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At least 397 records · Page 22Linked to original sources
PubMed1997

Engineering the human thyrotropin receptor ectodomain from a non-secreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves' patients' sera.

Previous attempts to generate autoantibody-reactive, secreted thyrotropin receptor (TSHR) ectodomain in mammalian cells have failed because of retention within the cell of material with immature carbohydrate. We have overcome this difficulty by performing progressive carboxyl-terminal truncations of the human TSHR ectodomain (418 amino acid residues including signal peptide). Three ectodomain variants (TSHR-261, TSHR-289, and TSHR-309) were truncated at residues 261, 289, and 309, respectively. Unlike the full ectodomain, ectodomain variants were secreted with an efficiency inversely proportional to their size. Secreted ectodomain variants contained approximately 20 kDa of complex carbohydrate. TSHR-261 was chosen for further study because it was secreted very efficiently and neutralized autoantibodies in Graves' patients' sera. This ectodomain variant was partially purified using sequential lectin and nickel-chelate chromatography, permitting the first direct visualization and quantitation of the mammalian TSHR. Most important, very small (nanogram) quantities of this material neutralized 70-100% of TSHR autoantibody activity in all 18 Graves' sera studied. In summary, carboxyl-terminal truncation of the human TSHR ectodomain generates a secreted protein with complex carbohydrate that neutralizes autoantibodies in Graves' patients' sera. Antigenically active TSHR will be valuable for future studies on the diagnosis, pathogenesis, and immunotherapy of Graves' disease.

Adsorption↗
PubMed2000

Leptin rapidly inhibits hypothalamic neuropeptide Y secretion and stimulates corticotropin-releasing hormone secretion in adrenalectomized mice.

Leptin may rapidly inhibit food intake by altering the secretion of hypothalamic neuropeptides such as neuropeptide Y (NPY), a stimulator of food intake, and/or corticotropin-releasing hormone (CRH), an inhibitor of food intake. We measured concentrations of NPY and CRH in specific hypothalamic regions [i.e., arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial nucleus and dorsomedial nucleus] of 7- to 8-wk-old lean and ob/ob mice at 1 or 3 h after intracerebroventricular leptin administration. No rapid-onset effects of leptin on hypothalamic NPY or CRH concentrations were observed in intact mice. The addition of leptin to hypothalamic preparations from intact mice also did not alter NPY or CRH secretion. Glucocorticoids may oppose leptin actions. Consistent with this, leptin administration to adrenalectomized mice markedly reduced CRH concentrations in the ARC within 3 h after injection. This rapid reduction in CRH concentration in the ARC after leptin administration is more likely due to stimulated CRH release from this region than to decreased synthesis/transport from the PVN because leptin stimulates CRH synthesis in the PVN. Within 20 min after exposure to leptin, NPY secretion from hypothalamic preparations obtained from adrenalectomized mice was lowered by 27% and CRH secretion was elevated by 51%. The current study demonstrates that leptin rapidly influences the secretion of hypothalamic NPY and CRH and that these actions of leptin within the hypothalamus are restrained by the presence of endogenous corticosterone.

Adrenalectomy↗
PubMed1999

Modulation of human cytotrophoblastic leptin secretion by interleukin-1alpha and 17beta-oestradiol and its effect on HCG secretion.

To investigate the role of leptin during pregnancy, we assessed leptin production by pure cultured human cytotrophoblastic cells (CTB), its regulation by cytokines and 17beta-oestradiol and its effects on human chorionic gonadotrophin (HCG) secretion. Purified CTB from first trimester placenta were incubated in duplicates in the presence or absence of cytokines or 17beta-oestradiol. Medium was harvested on day 2 and the culture stopped on day 4. Results were corrected for protein content of each individual well and expressed as percent of controls per day (mean +/- SEM). Basal CTB leptin production was 25.2 +/- 2.6 (ng/mg prot). In comparison with controls, leptin production was stimulated to 320 +/- 16% (P < 0.0001) and 195 +/- 3.2% (P < 0.0004) by 3 and 10 ng/ml of interleukin-1alpha respectively. 17beta-oestradiol 10(-6) to 10(-9) mol/l increased basal leptin production 5-9-fold, while 10(-5) mol/l had no such effect. Basal CTB HCG secretion was 5722 +/- 1055 (mIU/mg prot). There was a dose-dependent leptin-induced increase in HCG secretion (P = 0.0039) reaching a 5-fold increase with a leptin concentration of 1 microg/ml (P < 0.006). Gonadotrophin-releasing hormone (GnRH) 8.5 x 10(-8) mol/l significantly increased HCG secretion to 140 +/- 21% of controls (P = 0.031). Cetrorelix (0.1 microg/ml) inhibited leptin-induced HCG secretion (P = 0.0028).

Cells, Cultured↗
PubMed1984

A Bacillus subtilis secretion vector system derived from the B. subtilis alpha-amylase promoter and signal sequence region, and secretion of Escherichia coli beta-lactamase by the vector system.

A secretion vector system in Bacillus subtilis was constructed from the alpha-amylase promoter and signal sequence coding region of an alpha-amylase hyperproducing strain, B. subtilis NA64, and the major part of the plasmid pTUB4 which was derived from pUB110. When an Escherichia coli beta-lactamase gene, lacking its own promoter and signal sequence coding region, was introduced into the secretion vector system, beta-lactamase was expressed in B. subtilis. In addition, more than 95% of the enzyme synthesized was secreted into the culture medium via the secretion vector system. Secreted beta-lactamase crossreacted with rabbit antiserum raised against the E. coli enzyme.

Bacillus subtilis↗
PubMed1992

Biosynthesis and Secretion of Cryptogein, a Protein Elicitor Secreted by Phytophthora cryptogea.

The phytopathogenic fungi Phytophthora subspecies elicit hypersensitive-like necroses on their nonhost tobacco (Nicotiana tabacum), with the exception of the tobacco pathogen Phytophthora nicotianae. In culture, these fungi-except P. nicotianae-secrete proteins, called elicitins, that cause these remote leaf necroses and are responsible for the incompatible reaction. These proteins protect tobacco against invasion by the agent of the tobacco black shank, P. nicotianae, which is unable to produce such an elicitor. Cryptogein, secreted by Phytophthora cryptogea, has been purified, sequenced, and characterized as an elicitin, a novel family of 10 kilodalton holoproteins. In the present paper, we examined the secretion and biosynthesis of this protein elicitor from P. cryptogea culture. Results showed that the secretion of cryptogein began later than its synthesis and stopped earlier, simultaneously with mycelium growth, when the nitrogen source in the culture medium was nearly exhausted. Electrophoretic patterns of total protein from mycelium extracts and N-terminal sequence analysis showed that cryptogein accumulated in the mycelium in its mature form. The comparison of the immunoselected in vitro translation products with (35)S in vivo-labeled cryptogein showed that cryptogein was synthesized as a preprotein with a signal peptide removed cotranslationally before the secretion into the culture medium. Immunoselected in vitro-synthesized products were subjected to radiosequencing to clearly determine the N-terminal position and the size (20 amino acids) of the signal peptide. Cryptogein did not undergo any other posttranslational modification.

Journal Article↗
PubMed1987

Food-induced cortisol secretion is mediated by central alpha-1 adrenoceptor modulation of pituitary ACTH secretion.

Food ingestion stimulates cortisol secretion in man, but the mechanism of this effect is unknown. We have investigated the possible role of adrenoceptors in the mediation of this effect. Six normal males were given continuous 3 h i.v. infusions of normal saline, methoxamine (alpha-1 adrenoceptor agonist) and thymoxamine (alpha-1 adrenoceptor antagonist). Methoxamine enhanced and thymoxamine attenuated the ACTH and cortisol responses to a standard meal given 60 min after commencement of the infusion. The drugs had no effect on nutrient absorption. Four patients with recent onset of pituitary ACTH deficiency and normally responsive adrenal glands showed no ACTH or cortisol rises after the standard meal, demonstrating that postprandial cortisol secretion is mediated by pituitary rather than gut ACTH. Our previous investigations have demonstrated that alpha-1 adrenoceptors stimulate pituitary ACTH secretion in man by an action within the blood brain barrier. We therefore conclude that postprandial cortisol secretion is mediated by central stimulant alpha-1 adrenoceptors modulating pituitary ACTH secretion.

Adrenocorticotropic Hormone↗
PubMed1992

Preserved thyroidal secretion of thyroxine in acromegalic patients with suppressed hypophyseal secretion of thyrotrophin.

OBJECTIVE: We have assessed the mechanisms which maintain euthyroidism in acromegalic patients despite the suppression of thyrotrophin (TSH) secretion. MATERIALS: Fourteen untreated patients with acromegaly were analysed. Ten patients were also studied after pituitary surgery. METHODS: Thyroid hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and thyroidal uptake of radioactive iodine, thyrotrophin releasing hormone (TRH) test and basal metabolic rate (BMR) were measured before and after pituitary surgery. RESULTS: Nine patients had palpable goitres. The TSH response to TRH stimulation was suppressed in eight patients, who maintained normal serum levels of total T3, T4 and free T4. The patients with normal TSH response had lower levels of free and total T4 than controls. The response of TSH to TRH correlated inversely with the serum level of total and free T4, and also with the plasma level of IGF-I (r = -0.74, P less than 0.05, n = 9). After pituitary surgery, the serum levels of total and free T4 were elevated for at least up to 6 months, with a decrease in the T3/T4 ratio and the BMR. CONCLUSION: GH may have a direct stimulatory action on the thyroid secretion of T4 possibly via increased IGF-I, despite suppressed TSH secretion. The post-operative elevation of serum T4 suggests the persistent secretion of T4 from the thyroid gland, in spite of instantaneous normalization of the accelerated conversion of T4 to T3, even after reduction of excess GH secretion.

Acromegaly↗
PubMed1993

The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high-level secretion.

Mutations of the prsA gene of Bacillus subtilis have indicated that the gene is involved in protein secretion and it encodes a novel component of the cellular secretion machinery. We now demonstrate that the gene product is a membrane-associated lipoprotein, presumably bound to the outer face of the cytoplasmic membrane. Experiments to inactivate the prsA gene with insertions indicated that it is indispensable for viability. The cellular level of PrsA protein was shown to be decreased in prsA mutants with decreased level of exoproteins, consistent with an essential function in protein secretion. An increased amount of cellular PrsA protein was introduced by increasing the copy number of prsA in B. subtilis. This enhanced, from six- to twofold, the secretion of alpha-amylases and a protease in strains, which expressed high levels of these exoenzymes. This suggests that PrsA protein is the rate-limiting component of the secretion machinery, a finding that is of considerable biotechnological interest.

Bacillus subtilis↗
PubMed2005

Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system.

The process of DNA donation for natural transformation of bacteria is poorly understood and has been assumed to involve bacterial cell death. Recently in Neisseria gonorrhoeae we found that mutations in three genes in the gonococcal genetic island (GGI) reduced the ability of a strain to act as a donor in transformation and to release DNA into the culture. To better characterize the GGI and the process of DNA donation, the 57 kb genetic island was cloned, sequenced and subjected to insertional mutagenesis. DNA sequencing revealed that the GGI has characteristics of a horizontally acquired genomic island and encodes homologues of type IV secretion system proteins. The GGI was found to be incorporated near the chromosomal replication terminus at the dif site, a sequence targeted by the site-specific recombinase XerCD. Using a plasmid carrying a small region of the GGI and the associated dif site, we demonstrated that this model island could be integrated at the dif site in strains not carrying the GGI and was spontaneously excised from that site. Also, we were able to delete the entire 57 kb region by transformation with DNA from a strain lacking the GGI. Thus the GGI was likely acquired and integrated into the gonococcal chromosome by site-specific recombination and may be lost by site-specific recombination or natural transformation. We made mutations in six putative type IV secretion system genes and assayed these strains for the ability to secrete DNA. Five of the mutations greatly reduced or completely eliminated DNA secretion. Our data indicate that N. gonorrhoeae secretes DNA via a specific process. Donated DNA may be used in natural transformation, contributing to antigenic variation and the spread of antibiotic resistance, and it may modulate the host immune response.

Biological Transport↗
PubMed2007

Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors.

Shigella possess 220 kb plasmid, and the major virulence determinants, called effectors, and the type III secretion system (TTSS) are exclusively encoded by the plasmid. The genome sequences of S. flexneri strains indicate that several ipaH family genes are located on both the plasmid and the chromosome, but whether their chromosomal IpaH cognates can be secreted from Shigella remains unknown. Here we report that S. flexneri strain, YSH6000 encodes seven ipaH cognate genes on the chromosome and that the IpaH proteins are secreted via the TTSS. The secretion kinetics of IpaH proteins by bacteria, however, showed delay compared with those of IpaB, IpaC and IpaD. Expression of the each mRNA of ipaH in Shigella was increased after bacterial entry into epithelial cells, and the IpaH proteins were secreted by intracellular bacteria. Although individual chromosomal ipaH deletion mutants showed no appreciable changes in the pathogenesis in a mouse pulmonary infection model, the DeltaipaH-null mutant, whose chromosome lacks all ipaH genes, was attenuated to mice lethality. Indeed, the histological examination for mouse lungs infected with the DeltaipaH-null showed a greater inflammatory response than induced by wild-type Shigella, suggesting that the chromosomal IpaH proteins act synergistically as effectors to modulate the host inflammatory responses.

Animals↗
PubMed1982

Plasma secretion and pancreatic secretion in response to liver extract meal with varied pH and exogenous secretin in the dog.

1. In dogs with chronic gastric and pancreatic fistulas, a liver extract meal adjusted to various pH levels ranging from 7.0 to 2.0, was introduced into the stomach and the increments in plasma secretin levels were correlated with the pH of the liver extract meal and pancreatic bicarbonate outputs. 2. The pH threshold for both bicarbonate secretion and secretin release was found to be about 4.5. With a stepwise decrease in the pH of the meal below pH 4.5, there were stepwise increments in the plasma secretin concentrations and pancreatic bicarbonate outputs. 3. Exogenous secretin, given in graded doses ranging from 0.03 to 2.0 clinical unit/kg per hr, increased the plasma secretin concentrations and bicarbonate secretion in a dose-dependent fashion. 4. These results indicate that the pH threshold for release of endogenous secretin is 4.5 and suggests that, at pH levels below 4.5, pancreatic bicarbonate secretion depends upon the duodenal acid load and is linearly correlated to an increment in plasma secretin concentrations. 5. It is concluded that endogenous secretin is a major determinant of pancreatic bicarbonate secretion after a meal. 6. Pancreatic protein secretion by intragastric liver extract meal was greatly increased both in experiments with liver extract meal, pH 4.0 or below, and I.V. infusion of secretin at a dose of 0.12 u./kg per hr. It is questioned, however, whether this effect of secretin is physiological.

Animals↗
PubMed1998

Chemotactic activities in nonmastitic and mastitic mammary secretions: presence of interleukin-8 in mastitic but not nonmastitic secretions.

Due to its association with low-quality milk and a decrease in milk production in bovines, mastitis is a major cause of economic loss. Additionally, mastitis can be harmful to suckling newborns and can cause damage to the mammary gland. In mastitic mammary secretions there is a substantial increase in somatic cells, specifically neutrophils. In this study we examined the ability of mastitic and nonmastitic mammary secretions to cause in vitro neutrophil chemotaxis using a microchemotaxis assay. Also, the role of the inflammatory chemokine interleukin-8 (IL-8) in neutrophil recruitment during mastitis was addressed in these in vitro experiments. We found that both nonmastitic and mastitic mammary secretions were chemotactic, not chemokinetic, for neutrophils. The neutrophil chemotactic activity in mastitic, but not nonmastitic, mammary secretions was blocked by anti-IL-8 antibodies. Molecular mass separation of the active components showed that the chemotactic activity of the mastitic secretions was present in the 10-kDa-or-less fraction and was blocked by anti-IL-8 antibodies. These results indicate that IL-8 plays a major role in neutrophil recruitment during mastitis. An understanding of its role will be of help in designing strategies for immunomodulatory therapies for mastitis.

Animals↗
PubMed2006

Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1.

Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important pathogen causing food-borne gastroenteritis or septicemia. Recent genome sequencing of the RIMD2210633 strain (a Kanagawa phenomenon-positive clinical isolate of serotype O3:K6) revealed that the strain has two sets of gene clusters that encode the type III secretion system (TTSS) apparatus. The first cluster, TTSS1, is located on the large chromosome, and the second, TTSS2, is on the small chromosome. Previously, we reported that TTSS1 is involved in the cytotoxicity of the RIMD2210633 strain against HeLa cells. Here, we analyzed proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional gel electrophoresis and identified the proteins encoded by genes VP1680, VP1686, and VPA450. To investigate the roles of those secreted proteins, we constructed and analyzed a series of deletion mutants. Flow cytometry analysis using fluorescence-activated cell sorting with fluorescein isothiocyanate-labeled annexin V demonstrated that the TTSS1-dependent cell death was by apoptosis. The cytotoxicity to HeLa cells was related to one of the newly identified secreted proteins encoded by VP1680. Adenylate cyclase fusion protein studies proved that the newly identified secreted proteins were translocated into HeLa cells. Thus, these appear to be the TTSS effector proteins in V. parahaemolyticus.

Amino Acid Sequence↗
PubMed1991

Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism.

A 6.5-kb DNA fragment carrying the functions required for specific secretion of the extracellular alkaline protease produced by Pseudomonas aeruginosa was cloned. The whole 6.5-kb DNA fragment was transcribed in one direction and probably carried three genes involved in secretion. The expression in trans of these genes, together with the apr gene, in Escherichia coli allowed synthesis and secretion of the alkaline protease, which was extensively investigated by performing pulse-chase experiments under various conditions. We demonstrated the absence of a precursor form, as well as the independence of alkaline protease translocation from SecA. The absence of secretion genes impaired alkaline protease secretion; the protein then remained intracellular and was partially degraded.

Adenosine Triphosphatases↗
PubMed2001

SseBCD proteins are secreted by the type III secretion system of Salmonella pathogenicity island 2 and function as a translocon.

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is required for systemic infections and intracellular accumulation of Salmonella enterica. This system is induced by intracellular Salmonella and subsequently transfers effector proteins into the host cell. Growth conditions either inducing expression of the type III secretion system or the secretion of substrate proteins were defined. Here we report the identification of a set of substrate proteins consisting of SseB, SseC, and SseD that are secreted by the SPI2 system in vitro. Secretion was observed if bacterial cells were exposed to acidic pH after growth in minimal medium with limitation of Mg(2+) or phosphate. SseB, -C, and -D were isolated in a fraction detached from the bacterial cell surface by mechanical shearing, indicating that these proteins are predominantly assembled into complexes on the bacterial cell surface. The three proteins were required for the translocation of SPI2 effector proteins SspH1 and SspH2 into infected host cells. Thus, SseB, SseC, and SseD function as the translocon for effector proteins by intracellular Salmonella.

Animals↗
PubMed2001

Quantitation of the capacity of the secretion apparatus and requirement for PrsA in growth and secretion of alpha-amylase in Bacillus subtilis.

Regulated expression of AmyQ alpha-amylase of Bacillus amyloliquefaciens was used to examine the capacity of the protein secretion apparatus of B. subtilis. One B. subtilis cell was found to secrete maximally 10 fg of AmyQ per h. The signal peptidase SipT limits the rate of processing of the signal peptide. Another limit is set by PrsA lipoprotein. The wild-type level of PrsA was found to be 2 x 10(4) molecules per cell. Decreasing the cellular level of PrsA did not decrease the capacity of the protein translocation or signal peptide processing steps but dramatically affected secretion in a posttranslocational step. There was a linear correlation between the number of cellular PrsA molecules and the number of secreted AmyQ molecules over a wide range of prsA and amyQ expression levels. Significantly, even when amyQ was expressed at low levels, overproduction of PrsA enhanced its secretion. The finding is consistent with a reversible interaction between PrsA and AmyQ. The high cellular level of PrsA suggests a chaperone-like function. PrsA was also found to be essential for the viability of B. subtilis. Drastic depletion of PrsA resulted in altered cellular morphology and ultimately in cell death.

Bacillus subtilis↗
PubMed2005

An amino-terminal secretion signal is required for YplA export by the Ysa, Ysc, and flagellar type III secretion systems of Yersinia enterocolitica biovar 1B.

Yersinia enterocolitica biovar 1B maintains three distinct type III secretion (TTS) systems, which independently operate to target proteins to extracellular sites. The Ysa and Ysc systems are prototypical contact-dependent TTS systems that translocate toxic effectors to the cytosols of targeted eukaryotic host cells during infection. The flagellar TTS system is utilized during the assembly of the flagellum and is required for secretion of the virulence-associated phospholipase YplA to the bacterial milieu. When ectopically produced, YplA is also a secretion substrate for the Ysa and Ysc TTS systems. In this study, we define elements that allow YplA recognition and export by the Ysa, Ysc, and flagellar TTS systems. Fusion of various amino-terminal regions of YplA to Escherichia coli alkaline phosphatase (PhoA) lacking its native secretion signal demonstrated that the first 20 amino acids or corresponding mRNA codons of YplA were sufficient for export of YplA-PhoA chimeras by each TTS system. Export of native YplA by each of the three TTS systems was also found to depend on the integrity of its amino terminus. Introduction of a frameshift mutation or deletion of yplA sequences encoding the amino-terminal 20 residues negatively impacted YplA secretion. Deletion of other yplA regions was tolerated, including that resulting in the removal of amino acid residues 30 through 40 of the polypeptide and removal of the 5' untranslated region of the mRNA. This work supports a model in which independent and distantly related TTS systems of Y. enterocolitica recognize protein substrates by a similar mechanism.

Bacterial Outer Membrane Proteins↗
PubMed1983

Lung secretion sol-phase proteins: comparison of sputum with secretions obtained by direct sampling.

The protein content of tracheal secretions and of bronchial lavage and bronchoalveolar lavage fluid was compared with that of sputum in 33 patients who underwent fibreoptic bronchoscopy. The secretion-to-serum concentration ratios for albumin, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and immunoglobulin A (IgA) fell progressively as samples were obtained from further down the bronchial tree, probably reflecting greater sample dilution. The secretion-to-serum protein ratios when standardised for the corresponding albumin ratio were similar in all secretions studied. In particular, the IgA ratios were about eight times those of albumin and alpha 1-antichymotrypsin ratios were about twice those of albumin, suggesting similar degrees of "local production" of these proteins in all secretions studied. Some patients showed considerable differences in IgA ratios between sputum and bronchial lavage fluid. The significance of these differences is not clear.

Adult↗