The venom gland of queens of Apis mellifera (Hymenoptera, Apidae): morphology and secretory cycle

The venom gland of queens of Apis mellifera was examined through light and transmission electron microscopy and subjected to electrophoretic analyses. Virgin queens exhibited prismatic secretory cells containing large amounts of rough endoplasmic reticulum with dilated cisternae, open secretory spaces, numerous vacuoles and granules scattered in the cytoplasm, and spherical nuclei with numerous nucleoli. The secretion produced was non-refringent under polarized light and the electrophoretic analysis of glandular extracts revealed five main protein bands. In mated queens, the venom gland exhibited a high degree of degeneration. Its secretion was refringent under polarized light and one of the main bands was absent in the electrophoretic pattern obtained. The morphological aspects observed are in agreement with the function of this gland in queens, given that virgin queens use venom in battles for the dominance of the colony, a situation that occurs as soon as they emerge, while fertilized queens rarely use venom. (c) 2006 Elsevier Ltd. All rights reserved.

Light and temperature induce variations in the density and ultrastructure of the secretory spaces in the diesel-tree (Copaifera langsdorffii Desf.-Leguminosae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coil, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAD) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut CAD hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C265) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 angstrom, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. (C) 2012 Elsevier Ltd. All rights reserved.

LRP1 modulates APP trafficking and APP metabolism within compartments of the secretory pathway. Increased AICD generation is ineffective in nuclear translocation and transcriptional activation

LRP1 modulates APP trafficking and metabolism within compartments of the secretory pathway The amyloid precursor protein (APP) is the parent protein to the amyloid beta peptide (Abeta) and is a central player in Alzheimer’s disease (AD) pathology. Abeta liberation depends on APP cleavage by beta- and gamma-secretases. To date, only a unilateral view of APP processing exists, excluding other proteins, which might be transported together and/or processed dependent on each other by the secretases described above. The low density lipoprotein receptor related protein 1 (LRP1) was shown to function as such a mediator of APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. Therefore, we wanted to investigate whether LRP1 can mediate APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, we demonstrate that APP trafficking is strongly influenced by LRP1 transport through the endoplasmic reticulum (ER) and Golgi compartments. LRP1-constructs with ER- and Golgi-retention motifs (LRP-CT KKAA, LRP-CT KKFF) had the capacity to retard APP trafficking at the respective steps in the secretory pathway. Here, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases. Increased AICD generation is ineffective in nuclear translocation and transcriptional activity A sequence of amyloid precursor protein (APP) cleavages gives rise to the APP intracellular domain (AICD) together with amyloid beta peptide (Abeta) and/or p3 fragment. One of the environmental factors identified favouring the accumulation of AICD appears to be a rise in intracellular pH. This accumulation is a result of an abrogated cleavage event and does not extend to other secretase substrates. AICD can activate the transcription of artificially expressed constructs and many downstream gene targets have been discussed. Here we further identified the metabolism and subcellular localization of the constructs used in this well documented gene reporter assay. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely that cleaved from C83. Furthermore, the AICD surplus is not transcriptionally active but rather remains membrane tethered and free in the cytosol where it interacts with Fe65. However, Fe65 is still essential in AICD mediated transcriptional transactivation although its exact role in this set of events is unclear.

Analysis of cytokines in the peritoneal fluid of endometriosis patients as a function of the menstrual cycle stage using the Bio-Plex® platform

Endometriosis is a painful disease affecting 10-15% of reproductive-age women. Concentrations of several cytokines and angiogenic factors in peritoneal fluid (PF) have been found to correlate with the severity of the disease. However, levels of some analytes vary across the menstrual cycle, and an ideal biomarker of endometriosis has not yet been identified. We have compared the PF concentrations of different cytokines in proliferative and secretory phases in women with and without the disease using the Bio-Plex platform.

The immune geography of IgA induction and function

The production of immunoglobulin A (IgA) in mammals exceeds all other isotypes, and it is mostly exported across mucous membranes. The discovery of IgA and the realization that it dominates humoral mucosal immunity, in contrast to the IgG dominance of the systemic immune system, was early evidence for the distinct nature of mucosal immunology. It is now clear that IgA can function in high-affinity modes for neutralization of toxins and pathogenic microbes, and as a low-affinity system to contain the dense commensal microbiota within the intestinal lumen. The basic map of induction of IgA B cells in the Peyer's patches, which then circulate through the lymph and bloodstream to seed the mucosa with precursors of plasma cells that produce dimeric IgA for export through the intestinal epithelium, has been known for more than 30 years. In this review, we discuss the mechanisms underlying selective IgA induction of mucosal B cells for IgA production and the immune geography of their homing characteristics. We also review the functionality of secretory IgA directed against both commensal organisms and pathogens.

Defective pituitary function and gamete interactions in $\beta$1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.^ In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions. ^

ANAPHYLACTICALLY - MEDIATED EPITHELIAL CHLORINE ION SECRETORY RESPONSE INISOLATED JEJUNUM OF PARASITIZED GUINEA PIGS (TRICHINELLA SPIRALIS)

Electrophysiological studies were conducted to test the hypothesis that alterations in intestinal epithelial function are associated with immunological responses directed against the enteric parasite, Trichinella spirals. Trichinella antigens were used to challenge sensitized jejunum from infected guinea pigs while monitoring ion transport properties of the tissue in an Ussing-type chamber. The addition of antigen caused increases in transepithelial PD and I(,sc) that were rapidly induced, peaked at 1.5 to 2 min after antigen-challenge, and lasted 10 to 20 min thereafter. The increase in I(,sc) ((DELTA)I(,sc)) varied in a dose-dependent manner until a maximal increase of 40 (mu)A/cm('2) was obtained by the addition of 13 (mu)g of antigenic protein per ml of serosal fluid in the Ussing chamber. Trichinella antigen did not elicit alterations in either PD or I(,sc) of nonimmune tissue. Jejunal tissue from guinea pigs immunized with ovalbumin according to a protocol that stimulated homocytotropic antibody production responded electrically to challenge with ovalbumin but not trichinella antigen. Jejunal tissue which was passively sensitized with immune serum having a passive cutaneous anaphylaxis (PCA) titer of 32 for both IgE and IgG(,1) anti-trichinella anti-bodies responded electrically after exposure to trichinella antigen. Heat treatment of immune serum abolished the anti-trichinella IgE titer as determined by the PCA test but did not decrease either the electrical response of passively sensitized tissue to antigen or the anaphylactically mediated intestinal smooth muscle contractile response to antigen in the classical Schultz-Dale assay. These results strongly support the hypothesis that immunological responses directed against Trichinella Spiralis alter intestinal epithelial function and suggest that immediate hypersensitivity is the immunological basis of the response.^ Additional studies were performed to test the hypothesis that histamine and prostaglandins that are released from mucosal mast cells during IgE or IgG(,1) - antigen stimulated degranulation mediate electrophysiological changes in the intestinal epithelium that are reflective of Cl('-) secretion and mediated intracellularly by cAMP. Pharmacological and biochemical studies were performed to determine the physiological messengers and ionic basis of electrical alterations in small intestinal epithelium of the guinea pig during in vitro anaphylaxis. Results suggest that Cl('-) secretion mediated, in part, by cAMP contributes to antigen-induced jejunal ion transport changes and that histamine and prostaglandins are involved in eliciting epithelial responses. ^

Gi regulation of secretory vesicle swelling examined by atomic force microscopy

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the α subunit of a heterotrimeric GTP binding protein Gαi3 and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15–25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that Gαi3 protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

Sec61p Serves Multiple Roles in Secretory Precursor Binding and Translocation into the Endoplasmic Reticulum Membrane

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.

Homotypic Fusion of Immature Secretory Granules During Maturation Requires Syntaxin 6

Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroendocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) molecules are involved in the homotypic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocytosis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion. Subcellular fractionation studies and indirect immunofluorescence microscopy show that syntaxin 6 is sorted away during the maturation of ISGs to MSGs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SNAP-29/GS32, we could not find evidence that these target (t)-SNARE molecules are involved in homotypic ISG fusion. Nor could we find any involvement for the vesicle (v)-SNARE VAMP4, which is known to be associated with syntaxin 6. Importantly, we have shown that homotypic fusion requires the function of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t–t-SNARE interactions, either direct or indirect, may be required during fusion of ISG membranes.

Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.

Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.

Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta-lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such.

Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation.

The uterine expression of leukemia inhibitory factor (LIF) is essential for embryo implantation in the mouse. Here, we describe the expression of LIF, related members of this group of cytokines, oncostatin M and ciliary neurotrophic factor, and the LIF receptor beta and glycoprotein gp130 in normal human tissues and in the endometrium of fertile women. Our results show that LIF is the only one of these factors expressed at detectable levels in the endometrium of women of proven fertility. LIF expression is restricted to the endometrial glands during the secretory/postovulatory phase but is not present in the endometrium during the proliferative/preovulatory phase. The LIF receptor beta is expressed during the proliferative and secretory phases of the cycle and is restricted to the luminal epithelium. The associated signal-transducing component of the LIF receptor, gp130, is also expressed in both the luminal and glandular epithelium throughout the cycle. These results suggest that uterine expression of LIF in humans, like mice, may have a role in regulating embryo implantation, possibly through an autocrine/paracrine interaction between LIF and its receptor at the luminal epithelium.