The N terminus of the HasA protein and the SecB chaperone cooperate in the efficient targeting and secretion of HasA via the ATP-binding cassette transporter.

Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens. We reconstituted the HasA secretion pathway in Escherichia coli. In E. coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA. The secretion of the isolated C-terminal secretion signal was not SecB-dependent. We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal. Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent. The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein. We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemi by their specific ABC transporter. Here we show that this abortive interaction between HasA and the E. chrysanthemi metalloprotease ABC transporter required both SecB and the N terminus of HasA. N-terminal fragments of HasA displayed this abortive interaction in vivo and also interacted specifically in vitro with the ABC protein of the Prt system. SecB also interacted specifically in vitro with the ABC protein of the Prt system. Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system. We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.     

Feedback control of pancreatic secretion in rats. Role of gastric acid secretion.

Pancreatic secretion in rats is regulated by feedback inhibition of cholecystokinin (CCK) release by proteases in the gut lumen, but little is known about the role of gastric acid in this regulation. This study, carried out on conscious rats with large gastric fistulas (GF) and pancreatic fistulas, shows that diversion of pancreatic juice results in the progressive stimulation of pancreatic secretion only in rats with the GF closed. When the GF was kept open, the diversion resulted in only small increment in pancreatic secretion and this was accompanied by progressive increase in gastric acid outputs. Similar amounts of HCl instilled into the duodenum in rats with the GF open fully reproduced the increase in pancreatic secretion observed after the diversion of pancreatic juice. Pretreatment with omeprazole (15 mumol/kg) to suppress gastric acid secretion or with L-364,718 (5 mumol/kg) to antagonize CCK receptors in the diverted state, resulted in the decline in pancreatic secretion similar to that observed after opening the GF. CCK given s.c. (20-320 pmol/kg) failed to cause any significant rise in the post-diversion pancreatic secretion in rats with the GF closed, but stimulated this secretion dose-dependently when the GF was open. Camostate (6-200 mg/kg) in rats with pancreatic juice returned to the duodenum caused dose-dependent increase in pancreatic secretion, but after opening the GF or after omeprazole this increase was reduced by about 75%. This study provides evidence that gastric acid plays a crucial role in the pancreatic response to diversion of pancreatic juice or inhibition of luminal proteases, and that factors that eliminate gastric acid secretion reduce this response.     

Evaluation of the significance of malic acid secretion in chickpea

Leaves of chickpea ( Cicer urietinum L.) secrete malic acid hut the significance of this secretion is not known. Our studies have confirmed that secretion of malic acid was mostly confined to the day time. PEP carboxylase activity and the secretion of malic acid are associated with the presence of glands on the surface of leaves and fruit wall. PEP carboxylase activity increased while malic acid secretion and MDH activity decreased in water stressed plants. The secretion of malic acid did not influence the reflectance properties of the crop canopy thereby discounting the possibility of reducing radiation load. It is suggested that the significance of malic acid secretion in chickpea should be considered in relation to insect pests which are very few on this plant.     

Construction and expression of alpha 1-proteinase inhibitor mutants and the effects of these mutations on secretion of the variant inhibitors

Human alpha 1-proteinase inhibitor (A1Pi) deficiency, associated with the Z variant A1Pi gene, results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of replacement of Glu342 with Lys. To investigate the effect of the amino acid occupying position 342 on secretion of A1Pi, we have used oligonucleotide-directed mutagenesis of A1Pi cDNA to randomly change the codon specifying this amino acid. Since replacement of Glu342 by Lys leads to a change in the predicted secondary structure for this protein, we also tested the possibility that defective secretion of A1PiZ is the result of this type of alteration. For this purpose, site-directed mutagenesis was used to produce sequences encoding A1Pi retaining Glu342 but predicted to have A1PiZ type secondary structure. The effects of 10 different amino acids occupying position 342 on the secretion of A1Pi were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of medium from transiently transfected COS cells. Results of these studies show that secretion of A1Pi is most efficient when position 342 is occupied by a negatively charged amino acid, efficient but somewhat less so when occupied by a neutral amino acid, and least efficient when a positively charged residue is present. The mutation designed to alter secondary structure had no effect on the secretion of A1Pi. As indicated by immunofluorescence microscopy and mobility of intracellular A1Pi on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lowered secretion is accompanied by accumulation of A1Pi in the endoplasmic reticulum of the transfected cells. These results are compatible with the ideas that secretion of A1Pi is directly influenced by the amino acid occupying position 342, that a positively charged amino acid in this position is especially detrimental to secretion of this protein, and that the rate-limiting step in the secretion of the altered forms is transport from endoplasmic reticulum to Golgi.     

Mechanophysical stimulations of mucin secretion in cultures of nasal epithelial cells

Nasal epithelial cells secret mucins and are exposed in vivo to airflow-induced mechanophysical stresses, including wall shear stress (WSS), temperature, and humidity. In this work, human nasal epithelial cells cultured under air-liquid interface conditions were subjected to fields of airflow-induced oscillatory WSS at different temperature and humidity conditions. Changes in mucin secretion due to WSS were measured and the role of the cytoskeleton in mucin secretion was explored. Mucin secretion significantly increased in response to WSS in a magnitude-dependent manner with respect to static cultures and independently of the airflow temperature and humidity. In static cultures, mucin secretion decreased at high humidity with or without elevation of the temperature with respect to cultures at a comfortable climate. In cultures exposed to WSS, mucin secretion increased at high temperature with respect to cultures at comfortable climate conditions. The polymerization of actin microfilaments was shown to increase mucin secretion under WSS, whereas the dynamics of microtubule polymerization did not affect secretion. In conclusion, the data in this study show that mucin secretion is sensitive to oscillatory WSS as well as high temperature and humidity conditions.     

Influence of temperature on the secretion of insulin and glucagon induced by stimulation of cholinergic receptors in the presence of glucose]

In the presence of a glucose concentration of 1.5 g/1 the secretion of insulin from the isolated perfused rat pancreas is clearly weaker at 28 degrees C than at 37.5 degrees C. In response to cholinergic stimulation, the absolute increase of insulin secretion rate is less at 28 degrees C than at 37.5 degrees C. However, when evaluated in percentage in relation to the baseline value, this increase is more important at the lower temperature. As to glucagon secretion, lowering of the temperature from 37.5 degrees C to 28 degrees C modifies neither this secretion in the presence of glucose alone, nor the increased secretion provoked by the cholinergic stimulation.     

The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii

Type-IV secretion systems are devices present in a wide range of bacteria (including bacterial pathogens) that deliver macromolecules (proteins and single-strand-DNA) across kingdom barriers (as well as between bacteria and into the surroundings). The type-IV secretion systems were divided into two subgroups and Legionella pneumophila and Coxiella burnetii are the only two bacteria known today to utilize a type-IVB secretion system for pathogenesis. In this review we summarized the available information concerning the icm/dot type-IVB secretion systems by comparing the two bacteria that possess this system, the proteins components of their systems as well as the homology of proteins from type-IVB secretion systems to proteins from type-IVA secretion systems. In addition, the phenotypes associated with mutants in the L. pneumophila icm/dot genes, their relations to properties of specific Icm/Dot proteins as well as the protein substrates delivered by this system are described.     

Comparison of the secretory processes in the parotid and sublingual glands of the mouse. 1. Regulation of the secretory processes.

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.     

Computational analysis of the ESX-1 region of Mycobacterium tuberculosis: insights into the mechanism of type VII secretion system

Type VII secretion system (T7SS) is a recent discovery in bacterial secretion systems. First identified in Mycobacterium tuberculosis, this secretion system has later been reported in organisms belonging to the Actinomycetales order and even to distant phyla like Firmicutes. The genome of M. tuberculosis H37Rv contains five gene clusters that have evolved through gene duplication events and include components of the T7SS secretion machinery. These clusters are called ESAT-6 secretion system (ESX) 1 through 5. Out of these, ESX-1 has been the most widely studied region because of its pathological importance. In spite of this, the overall mechanism of protein translocation through ESX-1 secretion machinery is not clearly understood. Specifically, the structural components contributing to the translocation through the mycomembrane have not been characterized yet. In this study, we have carried out a comprehensive in silico analysis of the genes known to be involved in ESX-1 secretion pathway and identified putative proteins having high probability to be associated with this particular pathway. Our study includes analysis of phylogenetic profiles, identification of domains, transmembrane helices, 3D folds, signal peptides and prediction of protein-protein associations. Based on our analysis, we could assign probable novel functions to a few of the ESX-1 components. Additionally, we have identified a few proteins with probable role in the initial activation and formation of mycomembrane translocon of ESX-1 secretion machinery. We also propose a probable working model of T7SS involving ESX-1 secretion pathway.     

Influence of cimetidine on hypoglycemia-induced GH secretion]

Dopaminergic drugs inhibit prolactin and stimulate GH secretion. On the contrary antidopaminergic drugs stimulate prolactin and decrease hypoglycemia-induced GH secretion. According to the hypothesis that Cimetidine, an H2-receptor antagonist, decreases hypothalamic dopamine secretion, it was evaluated GH response to hypoglycemia after this drug. It was demonstrated that Cimetidine decreases hypoglycemia-induced GH secretion.     

Molecular and functional analysis of the type III secretion signal of the Salmonella enterica InvJ protein

Central to the pathogenicity of Salmonella enterica is the function of a type III secretion system (TTSS) encoded within a pathogenicity island at centisome 63 (SPI-1). An essential component of this system is a supramolecular structure termed the needle complex. Proteins to be delivered into host cells possess specific signals that route them to the type III secretion pathway. In addition, some bacterial proteins have signals that deliver them to the secretion complex to either become their structural components or exert their function at that location. One of these proteins is InvJ, which controls the length of the needle substructure of the needle complex. In this study, we have analysed the signal that targets InvJ to the TTSS. We found that amino acid residues 4 to 7 of InvJ are necessary and sufficient to mediate secretion of InvJ or a reporter protein in a TTSS-dependent manner. InvJ secretion was found to be essential for its function in needle length determination, effector protein secretion and bacterial invasion of epithelial cells. Frameshift mutagenesis analysis indicated that the InvJ type III secretion signal sequence tolerates significant alterations in its amino acid sequence without affecting InvJ secretion. Introduction of silent mutations in the secretion signal coding sequence that result in drastically different predicted mRNA folds had no effect on InvJ secretion or expression.     

Fine structure of the tegumentary glands secreting the protective "shield" in a sessile insect (Homoptera, Diaspiddae)

The tegumentary pygidial glands of Aonidiella aurantii (Homoptera, Diaspididae) produce a secretion forming the shield of these fixed parasites of plants. They are formed of seven cells: a principal unpaired secretory cell which produces an abundant glycoproteinaceous secretion; a small associated cell with a secondary reservoir for this secretion; two accessory secretory cells which have very abundant tubular extensions coming from the plasma membrane, and a flocculent secretion gathered in a large sub-cuticular space; two cells forming an enlarged part of the excretory canal, functioning like a spinneret; and finally a single cell forming the tubular duct of this complex gland. The cuticle of the secretory cells has a very special porous structure, through which the secretion passes. The final product is a ribbon formed by two hollow strands stuck together. The exact nature of this secretion is not clear. It is comparable to a silk secretion though it has its own particular characteristics.     

Ionic mechanism of forskolin-induced liquid secretion by porcine bronchi

cAMP-elevating agents such as forskolin and vasoactive intestinal peptide induce liquid secretion by tracheobronchial submucosal glands. This pathway is thought to be CFTR dependent and thus defective in cystic fibrosis; however, the ionic mechanism that drives this secretion process is incompletely understood. To better define this mechanism, we studied the effects of ion transport inhibitors on the forskolin-induced liquid secretion response (Jv) of porcine distal bronchi. The forskolin-induced Jv was driven by a combination of bumetanide-sensitive Cl- secretion and DIDS-sensitive HCO3- secretion. When Cl- secretion was inhibited with bumetanide, Na+/H+ exchange-dependent HCO3- secretion was apparently induced to compensate for the loss of Cl- secretion. The forskolin-induced Jv was significantly inhibited by the anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, and glibenclamide. We conclude that the forskolin-induced Jv shares many characteristics of cholinergically induced secretion except for the presence of a DIDS-sensitive component. Although the identity of the DIDS-sensitive component is unclear, we speculate that it represents a basolateral membrane Na+ -HCO3- cotransporter or an Na+-dependent anion exchanger, which could account for transepithelial HCO3- secretion.     

Control of fetal insulin secretion

In this study, we investigated the way in which fetal insulin secretion is influenced by interrelated changes in blood glucose and sympathoadrenal activity. Experiments were conducted in late gestation sheep fetuses prepared with chronic peripheral and adrenal catheters. The fetus mounted a brisk insulin response to hyperglycemia but with only a minimal change in the glucose-to-insulin ratio, indicating a tight coupling between insulin secretion and plasma glucose. In well-oxygenated fetuses, alpha(2)-adrenergic blockade by idazoxan effected no change in fetal insulin concentration, indicating the absence of a resting sympathetic inhibitory tone for insulin secretion. With hypoxia, fetal norepinephrine (NE) and epinephrine secretion and plasma NE increased markedly; fetal insulin secretion decreased strikingly with the degree of change related to extant plasma glucose concentration. Idazoxan blocked this effect showing the hypoxic inhibition of insulin secretion to be mediated by a specific alpha(2)-adrenergic mechanism. alpha(2)-Blockade in the presence of sympathetic activation secondary to hypoxic stress also revealed the presence of a potent beta-adrenergic stimulatory effect for insulin secretion. However, based on an analysis of data at the completion of the study, this beta-stimulatory mechanism was seen to be absent in all six fetuses that had been subjected to a prior experimentally induced hypoxic stress but in only one of nine fetuses not subjected to this perturbation. We speculate that severe hypoxic stress in the fetus may, at least in the short term, have a residual effect in suppressing the beta-adrenergic stimulatory mechanism for insulin secretion.     

革兰氏阴性细菌蛋白分泌系统研究进展

革兰氏阴性细菌由于具有复杂的双层膜结构,其蛋白质分泌能力较差.这使得革兰氏阴性细菌的典型菌株——大肠杆菌作为最常用的受体细胞在生物制药工程和其他生物技术产品生产中受到一定的限制.因此,革兰氏阴性细菌蛋白分泌系统的研究具有重要意义.本文详细地归纳了革兰氏阴性细菌已知的蛋白分泌系统,分别从分泌系统的分泌过程、分泌蛋白类别、...     

Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis

The mechanism of inhibition of the vascular-platelet stage of hemostasis by medicinal leech salivary gland secretion was studied. It was shown that the secretion blocks platelet adhesion on the surface of collagens belonging to different genetic classes, inhibits the primary attachment of platelets and completely suppresses their spreading on collagen surface. Whatever its antithrombin activity, the leech secretion inhibits platelet aggregation stimulated by various inductors, e. g., ADP, prostaglandin endoperoxide analog U-46619, Ca2+ ionophore A23187, arachidonic acid. The secretion possessing the antithrombin activity causes a greater inhibition of the thrombin-stimulated aggregation than that devoid of this activity. Leech secretion stimulates adenylate cyclase of platelet membranes in a receptor-mediated fashion and increases the level of cAMP. The active substance is a low molecular weight, thermostable trypsin-resistant fraction of the secretion. Stimulation of adenylate cyclase is not mediated by adenosine receptors. It is supposed that the mechanism of this activating effect involves platelet prostaglandin receptors.     

Neural regulation of glucagon-like peptide-1 secretion in pigs

Glucagon-like peptide (GLP)-1 is secreted rapidly from the intestine postprandially. We therefore investigated its possible neural regulation. With the use of isolated perfused porcine ileum, GLP-1 secretion was measured in response to electrical stimulation of the mixed, perivascular nerve supply and infusions of neuroactive agents alone and in combination with different blocking agents. Electrical nerve stimulation inhibited GLP-1 secretion, an effect abolished by phentolamine. Norepinephrine inhibited secretion, and phentolamine abolished this effect. GLP-1 secretion was stimulated by isoproterenol (abolished by propranolol). Acetylcholine stimulated GLP-1 secretion, and atropine blocked this effect. Dimethylphenylpiperazine stimulated GLP-1 secretion. In chloralose-anesthetized pigs, however, electrical stimulation of the vagal trunks at the level of the diaphragm had no effect on GLP-1 or GLP-2 and weak effects on glucose-dependent insulinotropic peptide and somatostatin secretion, although this elicited a marked atropine-resistant release of the neuropeptide vasoactive intestinal polypeptide to the portal circulation. Thus GLP-1 secretion is inhibited by the sympathetic nerves to the gut and may be stimulated by intrinsic cholinergic nerves, whereas the extrinsic vagal supply has no effect.     

A population analysis of VEGF transgene expression and secretion

The induction of therapeutic angiogenesis with gene therapy approaches has received considerable interest and some limited clinical success. A major drawback to this approach is a lack of understanding of the pharmacokinetics of therapeutic protein delivery. This has become increasingly more relevant as recent studies have illustrated a defined therapeutic window for angiogenic protein secretion into the local microenvironment. For cell based gene therapies, with cells widely distributed throughout the tissue, this implies that any individual cell must attain a specific secretion rate to produce a local angiogenic response. Here we report a reproducible technique enabling the study of growth factor secretion from individual cells following transient plasmid transfection. We demonstrate significant variability in single cell vascular endothelial growth factor (VEGF) secretion with the majority of total protein secretion arising from a small subpopulation of transfected cells. We demonstrate that VEGF secretion is linearly correlated to intracellular plasmid copy number and protein secretion does not appear to reach saturation within the cell population. The selection of gene therapy approaches that optimize individual cell secretion profiles may be essential for the development of effective gene therapies.     

Influence of VIP on thyroid hormone secretion

Since VIP occurs in intrathyroidal nerves its role in thyroid hormone secretion has been investigated. It has been found that VIP is a stimulator of iodothyronine secretion in mice. In this respect VIP has a weaker potency than TSH, but shows a similar time characteristic. Also, VIP and TSH potentiate each others effects. In contrast to the effect of TSH, that of VIP is uninfluenced by alpha-adrenoceptor blockade. VIP, like TSH, stimulates thyroid cyclic AMP production. Thus, VIP nerves might, together with TSH, adrenergic and cholinergic nerves and other peptides such as somatostatin, participate in the complex regulation of iodothyronine secretion. Beside this, VIP has also been found to stimulate calcitonin secretion in rats. Other intrathyroidal neuropeptides, such as substance P and CCK-4, have been found to be without effects on iodothyronine secretion, but, like VIP, to stimulate calcitonin secretion.