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PubMed1996

Assessment of stimulated and spontaneous adrenocorticotropin secretory dynamics identifies distinct components of cortisol feedback inhibition in healthy humans.

Corticosteroids inhibit ACTH secretion through diverse cellular mechanisms, including direct pituitary and indirect suprapituitary effects. Although exogenous CRH provides a useful assessment of corticotroph function, the suprapituitary component of ACTH regulation has been difficult to assess in humans. Naloxone (NAL) has been reported to stimulate ACTH secretion indirectly through the release of endogenous hypothalamic CRH, suggesting its potential application in the examination of suprapituitary regulation of ACTH secretory dynamics. We sought to examine the inhibitory effects of corticosteroids on kinetic parameters of ACTH secretion, assessed by a deconvolution method, in healthy human subjects. We also sought to directly compare the ACTH responses to serial administration of human CRH and NAL as well as spontaneously occurring ACTH pulses to distinguish pituitary and suprapituitary components of hypothalamic-pituitary-adrenal regulation. Normal healthy subjects (n = 11) received hCRH (0.4 microgram/kg) at 1800 h and then NAL (65 micrograms/kg) at 1930 h, respectively, on 3 separate study days: placebo pretreatment plus CRH/NAL stimulation, metyrapone (MET) pretreatment plus CRH/NAL, or MET alone. Plasma ACTH and serum cortisol were assessed at frequent (every 10 min) intervals during CRH/NAL or placebo infusions (1800-2100 h) on all 3 study days, and deconvolution analysis was performed to determine kinetic parameters of endogenous and stimulated ACTH secretion. Suppression of endogenous cortisol secretion with MET significantly increased both continuous (basal secretion rate) and pulsatile CRH- and NAL-stimulated ACTH bursts (P < 0.05). The increase in total ACTH secreted per burst was related to two distinct effects of cortisol regulating the amplitude (maximum secretion rate) and half-duration (P < 0.05) of secretory bursts. The ACTH responses to CRH and NAL for individual subjects were significantly and positively correlated in both placebo pretreatment plus CRH/NAL stimulation and MET pretreatment plus CRH/NAL studies (P < 0.01). MET administration disproportionately increased the ACTH response to NAL, producing a significant increase (P < 0.01) in the slope of the regression relating ACTH responses to CRH and NAL. The following conclusions were made: 1) endogenous cortisol secretion, even at levels associated with relatively low serum cortisol concentrations, exerts a significant negative feedback effect on both continuous and pulsatile ACTH secretion; 2) cortisol inhibits pulsatile ACTH secretion through distinct regulatory mechanisms that independently modulate both the mass and the duration of ACTH secretory bursts; 3) the differential sensitivity of the CRH- and NAL-stimulated ACTH responses to MET administration suggests that both pituitary and suprapituitary components of the hypothalmic-pituitary-adrenal axis are sensitive to negative regulation over a rapid or intermediate temporal domain. Endogenous cortisol modulates multiple components of dynamical ACTH secretion through composite effects that appear to be mediated through structurally and functionally distinct regulatory domains.

Adrenocorticotropic Hormone↗
PubMed1990

Reconstitution of translocation activity for secretory proteins from solubilized components of Escherichia coli.

The protein translocation system of Escherichia coli was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E. coli phospholipids. SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF-Lpp. Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction. The effects of IgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined. IgGs specific to the amino- and carboxyl-terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation. Generation of a proton motive force due to the simultaneous reconstitution of F0F1-ATPase was also observed in the presence of ATP. An ATP-generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes. Collapse of the proton motive force thus generated partially inhibited the translocation.

Adenosine Triphosphate↗
PubMed1992

Protease resistant interleukin-3 stimulating components in excretory and secretory products from adult worms of Strongyloides ratti.

Excretory and secretory (ES) products collected from adult worms of Strongyloides ratti stimulated interleukin-3 (IL-3) production with mesenteric lymph node cells from infected C57BL/6 mice, but not with normal mesenteric lymph node cells. The IL-3 stimulating components were not major IgG binding antigens. Activity of the IL-3 stimulating components was stable by treatment with protease, although reduced by heating in boiling water.

Animals↗
PubMed1990

[Secretary IgA and the oral cavity: general review].

The authors present a synthesis of the knowledge concerning the nature and origin of secretory IGA as well as its main components (J chain and secretory component) in the oral cavity. This IgA is the main mediator of humoral immunity and is the most frequently identified immunoglobulin in saliva. Its role in the protection of mucosae as well as in the anti-caries immunity is presented.

Dental Caries↗
PubMed1999

Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa.

The general secretory pathway of Pseudomonas aeruginosa is required for the transport of signal peptide-containing exoproteins across the cell envelope. After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane. This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein. XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium. Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane. This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP. An xcpP mutant demonstrated that the product of this gene is indeed an essential element of the P. aeruginosa secretion machinery. Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein. Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.

Amino Acid Sequence↗
PubMed2002

Post-secretory fate of host defence components in mucus.

Airway mucus is a complex mixture of secretory products that provide a multifaceted defence against infection. Among many antimicrobial substances, mucus contains a peroxidase identical to milk lactoperoxidase (LPO) that is produced by goblet cells and submucosal glands. Airway secretions contain the substrates for LPO, namely thiocyanate and hydrogen peroxide, at concentrations sufficient for production of the biocidal compound hypothiocyanite, a fact confirmed by us in vitro. In vivo, inhibition of airway LPO in sheep significantly inhibits bacterial clearance, suggesting that the LPO system is a major contributor to host defences. Since secretory products including LPO are believed to be steadily removed by mucociliary clearance, their amount and availability on the surface is thought to be controlled solely by secretion. In contrast to this paradigm, new data suggest that LPO and other substances are retained at the ciliary border of the airway epithelium by binding to surface-associated hyaluronan, thereby providing an apical, fully active enzyme pool. Thus, hyaluronan, secreted from submucosal gland cells, plays a previously unrecognized pivotal role in mucosal host defence by retaining LPO and possibly other substances important for first line host defence at the apical surface 'ready for use' and protected from ciliary clearance.

Animals↗
PubMed1987

Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker.

By using a conventional spectrophotometric assay with hippuryl-L-phenylalanine as the substrate, 10(6) BALB/c mouse serosal mast cells possessed 1.5 +/- 0.43 U (mean +/- SE, n = 5, range = 0.48 to 2.5) of carboxypeptidase A activity, while T cell factor-dependent, mouse bone marrow-derived mast cells (BMMC) had barely detectable levels of 0.01 +/- 0.001 U/10(6) cells (mean +/- SE, n = 3). In order to characterize the carboxypeptidase A present in the BMMC, a sensitive assay was developed that used angiotensin I as the substrate and reverse phase-high performance liquid chromatography to separate and quantify production of the cleavage product des-leu-angiotensin I. Using this assay, mouse BMMC carboxypeptidase A had a neutral to basic pH optimum and hydrolyzed angiotensin I with a Km of 0.78 mM. The antigen-induced net percent release of carboxypeptidase A from IgE-sensitized BMMC was proportional to that of the secretory granule component beta-hexosaminidase which indicates a secretory granule location for the exopeptidase. As defined by exclusion during Sepharose CL-2B chromatography, carboxypeptidase A was exocytosed as a greater than 1 X 10(7) m.w. complex bound to proteoglycans. Because BMMC cocultured with mouse skin-derived 3T3 fibroblasts are known to undergo an increase in histamine content and biosynthesis of 35S-labeled heparin proteoglycans, carboxypeptidase A activity was measured during BMMC/fibroblast coculture for 0 to 28 days. The carboxypeptidase A activity increased progressively during 28 days of co-culture from 0.004 +/- 0.002 U/10(6) starting BMMC (mean +/- SE, n = 3) to 0.36 +/- 0.10 U/10(6) co-cultured mast cells. These findings indicate that carboxypeptidase A, a neutral protease, is exocytosed from the secretory granules of mouse mast cells bound to proteoglycan and is increased during the in vitro differentiation of mouse BMMC from mucosal-like mast cells to serosal-like mast cells.

Angiotensin I↗
PubMed1983

Dual role of the liver in regulating circulating polymeric IgA in man: studies on patients with liver disease.

The human liver probably removes circulating polymeric IgA by two routes: (1) secretory component-mediated endocytic transport of polymeric IgA from portal tract blood vessels into bile across biliary epithelial cells and (2) uptake with possible catabolism by hepatocytes and/or sinusoidal phagocytic cells by uncharacterized receptor(s). Failure of either clearance mechanism due to liver disease results in elevated serum polymeric IgA levels. In patients with cholestasis, the raised serum polymeric IgA concentration is due to reflux of biliary secretory immunoglobulin into the blood.

Alkaline Phosphatase↗
PubMed1983

Ca2+-dependent binding of cytosolic components to insulin-secretory granules results in Ca2+-dependent protein phosphorylation.

1. Incubation of a rat islet cell tumour homogenate with [gamma-32P]ATP resulted in the Ca2+-dependent phosphorylation of 100000-, 57000-, 29000-, 26000- and 14000-Mr proteins. The Ca2+ concentration required was in the low-microM range. 2. Isolated insulin granules did not exhibit Ca2+-dependent protein phosphorylation, whereas a soluble protein fraction showed the Ca2+-dependent phosphorylation of a 57000-Mr protein. Combination of insulin granules with the soluble protein fraction resulted in the additional Ca2+-dependent phosphorylations of 100000-, 29000- and 10000-Mr proteins. The latter phosphorylations were not enhanced by exogenous calmodulin, but nevertheless were inhibited by trifluoperazine. Removal of endogenous calmodulin from the soluble protein fraction before incubation with insulin granules did not abolish the Ca2+-dependent phosphorylations of the 100000-, 29000- and 10000-Mr proteins but rendered the Ca2+-dependent phosphorylation of the 57000-Mr soluble protein dependent on exogenous calmodulin. 3. The components of the soluble protein fraction responsible for the interaction with insulin granules bound to intact granules in a Ca2+-dependent manner. 4. After phosphorylation, the 29000-Mr protein remained attached to granules, whereas the 100000- and 10000-Mr proteins dissociated from granules. 5. These Ca2+-dependent phenomena may be of regulatory importance in the secretory mechanism.

Adenoma, Islet Cell↗
PubMed2005

Modulation of calcium currents is eliminated after cleavage of a strategic component of the mammalian secretory apparatus.

Adenosine inhibits neurotransmitter secretion from motor nerves by an effect on the secretory apparatus in amphibia. In contrast, the inhibitory effect of adenosine is associated with decreases in calcium currents at mouse motor nerve endings. To determine if the action of adenosine in the mouse is mediated thorough a direct effect on calcium channels or through the secretory machinery, the effects of cleavage of the SNARE proteins on the action of adenosine were examined. Cleavage of the SNARE syntaxin with botulinum toxin type C (Botx/C) prevented the inhibitory effect of adenosine on nerve terminal calcium currents. Cleavage of the other SNAREs (synaptobrevin with Botx/D or SNAP-25 with Botx/A) failed to affect the inhibitory action of adenosine. The results provide evidence for an intimate coupling of nerve terminal calcium channels with a plasma membrane component of the SNARE complex, such that modulation of calcium currents by a G-protein coupled receptor cannot occur when syntaxin is cleaved.

Adenosine↗
PubMed1991

IgE antibody production in rats against multiple components of excretory-secretory products of the nematode Nippostrongylus brasiliensis.

Infection with the nematode Nippostrongylus brasiliensis (NB) induces the intense production of specific and non-specific immunoglobulin E (IgE) in rats. In the present study, we analysed NB-derived allergenic substances and the variability of IgE antibody production in response to these allergens in outbred Sprague-Dawley rats. Two kinds of crude allergens were used: the excretory-secretory products (ES) of adult NB, and an extract of homogenized adult worms (AW). ELISA showed that IgE antibody titres to ES were more than five times higher than the titres to AW. In the homologous passive cutaneous anaphylaxis (PCA) reaction using serum from infected rats, as little as 50 micrograms of ES had a maximal PCA activity, while even 1 mg of AW still gave a slightly lower PCA titre. Thus, it appeared that ES contained more allergen than AW at the same amount of total proteins. By immunoblot analysis, at least six components were recognized by IgE antibodies from infected animals, and these components were exactly the same in both ES and AW. The results indicated that the allergenic components in ES and AW were the same molecules, and that only those molecules which could be excreted or secreted from living worms were allergenic. Among the array of allergens, 130,000 and 70,000 molecular weight (mw) molecules were commonly recognized by IgE from all serum samples examined, while other components of the allergens were recognized variably by IgE antibodies from individual animals. These findings suggested that individual animals varied considerably in their IgE antibody production to the different constituents of the nematode allergens.

Animals↗
PubMed1992

Immunoglobulin A-induced shift of Epstein-Barr virus tissue tropism.

Increased immunoglobulin A (IgA) antibodies to the Epstein-Barr virus (EBV) appear months to years before the clinical onset of nasopharyngeal carcinoma and define populations at high risk for this EBV-associated epithelial cancer common in south China. In the human HT-29 epithelial cell line, polymeric IgA (pIgA) specific for EBV promoted infection of the otherwise refractory epithelial cells. When bound to pIgA, EBV entered epithelial cells through secretory component-mediated IgA transport but no longer infected B lymphocytes. Such an immune-induced shift in EBV tissue tropism provides a paradigm for endogenous spread of EBV in the immune host that predicts infectious sequelae of epithelium.

B-Lymphocytes↗
PubMed1989

Virus-specific IgA in serum, saliva, and tears of children with measles.

Measles-specific IgA antibody titres were determined by radioimmunoassay (RIA) for serial serum, saliva and tear samples obtained from 21 children with measles infection, from onset of rash until up to 14 months later. Serum IgA titres rose rapidly after onset of illness and remained detectable throughout the follow-up period. Virus-specific salivary IgA titres peaked at 4 to 7 days after onset of rash and decreased thereafter. Measles-specific lacrimal fluid IgA antibodies remained elevated for long periods of time; however, secretory component-bearing measles-specific antibodies in tears became for the most part undetectable by 1 month after onset of rash. These data raise anew the question of whether some form of viral latency is associated with the presence of virus-specific IgA antibody, or whether such antibody is simply a reflection of immune memory.

Adolescent↗
PubMed1996

Growth, growth hormone (GH)-binding protein, and GH receptors are differentially regulated by peak and trough components of the GH secretory pattern in the rat.

Body growth, GH secretory pattern, hepatic GH receptor (GHR), and plasma GH-binding protein (GHBP) levels are all sexually dimorphic in the rat. Male rats grow faster than females, and in GH-deficient animals, GH therapy is more effective when given in a pulsatile pattern rather than a continuous infusion. This contrasts with GHBP and hepatic GHR levels, which are lower in males than in females and raised by continuous but not pulsatile GH therapy. One possible explanation is that growth is primarily regulated by GH pulses, whereas GHR and GHBP are regulated mostly by the trough levels (which are lower in males than in females). To test this hypothesis directly, GH-deficient dwarf rats were given patterned iv infusions of hGH in which the relative contributions of the peak and trough components of the GH pattern were systematically varied, independently of dose, and their effects on weight and length gain, plasma GHBP, and hepatic GHR binding were measured. We found that the dose-response curves for GH given by pulsatile vs. continuous infusion were significantly nonparallel, and that growth was primarily stimulated by the pulsatile component of a mixed GH infusion pattern; doubling the GH dose by adding a continuous (c) infusion to a series of pulses (p) neither enhanced nor inhibited weight gain (36 micrograms hGH/day pulses (36p) vs. 36 micrograms hGH/day pulses + 36 micrograms hGH/day continuously (36p + 36c):0.9 +/- 0.2 g/day vs. 1.1 +/- 0.2 g/day), whereas doubling the GH dose by adding a pulsatile component significantly enhanced growth (72p:2.1 +/- 0.2 g/day, P < 0.01). Conversely, hepatic GHR and plasma GHBP levels were highly sensitive to the continuous element of the mixed infusion pattern and were totally unaffected by varying the pulsatile component over a wide range of doses. These results strongly suggest that growth and hepatic GHR/plasma GHBP respond differentially to the peak and trough components of the GH secretion pattern in the rat.

Animals↗
PubMed1990

The occurrence and sources of natural antibody in human bile and serum against the O antigens of two Escherichia coli serotypes.

Paired serum and bile samples from normal subjects as well as patients with biliary disease were tested for natural antibody to two individual Escherichia coli O antigens by ELISA. Serum antibody was most commonly of IgM and IgG class. Antibody was less frequently detected in bile and was more commonly IgM than IgA, with IgG activity detected infrequently. Little relation was apparent between antibody in paired samples; activity could be present in both serum and bile or in either fluid alone. Titres in paired samples also did not correspond when 'normalized' with respect to the concentrations of relevant isotypes; bile was frequently enriched for natural antibody as a proportion of total immunoglobulin compared with serum. Secretory component-bound antibody was detectable in some biles that contained IgA and/or IgM activity and in the serum of 33% of subjects with biliary disorders but not in normal sera. A series of paired samples taken from three individuals was also examined for antibody against each subject's own intestinal commensal E. coli. Serum IgM and IgG activity was present in all samples, but antibody in bile was less frequent and was of IgM or IgA class. These results suggest that natural antibody in human bile occurs independently of antibody in serum and that it is substantially derived from local sources.

Adolescent↗
PubMed1993

Transcytosis of the polymeric Ig receptor requires phosphorylation of serine 664 in the absence but not the presence of dimeric IgA.

MDCK cells expressing the polymeric immunoglobulin (poly-Ig) receptor, cocultured with IgA-producing hybridoma cells, transported dimeric IgA (dIgA) from the basolateral into the lumenal compartment, where it was recovered as secretory component-dIgA complexes. The tail of the receptor was phosphorylated on serines 664 and 726. Each serine was mutated to alanine. Appearance of A726 receptor at the basolateral surface was reduced approximately 5-fold. This was accompanied by a approximately 5-fold reduction in dIgA transcytosis. Basolateral delivery of receptor was not affected by mutation A664, and in the absence of dIgA, the receptor accumulated in recycling basolateral endosomes. In coculture, however, dIgA transcytosis by A664 receptor was normal. Thus, entry of receptor into the transcytotic pathway requires Ser-664 phosphorylation only in the absence of dIgA.

Amino Acid Sequence↗
PubMed1993

A component of the catecholamine secretory response in the bovine adrenal gland is resistant to dihydropyridines and omega-conotoxin.

Acetylcholine (ACh, 100 microM)-evoked catecholamine release from perfused bovine adrenal glands was unaffected by CTX (0.3 microM) and depressed 70% by nisoldipine (1 microM). Combined CTX plus nisoldipine did not inhibit further the secretory response K+ (150 mM)-evoked secretion was diminished 30% by CTX and 20% by nisoldipine. Combined CTX plus nisoldipine inhibited secretion by 35%. Cd2+ blocked the K(+)-evoked secretory response by over 90%. Bay K 8644 (1 microM) enhanced the basal output of catecholamines and clearly potentiated the secretory responses to mild concentrations of ACh (3 microM) or K+ (17.7 mM). The results suggest that in addition to CTX- and DHP-sensitive Ca2+ channels, a third Ca2+ pathway might contribute to the entry of external Ca2+ into chromaffin cells necessary to trigger the exocytotic catecholamine release response in the bovine adrenal gland.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Processing and methylation of PuIG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca.

The signal sequence of the Klebsiella oxytoca puIG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the puIO gene product processes prePuIG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PuIG signal sequence. Like most type IV pilins, processed PuIG was found to have a methylated amino-terminal phenylalanine residue. Site-directed mutagenesis was used to replace amino acids in prePuIG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePuIG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11(+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently N-methylated. Cleavage of prePuIG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the carboxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePuIG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.

Amino Acid Sequence↗