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Electron microscopy of watery vacuole formation in rat parotid acinar cells during reflex stimulation.

Reflex stimulation from eating hard chow caused variable degrees of watery vacuolation, most conspicuously in animals that had eaten the most food; this was accentuated by eating in the cold. Some vacuolation occurred in the absence of sympathetic impulses but there was much more in glands receiving both parasympathetic and sympathetic impulses. Vacuole formation was often associated with, but not dependent upon, a degree of acinar degranulation. The vacuoles appeared to originate either from the inner side of the Golgi apparatus, near condensing vacuoles, or from ballooning within the rough endoplasmic reticulum; some vacuoles appeared to arise close to secretory granules. Certain cellular components, including secretory granules, entered the contents of vacuoles, which also often had connections with lumina. Many basally-located vacuoles were large and bounded only by a tenuous layer of cytoplasm which was contained by the basal lamina. Vacuole formation may be related to strong stimulation of susceptible cells, which occurs to a variable extent during natural reflex secretion, as well as during artificial stimulation. Thus, under normal conditions, certain macromolecular components might enter saliva via this route and not solely by exocytosis. Leakages from acinar vacuoles could also occur into the glandular interstices, accounting for some of the parotid amylase found in peripheral blood.

Animals↗

Sequencing and analysis of the Edwardsiella ictaluri plasmids.

To determine possible functions of the Edwardsiella ictaluri plasmids, pEI1 and pEI2, we analyzed the sequence of both plasmids. Plasmid pEI1 is 4807 bp, with 51% G + C, and 23 possible open reading frames of 40 amino acids or greater. Plasmid pEI2 is 5643 bp, with 51% G + C, and 24 possible reading frames. Database searches indicated that pEI1 contains an insertion element and a ROM analog. In addition, pEI1 possesses an open reading frame with strong homology to SlrP, SspH1, and SspH2 of Salmonella typhimurium and IpaH of Shigella flexnari, which have leucine-rich repeat regions and are components of type III secretory systems. pEI2 has a frame with weak homology to Spa15 of S. flexnari 5 and InvB of S. sonnei and S. typhimurium, which are also type III secretory system components, three origins of replication, a Rep analog, and a multimer resolution site.

Bacterial Proteins↗

Crystallization and preliminary diffraction studies of TraF, a component of the Escherichia coli type IV secretory system.

TraF, a component of the Escherichia coli type IV secretory system, has been crystallized and preliminary X-ray diffraction data have been collected. TraF is a 26 kDa protein encoded by the E. coli F plasmid and is required for conjugative plasmid transfer and the formation of sex pili. The N-terminal domain of TraF has no recognizable sequence features, whereas the C-terminal domain is believed to adopt a thioredoxin fold. However, since the active-site cysteines of thioredoxin-like proteins are not conserved in TraF, its biochemical role remains unclear. TraF crystallizes in space group C2, with unit-cell parameters a = 119.87, b = 34.36, c = 46.21 A, beta = 90.40 degrees , and crystals diffract to 2.3 A resolution.

Crystallization↗

Interactions of galactosyltransferase with serum and secretory immunoglobulins and their component chains.

Assay of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of beta-1,4-GT activity have shown that beta-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of beta-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in beta-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the beta-1,4-GT activity was detected in fractions that contained lactoferrin and alpha-lactalbumin. Interactions of beta-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and alpha-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the beta-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of beta-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the beta-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral beta-1,4-GT in the presence of Mn2+. Beta-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely alpha, mu and J chains, bound beta-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

Elevated serum secretory immunoglobulin A levels in patients with head and neck carcinoma.

Secretory immunoglobulin A (SIgA) is a major component of the secretory immune system and has been demonstrated in the sera of patients with various pathologic conditions. Current studies were undertaken to quantitate secretory components (SC) in sera of patients with squamous cell carcinoma in the head and neck region by means of a sensitive double-antibody radioimmunoassay. Patients with head and neck carcinoma were found to have threefold higher levels of SC (1.54 +/- 0.74 mg/ml) compared to normal controls (0.51 +/- 0.22 mg/ml, P less than .0001). Of interest was that the sera of patients who had successfully been treated 2 to 6 months prior to the study demonstrated statistically lower levels than those of patients with untreated carcinoma or recurrent disease. SC in sera appears to be in the form of SIgA. The origin and diagnostic significance of SC are briefly discussed.

Aged↗

Hijacking components of the cellular secretory pathway for replication of poliovirus RNA.

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase approximately 4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

3C Viral Proteases↗

SecY is an indispensable component of the protein secretory machinery of Escherichia coli.

Using a reconstitution system for protein translocation, the involvement of SecY in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli was studied. Anti-SecY antibodies raised against the N- and C-terminal sequences prevented the functional reconstitution of the translocation system. Depletion of SecY from the solubilized membrane preparation was performed by treatment with anti-SecY IgG, followed by removal of IgG with protein A-agarose. The SecY-depleted preparation was inactive as to functional reconstitution. However, reconstitution with it was demonstrated in the presence of a protein fraction, which was released from the anti-SecY immunoprecipitate upon addition of the SecY fragment used to raise the antibody. Reconstitution with the SecY-depleted membrane fraction was also demonstrated in the presence of a purified SecY preparation. OmpT proteinase specifically cleaved SecY in the solubilized membrane preparation. The cleavage was accompanied by a decrease in the reconstituted activity. Based on these findings we conclude that SecY is an indispensable component of the secretory machinery.

Amino Acid Sequence↗

Expression profiling of pancreatic beta cells: glucose regulation of secretory and metabolic pathway genes.

Pancreatic beta cells respond to changes in blood glucose by secreting insulin and increasing insulin synthesis. To identify genes used in these responses, we have carried out expression profiling of beta cells exposed to high (25 mM) or low (5.5 mM) glucose by using oligonucleotide microarrays. Functional clustering of genes that averaged a 2.2-fold or greater change revealed large groups of secretory pathway components, enzymes of intermediary metabolism, cell-signaling components, and transcription factors. Many secretory pathway genes were up-regulated in high glucose, including seven members of the endoplasmic reticulum (ER) translocon. In agreement with array analysis, protein levels of translocon components were increased by high glucose. Most dramatically, the alpha subunit of the signal recognition particle receptor was increased over 20-fold. These data indicate that the translocon and ribosome docking are major regulatory targets of glucose in the beta cell. Analysis of genes encoding enzymes of intermediary metabolism indicated that low glucose brought about greater utilization of amino acids as an energy source. This conclusion was supported by observations of increased urea production under low-glucose conditions. The above results demonstrate genome-wide integration of beta-cell functions at the level of transcript abundance and validate the efficacy of expression profiling in identifying genes involved in the beta-cell glucose response.

Animals↗

An immunohistochemical study of the secretory immune system in human fetal membranes and decidua of the first trimester of pregnancy.

PROBLEM: We analyzed the presence and distribution of components of the secretory immune system (SIS) in human fetal membranes (amnion, yolk sac, chorion) and decidua from the first trimester of pregnancy. MATERIALS AND METHODS: Specimens from 17 embryos (4-8 weeks of pregnancy) and nine fetuses (9-12 weeks) were divided into those that had not been exposed to massive foreign antigenic effects (Group I, n = 18) and those that had suffered acute chorioamnionitis (Group II, n = 8). RESULTS: Positive immunostaining for secretory component (SC), joining (J) chain, IgG, IgA, and macrophages was seen from 4 to 5 weeks of development and then during the whole first trimester of pregnancy in the syncytio- and cytotrophoblasts, the amniotic epithelium, the yolk sac endoderm and decidual cells. Macrophages with J chain, IgG and IgA were found in embryonic tissues on week 4, whereas lymphocytes, including those synthesizing IgA and IgM, appeared only at the end of the first trimester of pregnancy. In the decidua, lymphocytes and macrophages were recognized during the whole period of study. In cases with chorioamnionitis (Group II), reactivity of IgG and IgA in the mentioned cells of fetuses decreased sharply while the rate of immunoreactivity of SC and J chain as well as the number of T and B lymphocytes did not change. In the decidua, the number of immune reactive cells sharply increased with the appearance of plasma cells. CONCLUSIONS: In the fetal membranes and decidua all the SIS components are present. We suggest that two SIS, maternal and fetal, participate in the formation of the barrier between a mother and the fetus. Both these systems have different origin and cellular content as well as different immune reactions.

B-Lymphocytes↗

Hormone secretory patterns, pituitary characteristics and selected blood metabolites in feedlot steers implanted with growth stimulants.

Twelve Charolais-crossbred steers (256 kg) received one of three treatments: nonimplanted controls (C), implanted initially and at 84 days with 36 mg zeranol (Ralgro, R) and implanted initially and at 84 days with 200 mg of progesterone and 20 mg of estradiol benzoate (Synovex-S,S). All steers were fed a corn-based diet (calculated metabolizable energy 2.89 Mcal/kg dry matter) ad libitum. In a parallel comparative slaughter trial, rates of empty body protein accretion were increased 14% in R and 24% in S steers (P less than .01). R and S steers in the present study had heavier pituitary weights (P less than .001), more pituitary growth hormone content (P less than .04) and more pituitary weight/unit live weight (P less than .05) than did C steers. Cattle implanted with R or S exhibited an increased growth hormone (GH) secretory response to a pituitary challenge with thyrotropin releasing hormone (TRH). Plasma insulin profiles were not significantly altered, but tended to be greater for steers given implants. Overall 9-hr GH secretory profiles were not affected by implantation. Plasma urea N at 94 days post-implantation was decreased (P less than .01) by implantation. Plasma glucose was increased (P less than .04) at both 94 and 199 days in R and S vs C steers. Overall mean and total (integrated area) plasma GH, as well as secretory profile components (baseline mean, amplitude of secretory spikes) were negatively correlated with body weight and size on days 94 and 199. Overall mean, baseline and integrated area of plasma insulin on days 94 and 199 were positively related to body weight and size. Thus positive protein anabolic growth responses from implantation (parallel comparative slaughter trial) were coupled with increased pituitary GH content and little change in circulating plasma GH concentrations between implanted and control steers. This may suggest that changes in tissue sensitivity, an increased plasma clearance rate of GH and/or a direct effect on target tissues may be involved in the improved growth performance of cattle implanted with R or S.

Animals↗

Movement of IgM antibody from blood to bile in rats.

The movement from blood to bile of passively injected autologous IgM antibody against horse erythrocytes was studied in rats. Both native and neuraminidase-treated antibody entered bile intact, with the peak titers for both measured between 60 and 90 min after injection. A small part (0.38%) of the injected dose of native antibody and 1.05% of the asialo-IgM antibody appeared in bile over 24 hr. These recoveries represented only a small fraction of the activity, which apparently disappeared from serum over the period. The pathways used by IgM antibody to enter bile were partially assessed. Prior injection of fetuin was found to abrogate the biliary secretion of native antibody and to significantly reduce the recovery of asialo-antibody in bile. In contrast, the presence of asialofetuin reduced (but not significantly) the secretion of native antibody and markedly changed the kinetics of appearance of asialo-IgM activity in bile. Some candidate routes to bile can be excluded as unlikely. The parahepatocellular pathway to bile canaliculi appears uninvolved because the secretion of antibody is slow and the inhibitory effects of fetuin argue against secretory component-mediated transport. Instead, two secretory pathways appear to be present. The first appears to be available to both native and asialo-IgM antibody, and may involve the peribiliary capillary plexus. The second route to bile is available to asialo-IgM antibody alone and is probably initiated by binding to the hepatic asialoglycoprotein receptor.

Animals↗

Biogenesis of secretory granules in the trans-Golgi network of neuroendocrine and endocrine cells.

Secretory granule formation requires selection of soluble and membrane proteins into nascent secretory granules, and exclusion of proteins not required for the function of secretory granules. Both selection and exclusion presumably can occur in the compartment where assembly of the secretory granule begins, the trans most cisternae of the Golgi complex. Current research focused on the initial stages of secretory granule formation includes a search for the 'signals' which may mediate active sorting of components into secretory granules, and the role of aggregation of regulated secretory proteins in sorting. In addition, the temporal sequence of the sorting events in the Golgi, and post-Golgi compartments has gained much attention, as summarized by the alternative but not mutually exclusive 'sorting for entry' vs. 'sorting by retention' models. 'Sorting for entry' which encompasses the most popular models requires selection of cargo and membrane and exclusion of non-secretory granule proteins in the TGN prior to secretory granule formation. 'Sorting by retention' stipulates that protein selection or exclusion may occur after secretory granule formation: secretory granule specific components are retained during maturation of the granule while non-secretory granule molecules are removed in vesicles which bud from maturing secretory granules. Finally, some progress has been made in the identification of cytosolic components involved in the budding of nascent secretory granules from the TGN. This review will focus on the recent data concerning the events in secretory granule formation which occur, in the trans-Golgi network.

Animals↗

Secretory immune system in human embryonic and fetal development: joining chain and immunoglobulin transport (Review).

The role of joining (J) chain, one of the protein components of the secretory immune system (SIS), in the immune reactions of the human embryo and fetus was analyzed on the basis of data from the literature and our previous studies. All organs and structures, including extra-corporeal ones, of 18 embryos (4-8 weeks of development) and 45 fetuses (9-38 weeks) were studied using methods of pathomorphology, immunohistochemistry and morphometry. This approach enabled us to analyze the problem in the whole organism throughout its embryonic and fetal development. J chain, as well as polymeric immunoglobulin (Ig) receptor-secretory component (pIgR/SC) and Igs, are already widely distributed in 4-week-old embryos before the appearance of the common immune system. The whole complex of protein components of the SIS was seen in mucous layers, and in blood-tissue and tissue-tissue barrier structures. Therefore, we can consider two parts of the SIS: mucosal and barrier. Already in embryos, an increase in the functional activity of the SIS following massive antigenic attack in cases of acute chorioamnionitis reflects the increased exocrine secretion of Igs. The J chain appears to participate in the endocytosis but not exocytosis of Igs. J chain and Igs, but not pIgR/SC, were present in cells of the heart, endocrine glands, gonads and some other organs. The exocrine secretion of Igs, the main function of the SIS, is absent in these organs, and, they are therefore, not considered part of the SIS.

Embryonic Development↗

Contribution of secretory IgA, polymeric IgA and IgA/secretory component-containing circulating immune complexes to the serum hyper-IgA in diabetes mellitus.

The relative contribution of secretory IgA, monomeric and polymeric IgA and IgA/secretory component-containing immune complexes was investigated in sera of diabetic patients. Secretory IgA and immune complexes containing IgA and secretory component seem to participate in the hyper-IgA of patients with Type 2 (non-insulin-dependent) diabetes only, suggesting an altered hepatic clearance via secretory component receptors on hepatocytes. In Type 1 (insulin-dependent) diabetes, the high serum IgA levels might be explained by an increase in IgA production in response to antigenic stimuli. Evidence is also accumulated that immune complexes containing IgA of mucosal origin may be involved in microangiopathy production in Type 2 diabetes.

Adolescent↗

Secretory immunoglobulin A is a component of the human milk fat globule membrane.

Secretory immunoglobulin A (sIgA), the predominant antibody fraction of human milk, represents a major protective factor against neonatal infection. Until now, sIgA had been identified only in the humoral fraction of human milk. The aim of this study was to assess whether in human milk sIgA is also associated with the milk fat globule (MFG). Using anti-sIgA-agglutinated human MFG and immune fluorescence microscopy, we could demonstrate that sIgA is, in fact, associated with human MFG. In human MFG membranes separated by Western blotting, sIgA bands were specifically stained, suggesting that sIgA is strongly associated with the human MFG membrane. This may be of physiologic relevance, inasmuch as earlier we could show that a quantity of undigested and functional human MFG are in fact found in the stools of the newborn. This would allow an additional extension of the protective mechanisms of sIgA throughout the whole intestine.

Agglutination Tests↗

Human glands of Moll: histochemical and ultrastructural characterization of the glands of Moll in the human eyelid.

The function of the human gland of Moll of the eyelid is not exactly known. We studied the secretory and cytoskeletal components of these apocrine glands in males and females by immunohistochemical methods, and the ultrastructural organization of the glandular cells with an electron microscope. The glands of Moll are exclusively located at the margin of the eyelids and their ducts empty into the lash follicle. Immunohistochemical staining for actin and cytokeratins CK19 and CK7 points to the involvement of actin in the pinching-off mechanism of the apical cell protrusion during apocrine secretion and to a stabilizing role for the cytokeratins in this apical region of the glandular cells. The presence of the bacteriolytic enzyme lysozyme, the membrane-associated mucin 1, and the immunoglobulin A and its secretory component within the gland suggest a function in local immune defense. The presence of a variety of sugar components in the secretory product was verified by lectin histochemistry and periodic acid Schiff and Alcian blue stain. We suppose that these apocrine glands are active from birth in producing agents against pathogenic microorganisms in the eyelid shaft and on the ocular surface.

Actins↗

Sequences translated by Balbiani ring 75S RNA in vitro are present in giant secretory protein from Chironomus tentans.

The 75S RNA originating in the large Balbiani rings 1 and 2 (BRI and 2) was isolated and used for in vitro translation in the mRNA dependent reticulocyte lystate. Conditions (K+-concentration, temperature, time etc). were optimized for obtaining translation products of maximal size. Polypeptide chains up to about 500,000 D were obtained but no complete translation products. Trypic fingerprints were performed on the in vitro products as well as on the secretory protein components nos. I and II+III labelled with 35S-methionine. There was a large degree of correspondence between the fingerprint of the in vitro product and that of component I but less to that of component II+III. The results suggest that 75S RNA with an origin in the BR1 and BR2 codes for the giant secretory protein component I.

Amino Acid Sequence↗

An immunohistochemical study of the secretory immune system in human fetal endocrine glands and their precursors.

We examined the presence and distribution of components of the secretory immune system (SIS) in fetal endocrine organs and their embryonic precursors. Specimens from 16 embryos (4 to 8 weeks of development) and 32 fetuses (9 to 38 weeks) were divided into those that had not been exposed to massive foreign antigenic effects (Group I, n=28) and those that had suffered from chorioamnionitis (Group II, n=20). An immunohistochemical study was performed using antibodies against the secretory component (SC), joining (J) chain, IgA, IgM, IgG, subsets of T and B lymphocytes, and macrophages. Positive immunostaining for SIS components in the precursors of endocrine organs was seen from 4 to 6 weeks of development, and was present thereafter in the pituitary body, thyroid, pancreatic islets and adrenals. J chain and immunoglobulins were found in all endocrine cells throughout intrauterine development, but the massive antigenic influence caused by chorioamnionitis decreased the latters immunoreactivity. The presence of SC in the precursors of adenohypophysis and pancreatic islet cells decreased significantly after their transformation into definitive endocrine organs. In the thyroidal follicular epithelium and the pars intermedia of the pituitary body cells, SC was present during the entire period of pregnancy. In adrenals, SC was not found. Maternal immunoglobulins, together with SC and J chain, are accumulated in endocrine gland cells from the early stages of intrauterine life. They are the major mechanism of endocrine cell defense during the early prenatal period when the common immune system is still structurally and functionally incompetent.

B-Lymphocytes↗