How the stimulus defines the dynamics of vesicle pool recruitment, fusion mode, and vesicle recycling in neuroendocrine cells.

The pattern of stimulation defines important characteristics of the secretory process in neurons and neuroendocrine cells, including the pool of secretory vesicles being recruited, the type and amount of transmitters released, the mode of membrane retrieval, and the mechanisms associated with vesicle replenishment. This review analyzes the mechanisms that regulate these processes in chromaffin cells, as well as in other neuroendocrine and neuronal models. A common factor in these mechanisms is the spatial and temporal distribution of the Ca(2+) signal generated during cell stimulation. For instance, neurosecretory cells and neurons have pools of vesicles with different locations with respect to Ca(2+) channels, and those pools are therefore differentially recruited following different patterns of stimulation. In this regard, a brief stimulus will induce the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels, whereas longer or more intense stimulation will provoke a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location. The pattern of stimulation, and therefore the characteristics of the Ca(2+) signal generated by the stimulus also influence the mode of exocytosis and the type of endocytosis. Indeed, low-frequency stimulation favors kiss-and-run exocytosis and clathrin-independent fast endocytosis, whereas higher frequencies promote full fusion and clathrin-dependent endocytosis. This latter type of endocytosis is accelerated at high-frequency stimulation. Synaptotagmins, calcineurin, dynamin, complexin, and actin remodeling, appear to be involved in the mechanisms that determine the response of these processes to Ca(2+) . In chromaffin cells, a brief stimulus induces the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels (A), whereas longer or high-frequency stimulation provokes a global Ca(2+) increase, promoting exocytosis irrespective of vesicle location (B). Furthermore, low-frequency stimulation favors kiss-and-run exocytosis (A), whereas higher frequencies promote full fusion (B). In this review, we analyze the mechanisms by which a given stimulation pattern defines the mode of exocytosis, and recruitment and recycling of neurosecretory vesicles. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

Triiodothyronine rapidly alters the TSH content and the secretory granules distribution in male rat thyrotrophs by a cytoskeleton rearrangement-independent mechanism.

Rapid actions of T3 on TSH synthesis in posttranscriptional steps, such as polyadenylation and translation rate, have already been described. The focus of this paper was to characterize rapid actions of T3 on TSH secretion and the involvement of actin and microtubule cytoskeleton in this process. For that, sham-operated (SO) and thyroidectomized (Tx) rats were subjected to acute or chronic treatment with T3. We observed a disarrangement in microtubule and actin cytoskeletons and an increase in Tshb mRNA levels in Tx rats, whereas the total TSH protein content was reduced in the pituitary gland as a whole, but increased in the secretory granules close to the plasma membrane of thyrotrophs, as well as in the extracellular space. The acute T3 dose promoted a rapid increase and redistribution of TSH secretory granules throughout the cytoplasm, as well as a rearrangement in actin and microtubule cytoskeletons. The T3 chronic treatment outcome reinforces the acute effects observed and, additionally, evinces an increase in the α-tubulin content and a rearrangement in microtubule cytoskeleton. Thus, T3 is able to rapidly suppress TSH secretion and, in parallel, to promote a rearrangement in actin and microtubules assembly throughout the pituitary gland, effects that seem to be independent from each other.

Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis.

Two so-called "secretory Rabs," Rab3 and Rab27, regulate late steps during dense-core vesicle exocytosis in neuroendocrine cells. Sperm contain a single large dense-core granule that is released by regulated exocytosis (termed the acrosome reaction) during fertilization or on exposure to inducers in vitro. Sperm exocytosis uses the same fusion machinery as neurons and neuroendocrine cells, with an additional requirement for active Rab3. Here we show that Rab27 is also required for the acrosome reaction, as demonstrated by the inability of inducers to elicit exocytosis when streptolysin O-permeabilized human sperm were loaded with inhibitory anti-Rab27 antibodies or the Rab27-GTP binding domain of the effector Slac2-b. The levels of GTP-bound Rab27 increased on initiation of exocytosis, as did the proportion of GTP-bound Rab3A. We have developed a fluorescence microscopy-based method for detecting endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Challenge with an inducer increased the population of cells exhibiting GTP-bound Rabs in this subcellular domain. Interestingly, introducing recombinant Rab27A loaded with GTP-γ-S into sperm elicited a remarkable increase in the number of cells evincing GTP-bound Rab3A. In the converse condition, recombinant Rab3A did not modify the percentage of Rab27-GTP-containing cells. Furthermore, Rab27A-GTP recruited a Rab3 GDP/GTP exchange factor (GEF) activity. Our findings suggest that Rab27/Rab3A constitutes a Rab-GEF cascade in dense-core vesicle exocytosis.

Óleo essencial de Baccharis tridentata Vahl: composição química, atividade antioxidante e fungitóxica, e caracterização morfológica das estruturas secretoras por microscopia eletrônica de varredura / Baccharis tridentata Vahl essential oil: chemical composition, and antioxidant and fungitoxic activities and morphological characterization of secretory structures by scanning electron microscopy

Este trabalho teve como objetivos avaliar a composição química do óleo essencial de Baccharis tridentata Vahl, as atividades antioxidante e fungitóxica, e estudar a morfologia das estruturas secretoras do óleo essencial presentes na superfície foliar por meio de microscopia eletrônica de varredura (MEV). A extração do óleo essencial foi realizada por hidrodestilação, as análises quantitativas e qualitativas foram executadas por meio de cromatografia em fase gasosa com detector de ionização de chamas (FID) e acoplada à espectrometria de massas, respectivamente. A atividade antioxidante foi realizada empregando-se os métodos de redução do radical estável DPPH e o ensaio de oxidação do sistema β-caroteno/ácido linoleico. As atividades fungitóxicas foram avaliadas utilizando o teste bioanalítico in vitro, sobre a inibição do crescimento micelial dos fitopatógenos Fusarium oxysporum, Colletotrichum gloeosporioides e Rhizoctonia solani. A composição química revelou a presença de 28 compostos, sendo o α-tujeno (22,93 por cento) o constituinte majoritário; não foi observada atividade antioxidante por meio dos ensaios utilizados, no entanto, observou-se atividade fungitóxica sobre o crescimento micelial dos fitopatógenos estudados. Já os estudos da superfície foliar por MEV revelaram a presença de tricomas glandulares em ambas as superfícies abaxial e adaxial.

This study aimed to evaluate the chemical composition and the antioxidant and fungitoxic activities of Baccharis tridentata essential oil, as well as to study the morphology of its secretory structures present on the leaf surface by scanning electron microscopy (SEM). The essential oil was extracted by hydrodistillation; the quantitative and qualitative analyses were performed on a gas chromatograph equipped with a flame ionization detector (FID) and coupled to a mass spectrometer, respectively. The antioxidant activity was determined by the methods of reduction of the DPPH stable radical and oxidation of the β-carotene/linoleic acid system. Fungitoxic activities were assessed by the in vitro bioanalytical test on the inhibition of the mycelial growth of the plant pathogens Fusarium oxysporum, Colletotrichum gloeosporioides and Rhizoctonia solani. The chemical composition revealed the presence of 28 compounds, with α-thujene (22.93 percent) as the major constituent. No antioxidant activity was observed in the tests used; however, there was fungitoxic activity against the mycelial growth of plant pathogens. Leaf surface studies by SEM revealed the presence of glandular trichomes on both abaxial and adaxial surfaces.

The Amazonian toad Rhaebo guttatus is able to voluntarily squirt poison from the paratoid macroglands

Amphibian defence mechanisms commonly rely on cutaneous toxins produced in either isolated or clusteredglands, such as toad parotoid macroglands. In contrast to the passive mechanism of poison liberation in other amphibians,we discovered that the Amazonian toad Rhaebo guttatus is unique because it can voluntarily squirt jets of poison from itsparotoids.

Human platelets express and are activated by galectin-8.

Gals (galectins) are proteins with glycan affinity that are emerging as mediators of atherosclerosis. Despite the similarities in structure and sequence, different Gals exert distinct effects on their target cells. We have shown that Gal-1 triggers platelet activation, suggesting a role for Gals in thrombus formation. Since Gal-8 is expressed upon endothelial activation and also contributes to inflammation, to understand further the role of these lectins in haemostasis, we evaluated the effect of Gal-8 on human platelets. Gal-8 bound specific glycans in the platelet membrane and triggered spreading, calcium mobilization and fibrinogen binding. It also promoted aggregation, thromboxane generation, P-selectin expression and granule secretion. GP (glycoprotein) αIIb and Ib-V were identified as putative Gal-8 counter-receptors by MS. Studies performed using platelets from Glanzmann's thromboasthenia and Bernard-Soulier syndrome patients confirmed that GPIb is essential for transducing Gal-8 signalling. Accordingly, Src, PLC2γ (phospholipase C2γ), ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3-kinase)/Akt downstream molecules were involved in the Gal-8 signalling pathway. Gal-8 fragments containing either the N- or C-terminal carbohydrate-recognition domains showed that activation is exerted through the N-terminus. Western blotting and cytometry showed that platelets not only contain Gal-8, but also expose Gal-8 after thrombin activation. These findings reveal Gal-8 as a potent platelet activator, supporting a role for this lectin in thrombosis and inflammation.

Tumor neuroendocrino de la mama: a propósito de un caso / Neuroendocrine tumor of the breast: a purpose of a case

Los tumores neuroendocrinos en la glándula mamaria, representan menos del 2 por ciento de las lesiones malignas que se presentan en la mama, un 30 por ciento pueden ser metastásicos, principalmente de tumores carcinoides intestinales. Se presenta el caso de una paciente femenina 64 años con el antecedente de carcinoma de mama izquierda pT1N1Mo Estadio II A, se le practicó en el año 2000 cirugía preservadora, recibió tratamiento sistémico y radioterapia, presentando recaída local, histológicamente y por inmuhistoquímica, como tumor neuroendocrino, en mayo de 2008 se le realiza mastectomía simple izquierda. Actualmente viva y sin enfermedad. Los tumores neuroendocrinos pueden presentarse en localizaciones extra intestinales. El diagnóstico debe realizarse por histología y confirmado con técnicas de inmunohistoquímica, son tumores de baja agresividad biológica, no se presentan síntomas sistémicos por liberación de hormonas como en otras localizaciones, y el tratamiento debe basarse en el estadio clínico de la enfermedad al momento del diagnóstico.

Neuroendocrines in the mammary gland tumors represent less than 2 percent of malignant injuries, which 30 percent can be source metastatic, mainly tumors intestinal carcinoid. The clinical of a female patient case 64 years old are presented with the antecedent of pT1N1Mo Stadium II A left breast carcinoma who was practiced in the year 2000 sparing surgery, received systemic therapy and radiotherapy, featuring local relapse, histological and inmuhistochemestry, as neuroendocrines, practicing it in May of 2008 mastectomy left, currently living and without disease tumor. Neuroendocrines tumors can occur in extra intestinal in lung, uterine and less common in the mammary gland neck locations. The diagnosis must be made by histology and confirmed with Immunohistochemistry techniques, are tumors of low biological aggression, not have systemic symptoms by release of hormones as in other locations, and treatment must be based on the clinical stage of the disease at the time of diagnosis.

Secretory organelles of pathogenic protozoa

Processos de secreção celular desempenham papel relevante na biologia e no ciclo de vida de protozoários patogênicos. A presente revisão analisa, sob uma perspectiva de biologia celular, o processo de secreção em (a) micronemas, roptrias e grânulos densos encontrados em membros do grupo Apicomplexa, onde essas estruturas participam da penetração do protozoário no interior da célula hospedeira, na sua sobrevivência intravacuolar e no posterior egresso da célula hospedeira, (b) a fenda de Maurer, encontrada em Plasmodium, uma estrutura envolvida na secreção de proteínas sintetizadas pelo protozoário intravacuolar e transportada, através de vesículas, para a superfície do eritrócito, (c) a secreção de macromoléculas na bolsa flagelar de tripanosomatídeos, e (d) a secreção de proteínas que fazem parte da parede cística de Giardia e Entamoeba e que se concentram nas vesículas de encistamento.

Secretory organelles of pathogenic protozoa.

Secretory processes play an important role on the biology and life cycles of parasitic protozoa. This review focus on basic aspects, from a cell biology perspective, of the secretion of (a) micronemes, rhoptries and dense granules in members of the Apicomplexa group, where these organelles are involved in the process of protozoan penetration into the host cell, survival within the parasitophorous vacuole and subsequent egress from the host cell, (b) the Maurer's cleft in Plasmodium, a structure involved in the secretion of proteins synthesized by the intravacuolar parasite and transported through vesicles to the erythrocyte surface, (c) the secretion of macromolecules into the flagellar pocket of trypanosomatids, and (d) the secretion of proteins which make the cyst wall of Giardia and Entamoeba, with the formation of encystation vesicles.

Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia.

The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.

Morphology of the sternal gland in workers of Coptotermes gestroi (Isoptera, Rhinotermitidae).

The sternal gland is considered the only source of trail pheromones in termites. The morphology of the sternal gland was investigated in workers of Coptotermes gestroi using transmission and scanning electron microscopy. The results showed a small bilobed gland at the anterior part of the fifth abdominal sternite. The cuticular surface of the sternal gland showed a V-shaped structure with two peg sensilla in elevated socket and various campaniform sensilla. Pores and cuticular scale-like protuberances also occur in the glandular area. The ultrastructure showed a gland composed of class 1 cells and two different types of class 3 cells distinguished by location, different size and electron-density of secretory vesicles. Small class 3 cells (type 1) of the anterior lobe are inserted among class 1 cells and have weakly electron-dense vesicles associated with mitochondria, glycogen and smooth endoplasmic reticulum. The class 3 cells (type 2) of posterior lobe showed many round electron-lucent vesicles of secretion, abundant free ribosomes and a well-developed Golgi apparatus. Each class 3 cell is connected to the cuticle by a cuticular duct constituted by the receiving canal and the conducting canal. The secretion of class 1 cells is stored in an inner subcuticular reservoir that is delimited by the microvilli of these cells. This inner reservoir is large and crossed by the campaniform sensilla and ducts of two types of class 3 cells that open outside of the insect body. An exterior reservoir also is present between the fourth and fifth sternite. The complex structure of the sternal gland suggests multicomponents for the trail pheromone in the worker of C. gestroi.

Seasonal variations of gonadotropins in the pars distalis male viscacha pituitary. Effect of chronic melatonin treatment.

The gonadotropes, LH and FSH cells, were immunohistochemically identified in the pituitary pars distalis of the adult male viscacha (Lagostomus maximus maximus) using specific antibodies against hLHbeta and hFSHbeta with the streptavidin-biotin-peroxidase complex. The distribution, size and percentage immunopositive area of these cells were analyzed by image analysis in viscachas captured during the annual reproductive cycle and after the chronic administration of melatonin. The LHbeta and FSHbeta cells showed seasonal changes in the distribution, size and percentage immunopositive area. The LHbeta cells were found widely distributed throughout the pars distalis during the reproductive period, and they were found in the ventro-medial region in the pars distalis during the gonadal regression and gonadal recovery periods. The LHbeta cells reached the largest size and immunopositive area during the reproductive period and the smallest size and immunopositive area during the gonadal regression period. The FSHbeta cells were found in the ventro-medial region during reproductive and gonadal regression periods. The FSHbeta cells were found widely distributed throughout the pars distalis during the gonadal recovery period when they showed the maximum percentage immunopositive area. A decrease in the size of LHbeta and FSHbeta cells was observed after the chronic administration of melatonin. Moreover, it produces a decrease in the immunopositive area occupied by the LHbeta cells but not in the immunopositive area occupied by the FSHbeta cells. Our results show great activity of LHbeta and FSHbeta cells in different moments of the annual reproductive cycle demonstrating that these cells do not secrete in parallel. Moreover, melatonin acts differentially on the activity of the gonadotrope cells.

Comparative study of mandibular glands of Melipona bicolor queens obtained from polygynic and monogynic colonies.

The aim of the present study was analyze, by histological and morphometrical studies, mandibular glands of Melipona bicolor queens collected from monogynic and polygynic colonies and compare their level of development. The results showed that the glands ofphysogastric queens from monogynic colony present a higher level of activity in relation to the queens of polygynic colonies; this is explained by the fact that just a unique queen controls the monogynic colony. In the polygynic colonies, the queens may divide such control to each other.

A unique mechanism of nuclear division in Giardia lamblia involves components of the ventral disk and the nuclear envelope

The fine structure of the binucleate, parasitic protist Giardia lamblia during interphase and divisional stages was studied by serial thin sectioning and three-dimensional reconstructions. The earlier sign of nuclear division is the development of a few peripheral areas of densely packed chromatin directly attached to the inner nuclear envelope. An intracytoplasmic sheet of ventral disk components grows from the cell periphery towards one of the nuclei, apparently constricting this nucleus, which becomes located at a ventral bulge. After the basal bodies become duplicated, a full nuclear division occurs in trophozoites, giving two pairs of parent-daughter nuclei. This full division occurs in a dorsal-ventral direction, with the resulting nuclear pairs located at the sides of the two sets of basal bodies. A new ventral disk is formed from the disk-derived sheets in the cell harboring the four nuclei. Cytokinesis is polymorphic, but at early stages is dorsal-to-dorsal. Encysting trophozoites show the development of Golgi cisternae stacks and dense, specific secretory granules. 3-D reconstructions show that cysts contain a single pair of incompletely strangled nuclei. The dividing Giardia lacks a typical, microtubular spindle either inside or outside the nuclei. The nuclear envelope seems to be the only structure involved in the final division of the parent-daughter nuclei. (AU)

A unique mechanism of nuclear division in Giardia lamblia involves components of the ventral disk and the nuclear envelope

The fine structure of the binucleate, parasitic protist Giardia lamblia during interphase and divisional stages was studied by serial thin sectioning and three-dimensional reconstructions. The earlier sign of nuclear division is the development of a few peripheral areas of densely packed chromatin directly attached to the inner nuclear envelope. An intracytoplasmic sheet of ventral disk components grows from the cell periphery towards one of the nuclei, apparently constricting this nucleus, which becomes located at a ventral bulge. After the basal bodies become duplicated, a full nuclear division occurs in trophozoites, giving two pairs of parent-daughter nuclei. This full division occurs in a dorsal-ventral direction, with the resulting nuclear pairs located at the sides of the two sets of basal bodies. A new ventral disk is formed from the disk-derived sheets in the cell harboring the four nuclei. Cytokinesis is polymorphic, but at early stages is dorsal-to-dorsal. Encysting trophozoites show the development of Golgi cisternae stacks and dense, specific secretory granules. 3-D reconstructions show that cysts contain a single pair of incompletely strangled nuclei. The dividing Giardia lacks a typical, microtubular spindle either inside or outside the nuclei. The nuclear envelope seems to be the only structure involved in the final division of the parent-daughter nuclei.

A unique mechanism of nuclear division in Giardia lamblia involves components of the ventral disk and the nuclear envelope.

The fine structure of the binucleate, parasitic protist Giardia lamblia during interphase and divisional stages was studied by serial thin sectioning and three-dimensional reconstructions. The earlier sign of nuclear division is the development of a few peripheral areas of densely packed chromatin directly attached to the inner nuclear envelope. An intracytoplasmic sheet of ventral disk components grows from the cell periphery towards one of the nuclei, apparently constricting this nucleus, which becomes located at a ventral bulge. After the basal bodies become duplicated, a full nuclear division occurs in trophozoites, giving two pairs of parent-daughter nuclei. This full division occurs in a dorsal-ventral direction, with the resulting nuclear pairs located at the sides of the two sets of basal bodies. A new ventral disk is formed from the disk-derived sheets in the cell harboring the four nuclei. Cytokinesis is polymorphic, but at early stages is dorsal-to-dorsal. Encysting trophozoites show the development of Golgi cisternae stacks and dense, specific secretory granules. 3-D reconstructions show that cysts contain a single pair of incompletely strangled nuclei. The dividing Giardia lacks a typical, microtubular spindle either inside or outside the nuclei. The nuclear envelope seems to be the only structure involved in the final division of the parent-daughter nuclei.

Identification and characterization of a novel secretory granule calcium-binding protein from the early branching eukaryote Giardia lamblia.

Giardia lamblia is a flagellate protozoan that infects humans and other mammals and the most frequently isolated intestinal parasite worldwide. Giardia trophozoites undergo essential biological changes to survive outside the intestine of their host by differentiating into infective cysts. Cyst formation, or encystation, is considered one of the most primitive adaptive responses developed by eukaryotes early in evolution and crucial for the transmission of the parasite among susceptible hosts. During this process, proteins that will assemble into the extracellular cyst wall (CWP1 and CWP2) are transported to the cell surface within encystation-specific secretory vesicles (ESVs) by a developmentally regulated secretory pathway. Cyst wall proteins (CWPs) are maintained as a dense material inside the ESVs, but after exocytosis, they form the fibrillar matrix of the cyst wall. Little is known about the molecular mechanisms involved in granule biogenesis and discharge in Giardia, as well as the assembly of the extracellular wall. In this work, we provide evidences that a novel 54-kDa protein that exclusively localizes to the ESVs is induced during encystation similar to CWPs, proteolytically processed during granule maturation, and able to bind calcium in vitro. The gene encoding this molecule predicts a novel protein (called gGSP for G. lamblia Granule-specific Protein) without homology to any other protein reported in public databases. Nevertheless, it possesses characteristics of calcium-sequestering molecules of higher eukaryotes. Inhibition of gGSP expression abolishes cyst wall formation, suggesting that this secretory granule protein regulates Ca(2+)-dependent degranulation of ESVs during cyst wall formation.

Acidic pH and increasing [Ca(2+)] reduce the swelling of mucins in primary cultures of human cervical cells.

BACKGROUND: Cervical mucus is a heterogeneous mixture of water, ions and mucins that form a hydrophilic polymer gel. Mucins, the main components of mucus, are condensed inside secretory granules and swell to become a hydrogel after exocytosis. Using human cervical secretory cell primary cultures, the effect of [Ca(2+)] and [H(+)] on the swelling velocity of mucin granules was investigated in vitro. METHODS AND RESULTS: Immunocytochemistry demonstrated that estrogen and progesterone receptors were expressed in cultured secretory cells along with mucins type 1, 4, 5AC and 5B. Exocytosis of secretory cells, recorded by videomicroscopy, showed that during swelling, the radius of the secretory granule matrix followed first-order kinetics. An increase in extracellular [Ca(2+)] from 1 to 4 mmol/l or a reduction in pH from 7.4 to 6.5 was seen to produce a significant decrease in the velocity of swelling of the secretory granule matrix. CONCLUSIONS: The inverse relationship observed between the diffusion of the granular matrix and the extracellular [Ca(2+)] or [H(+)] suggested that changes in cation concentration might drastically affect the swelling characteristics of mucins and provide a control mechanism for the observed viscoelastic properties of mucus.