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At least 73 records · Page 4Linked to original sources
PubMed1990

Soluble interleukin-2 receptor secretion defect in vitro in HLA-B8, DR3 positive subjects.

Several studies have shown that HLA-B8,DR3 positive subjects may display T cell dysfunctions. Recently, a soluble form of the receptor for IL-2 (sIL-2R) has been demonstrated in human sera and in vitro-stimulated culture supernatant from human T lymphocytes. In the present paper we report sIL-2R serum levels and sIL-2R production from peripheral blood mononuclear cells in HLA-B8,DR3 positive subjects. We found that HLA-B8,DR3 positive subjects have the highest values of serum sIL-2R, but comparing the values of these subjects with those of negative ones no significant difference was observed. As regards the in vitro production of sIL-2R, no difference exists for unstimulated cultures, whereas after stimulation, the HLA-B8,DR3 positive subjects showed the lowest values compared with negative ones. It is noteworthy that these changes are observed in autoimmune diseases linked to this HLA phenotype.

Adolescent↗
PubMed1999

Hypofibrinogenemia associated with a heterozygous missense mutation gamma153Cys to arg (Matsumoto IV): in vitro expression demonstrates defective secretion of the variant fibrinogen.

We genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified gamma-chain gene segment showed a novel nucleotide substitution. This heterozygous mutation encodes both Cys (TGT) and Arg (CGT) at residue 153. To examine the basis for the fibrinogen deficiency, we prepared expression vectors containing mutant gamma-chain DNAs encoding gamma153R and gamma153A for in vitro expression in Chinese hamster ovary (CHO) cells. Enzyme-linked immunosorbent assay and immunoblot analysis of the culture media and cell lysates showed that CHO cells transfected with gamma153R or gamma153A synthesized the variant gamma-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant gamma-chain was expressed. In cells expressing normal fibrinogen, assem- bly intermediates and intact fibrinogen were seen in cell lysates prepared after short (3 minutes) or long (1 hour) incubation with (35)S-methionine. Neither intermediates nor intact fibrinogen was seen with the variant gamma-chains. These data suggest that gamma-chains have an important early role in fibrinogen assembly. Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first step in assembly is the formation of alphagamma or betagamma dimers, or both. This model implies that gammaCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the gamma153Cys-->Arg substitution does not allow fibrinogen assembly and secretion, and this is manifest in vivo as a fibrinogen deficiency. We designated this variant as fibrinogen Matsumoto IV.

Adult↗
PubMed1992

A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus.

The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins. Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3438-3441; Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983) J. Biol. Chem. 258, 7141-7148). We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus. Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein. This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so. Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis. This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus.

Amino Acid Sequence↗
PubMed1993

Defective secretion of albumin encoded by exon-skipped mRNA found in aged analbuminemic rat liver.

Remarkable increases in the amount of exon H-skipped (delta H), exons G and H-skipped (delta GH), and exons H and I-skipped (delta HI) mRNAs of rat albumin have been found in the livers of aged or mutagen-treated analbuminemic rats (Kaneko, T., H. Shima, H. Esumi, M. Ochiai, S. Nagase, T. Sugimura, M. Nagao: Proc. Natl. Acad. Sci. USA 98, 2707-2711 (1991)), in association with increases in numbers of immunohistochemically albumin-positive hepatocytes and the accumulation of albumin(s) in the cells. To determine which type of mRNA is responsible for the accumulation of albumin(s), expression vector systems for intact mRNA and three kinds of exon-skipped albumin mRNAs were constructed and transfected into COS-1 and HepG2 cells. Transient expression and secretion of albumins were examined in these cells. delta HI albumin and intact albumin were efficiently expressed by both types of transfected cells. The delta HI albumin expressed had a similar molecular weight to the major albumin isolated from the microsome fraction of the liver of aged analbuminemic rats, as judged by SDS-PAGE. The rate of secretion of delta HI albumin was very low in both types of transfected cells, whereas those of delta H and delta GH albumins were normal. Immunocytochemical analyses of the transfected COS-1 cells with antiserum against rat albumin showed that the distribution of intact albumin had a compact Golgi-like pattern, whereas that of delta HI albumin had a diffuse endoplasmic reticulum-like pattern. These distribution patterns were similar to those in aged analbuminemic rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1995

Platelet aggregation at high shear is impaired in patients with congenital defects of platelet secretion and is corrected by DDAVP: correlation with the bleeding time.

Techniques measuring platelet aggregation in vitro under the high shear rate conditions that can be found in the microcirculation could reflect the status of primary hemostasis better than the turbidimetric technique. We studied platelet aggregation at high shear in patients with prolonged bleeding time caused by congenital platelet secretion defects such as delta-storage pool deficiency and primary secretion defect. Two different techniques were used: shear-induced platelet aggregation in a cone-and-plate viscometer and the filter aggregation test. With both techniques, platelet aggregation at high shear rate was defective in 14 patients with delta-storage pool deficiency and in 8 with primary secretion defect. There was a statistically significant correlation between platelet aggregation at high shear rate and the bleeding time. In patients with delta-storage pool deficiency, platelet aggregation at high shear rate and the bleeding time were significantly correlated with the platelet serotonin content. The intravenous infusion of 1-deamino-8-D-arginine vasopressin (DDAVP) (0.3 micrograms/kg) increased the plasma concentration of von Willebrand factor (vWf), shortened the bleeding time, and potentiated platelet aggregation at high shear rate in all patients. Because platelet aggregation at high shear rate requires vWf, the effect of DDAVP is probably due to the induced increase in plasma vWf. Therefore, platelet aggregation at high shear rate is defective in patients with congenital defects of platelet secretion and is potentiated by DDAVP. Potentiation of platelet aggregation at high shear rate may be one mechanism by which DDAVP shortens the prolonged bleeding time of patients with congenital defects of platelet secretion.

Bleeding Time↗
PubMed1997

Short stature and failure of pubertal development in thalassaemia major: evidence for hypothalamic neurosecretory dysfunction of growth hormone secretion and defective pituitary gonadotropin secretion.

UNLABELLED: In patients with beta-thalassaemia major, frequent blood transfusions combined with desferrioxamine chelation therapy lead to an improved rate of survival. Endocrine disorders related to secondary haemosiderosis such as short stature, delayed puberty and hypogonadism are major problems in both adolescent and adult patients. A total of 32 patients with beta-thalassaemia major undergoing treatment at the Children's Hospital, University of Göttingen were examined. Fourteen of these were short in stature. Growth hormone (GH) secretion was investigated in 13 patients exhibiting either a short stature or reduced growth rate. The stimulated GH secretion of 10 patients in this subgroup lay within the normal range. Studies of their spontaneous GH secretion during the night revealed that these patients had a markedly reduced mean GH and reduced amplitudes in their GH peaks. Low insulin-like growth factor (IGF)-I levels were seen in the growth-retarded thalassaemic patients. Eight were subjected to an IGF generation test and showed a strong increase in both IGF-I and insulin-like growth factor binding protein (IGFBP)-3 levels indicating intact IGF-I generation by the liver. Hypogonadotropic hypogonadism was found to be present in both the male and female patients with impaired sexual development. After priming with LH-releasing hormone (GnRH) per pump in 2 female and 5 male patients, no change in either their serum oestradiol or testosterone levels or in LH/FSH response to GnRH was observed suggesting that they were suffering from a severe pituitary gonadotropin insufficiency. Three male patients at the age of puberty but exhibiting short stature. low GH, low IGF-I and hypogonadism received low dose long-acting testosterone. After 3 12 months of therapy there was a marked growth spurt, higher nocturnal GH levels and an increase in both IGF-I and IGFBP-3. CONCLUSION: Reduced GH secretion and low IGF-I in thalassaemic patients are related to a neurosecretory dysfunction due to iron overload rather than to liver damage. Hypogonadotropic hypogonadism is caused by the selective loss of pituitary gonadotropin function. In patients with both GH deficiency and hypogonadism, low dose sexual steroid treatment should be considered either as an alternative or an additional treatment before starting GH therapy.

Adolescent↗
PubMed1992

In vivo T cell activation, in vitro defective IL-2 secretion, and response to influenza vaccination in elderly women.

IL-2 secretion in response to mitogenic stimulation, assayed in vitro, is significantly reduced in circulating T lymphocytes isolated from healthy old people, but the significance of this abnormality and how it relates to in vivo IL-2 secretion remain unclear. We found that IL-2 secretion in response to PHA plus PMA by peripheral blood T cells isolated from 10 out of 32 (31%) healthy old individuals (mean age 86 yr, range 74-97) was significantly decreased compared with results obtained in 23 younger individuals (mean age 34 yr, range 23-46). This IL-2 secretion defect in vitro was reversible after a 3-day incubation in the absence of activators. The 10 healthy old individuals who had defective IL-2 secretion in vitro also showed increased levels of serum IL-2. T cells from 22 healthy old and 22 young individuals, who had normal IL-2 secretion (geometric mean +/- log of 1 SD: 139 +/- 0.3 U/ml and 212 +/- 0.31 U/ml, respectively) in vitro, showed a remarkable transient T cell defect in IL-2 secretion (15 +/- 0.47 U/ml for the old, 54 +/- 0.28 U/ml for the young) 15 days after influenza vaccination. IL-2 secretion became normal again 30 days after vaccination. The T cell-IL-2 activity, expressed as a T cell-IL-2 activity score (calculated as the logarithm of the serum IL-2 U/ml divided by the logarithm of the IL-2 secretion U/ml, in vitro) was significantly increased in elderly non-responders after influenza vaccination (mean +/- 1 SD: 1.4 +/- 0.51) compared with elderly (0.44 +/- 0.13) and younger responders (0.3 +/- 0.2). Our data suggest that in vitro defective IL-2 secretion is a consequence of T cell activation which seems to occur in a significant proportion of healthy elderly individuals and may be clinically relevant inasmuch as it appears to prevent the normal vaccine-induced antibody response.

Aged↗
PubMed1999

A cyclic nucleotide PDE5 inhibitor corrects defective mucin secretion in submandibular cells containing antibody directed against the cystic fibrosis transmembrane conductance regulator protein.

A selective cyclic nucleotide PDE5 inhibitor corrected the defective mucin secretion response to the beta-agonist isoproterenol in submandibular acinar cells inhibited by antibody directed against the cystic fibrosis transmembrane conductance regulator. The PDE5 inhibitor was as effective as cpt-cyclic AMP or a selective PDE4 inhibitor. However, the PDE5 inhibitor had no effect on basal or isoproterenol-stimulated cyclic AMP levels and did not stimulate mucin secretion. The results showing, for the first time, correction of the CFTR mucin secretion defect by a PDE5 inhibitor, which may involve cyclic GMP, will have a major impact in development of a rational drug treatment for cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed2002

Molecular regulation of Na+-K+-2Cl- cotransporter (NKCC1) and epithelial chloride secretion.

Defective regulation of epithelial secretion underlies the clinical manifestations of a number of important human diseases ranging from cystic fibrosis to secretory diarrhea. Although much attention has focused on the role of apical membrane chloride (Cl-) channels in this process, emerging evidence highlights the importance of a basolateral Na+-K+-2Cl- cotransporter (NKCC1) as an independent regulatory site that may determine the overall rate of epithelial secretion. The cotransporter NKCC1 is expressed not only in epithelial cells but also in virtually all mammalian cells, where it plays a more generalized role in cell volume homeostasis, cell ionic composition, and, possibly, the control of cell growth. Emerging molecular evidence indicates that NKCC1 function is regulated in the short and long term at the level of protein phosphorylation, membrane targeting, and gene expression. An improved understanding of NKCC1 may lead to new therapeutic approaches to secretory diarrhea as well as diverse clinical conditions in which cell ion composition is disturbed.

Animals↗
PubMed1998

Defects in type III secretion correlate with internalization of Pseudomonas aeruginosa by epithelial cells.

Previous characterization of Pseudomonas aeruginosa clinical isolates has demonstrated an inverse correlation between cytotoxicity and internalization by epithelial cells. To further investigate this relationship, we tested PA103, a cytotoxic P. aeruginosa strain, and 33 isogenic noncytotoxic transposon mutants for internalization by Madin-Darby canine kidney cells. The majority of the mutants were not internalized, demonstrating that an inverse correlation between cytotoxicity and bacterial uptake by epithelial cells is not absolute. Six of the noncytotoxic mutants, however, demonstrated measurable levels of internalization by standard aminoglycoside exclusion assays even though internalization of wild-type strain PA103 was not detectable. All six had evidence of protein secretion defects involving two proteins, a 40-kDa protein and a 32-kDa protein. These proteins, designated PepB (for Pseudomonas exoprotein B) and PepD, respectively, each had characteristics of type III transported proteins. In addition, nucleotide sequencing studies demonstrated that PepB and PepD are homologs of YopB and YopD, respectively, type III secreted proteins of Yersinia spp. necessary for the translocation of effector molecules into the cytoplasmic compartment of eukaryotic cells. Thus, while many mutations in PA103 result in loss of cytotoxicity without an appreciable increase in internalization, defects in transport of type III secretion proteins PepB and PepD correlate with both loss of cytotoxicity and gain of internalization. These results are consistent with type III secretion of an inhibitor of internalization that requires PepB and PepD for translocation into the host cell.

Amino Acid Sequence↗
PubMed2003

Rescue of defective pancreatic secretion in cystic-fibrosis cells by suppression of a novel isoform of phospholipase C.

BACKGROUND: Cystic fibrosis is caused by mutations in the gene encoding an ion-transport protein, the cystic-fibrosis transmembrane conductance regulator (CFTR). Defective secretion of anions is the primary cause of many of the clinical manifestations of cystic fibrosis, including pancreatic insufficiency. We aimed to identify a molecular mechanism from which a new method to circumvent defective pancreatic secretion could be derived. METHODS: Multiple-human-tissue RT-PCR and semiquantitative RT-PCR analyses were used to examine gene expression. An antisense technique was used in conjunction with radioimmunoassay, Fura-2 spectrofluorometry, immunohistochemistry, and the short-circuit current technique (Ussing chamber) for elucidation of gene function and its application in rescuing defective pancreatic secretion. FINDINGS: We cloned a newly identified gene, NYD-SP27, which has structural similarity to an isoform of phospholipase C. NYD-SP27 was expressed endogenously in human pancreatic-duct cells and upregulated in cystic fibrosis. Suppression of NYD-SP27, by transfection of its antisense into human cystic-fibrosis pancreatic-duct cells, resulted in augmentation of phospholipase-C-coupled calcium-ion release and protein kinase C activity, improvement in the amount of mutated CFTR reaching the plasma membrane, and restoration of cAMP-activated pancreatic anion secretion. INTERPRETATION: NYD-SP27 exerts an inhibitory effect on phospholipase-C-coupled processes that depend on calcium ions and protein kinase C, including CFTR trafficking and function. Its upregulation in pancreatic-duct cells may reveal a previously unsuspected defect in cystic fibrosis contributing to pancreatic insufficiency, and thus represents a new target for pharmacological intervention in cystic fibrosis.

Antisense Elements (Genetics)↗
PubMed1995

Physiological and biochemical analysis of the effects of alkaline phosphatase overproduction in Escherichia coli.

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase (PhoA), on multicopy plasmids caused a severe defect in the precursor processing (secretion) of PhoA, beta-lactamase, and the outer membrane protein OmpA. This secretion defect continued even after the repression of phoA expression, indicating that protein secretion was irreversibly impaired in cells. Among the secretory proteins, only OmpA gradually secreted posttranslationally. The inverted inner membrane vesicles prepared from cells with the secretion defect showed appreciably reduced translocation activity in vitro. But the membrane vesicles retained the ability to generate a proton motive force which, together with ATP, is essential as an energy source for the efficient secretion of proteins in E. coli. An appreciable amount of incompletely translocated PhoA molecules was detected in the inner membranes of cells with the secretion defect.

Alkaline Phosphatase↗
PubMed2004

Pathophysiology of insulin secretion.

Defects in pancreatic islet beta-cell function play a major role in the development of diabetes mellitus. Type 1 diabetes is caused by a more or less rapid destruction of pancreatic beta cells, and the autoimmune process begins years before the beta-cell destruction becomes complete, thereby providing a window of opportunity for intervention. During the preclinical period and early after diagnosis, much of the insulin deficiency may be the result of functional inhibition of insulin secretion that may be at least partially and transiently reversible. Type 2 diabetes is characterized by a progressive loss of beta-cell function throughout the course of the disease. The pattern of loss is an initial (probably of genetic origin) defect in acute or first-phase insulin secretion, followed by a decreasing maximal capacity of insulin secretion. Last, a defective steady-state and basal insulin secretion develops, leading to almost complete beta-cell failure requiring insulin treatment. Because of the reciprocal relation between insulin secretion and insulin sensitivity, valid representation of beta-cell function requires interpretation of insulin responses in the context of the prevailing degree of insulin sensitivity. This appropriate approach highlights defects in insulin secretion at the various stages of the natural history of type 2 diabetes and already present in individuals at risk to develop the disease. To date none of the available therapies can stop the progressive beta-cell defect and the progression of the metabolic disorder. The better understanding of the pathophysiology of the disease should lead to the development of new strategies to preserve beta-cell function in both type 1 and type 2 diabetes mellitus.

Animals↗
PubMed2001

Isolation and characterization of Escherichia coli tolC mutants defective in secreting enzymatically active alpha-hemolysin.

This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed alpha-helical domain, while the remaining two mapped within the outer membrane-embedded beta-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.

Bacterial Outer Membrane Proteins↗
PubMed1997

Intracellular degradation of secretion defect-type mutants of antithrombin is inhibited by proteasomal inhibitors.

To examine the cellular basis for secretion defect-type antithrombin deficiency, we expressed two mutants, P --> stop (Pro429 to stop codon) and deltaGlu (deletion of Glu313). Pulse-chase experiments using stably transfected BHK cells showed that little (< 5%) of P --> stop mutant as well as deltaGlu mutant was secreted and the total amount of radioactivity was significantly reduced, suggesting an intracellular degradation. The degradation was not inhibited by brefeldin A, indicating it occurring in a preGolgi apparatus. However, the degradation was strongly inhibited by proteasomal inhibitors, such as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL), carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV) and lactacystin. By endoglycosidase H digestion and immunofluorescence staining, these mutants were shown to localize in the endoplasmic reticulum (ER). These results suggest that the secretion defect-type mutants of antithrombin are degraded by proteasome through the ER-associated quality control mechanism in the cells.

Acetylcysteine↗