phaseolin ingestion affects vesicular traffic causing oxidative stress in the midgut of Callosobruchus maculatus larvae.

It has been reported that phaseolin, the major storage globulin of the common bean (Phaseolus vulgaris), is toxic to Callosobruchus maculatus larvae, an Old World bruchid beetle that is not capable of infesting this New World edible bean. It has also been demonstrated that vicilin, the major storage globulin found in cowpea (Vigna unguiculata) seeds, is absorbed through receptor-mediated endocytosis in the insect midgut. A putative vicilin receptor has been purified and showed high homology to α-tocopherol transfer protein. However, the ingestion of a variant vicilin purified from C. maculatus resistant seeds inhibits transcytosis, resulting in the accumulation of vicilins in the midgut cells and ultimately antibiosis. In the present work, we studied the cellular up-take of phaseolin in C. maculatus larvae with the aim of discovering if this protein is also capable of inhibiting endocytic traffic in the enterocytes. FITC-labelled vicilin and FITC-labelled phaseolin were incorporated into the diet of the larvae at a physiological concentration of 0.5% w/w. The fate of labelled and non-labelled globulins was monitored by confocal microscopy. Here we demonstrated that phaseolin is also endocytosed by enterocytes causing an accumulation of endocytic vesicles in the midgut when compared to the ingestion of vicilin obtained from a susceptible V. unguiculata cultivar. From the results obtained for HNE, MDA and TBARS, a pro-oxidative scenario was established in the intestinal epithelial cells of the larvae, which may explain the deleterious effect observed in larvae developing inside P. vulgaris seeds.

Morphofunctional description of mucous cells in the gills of the Arapaimidae Arapaima gigas (Cuvier) during its development.

The gill structure of the Amazonian fish Arapaima gigas (Cuvier 1829) shows ontogenetic changes during development, particularly due the transition from the aquatic to the obligatory air breathing mode of respiration. However, three main cell types can be found in the gills: mitochondrial rich cells, pavement cells and mucous cells (MCs). The MCs are involved in the secretory pathway. The functions of the secreted molecules include mechanical protection of epithelia, protection against parasites and bacterial infection, and role on ion regulation. In this study, we analysed mucous cell location and mucous cell type, based on pH, during the development of A. gigas. Using samples obtained from the environment, gills were collected and fixed in buffered solution. Histological techniques for the identification of MCs were performed Alcian Blue (AB) and periodic acid-Schiff (PAS). The results showed the presence of PAS+ and AB+ cells in the whole filament in all examined fish. In animals less than 50 g, few MCs were present, and no differences were observed in AB+ and PAS+ cells. In animals weighing close to 500 g, more PAS+ cells than AB+ cells were observed, and in animals that weighed more than 1,000 g, more AB+ cells than PAS+ cells were observed. These observations may be a result of the ontogenetic changes in the gill epithelia, which can change the osmorespiratory compromise in ion regulation functions as well the glycosaminoglycans secreted by PAS cells, which in large animals can play a role in the protection against parasites and bacterial infection.

Contemporary understanding of the secretory granules in human eosinophils.

Eosinophil secretory (specific) granules have a unique morphology and are both a morphologic hallmark of eosinophils and fundamental to eosinophil-mediated responses. Eosinophil mediators with multiple functional activities are presynthesized and stored within these granules, poised for very rapid, stimulus-induced secretion. The structural organization and changes of eosinophil specific granules are revealing in demonstrating the complex and diverse secretory activities of this cell. Here, we review our current knowledge on the architecture, composition, and function of eosinophil specific granules as highly elaborated organelles able to produce vesiculotubular carriers and to interplay with the intracellular vesicular trafficking. We reconsider prior identifications of eosinophil cytoplasmic granules, including "primary," "secondary," "microgranules," and "small granules"; and consonant with advances, we provide a contemporary recognition that human eosinophils contain a single population of specific granules and their developmental precursors and derived secretory vesicles.