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PubMed1996

Mutational analysis of the Streptomyces scabies esterase signal peptide.

Ten site-directed mutations affecting the predicted 39-amino-acid signal peptide of the Streptomyces scabies esterase were used to examine start-codon usage and esterase secretion in S. lividans. The first of two in-frame AUG codons was preferred for translation initiation. Removal of 2 of the 4 positively charged amino acids at the amino terminus of the signal peptide reduced esterase expression more than 100-fold; however, deletion of all 4 charged residues reduced expression by only 2- to 5-fold. Deletion of 4 or 8 amino acids from the hydrophobic core of the signal peptide reduced esterase production more than 200-fold, and a signal peptide processing site deletion completely disrupted esterase expression. For all constructs in which a mutation in the signal sequence decreased esterase production, esterase mRNA levels were also reduced, suggesting that a defect in secretion or processing affected esterase transcript abundance.

Amino Acid Sequence↗
PubMed1991

Mutagenesis and isolation of Aeromonas hydrophila genes which are required for extracellular secretion.

Transposon mutagenesis was used to isolate mutants of Aeromonas hydrophila which were deficient in the production of extracellular proteins. The culture supernatants of two of the mutants were essentially devoid of the proteins normally secreted by the parent strain, despite their continued synthesis. Western immunoblot analysis of one of these proteins indicated that normal signal sequence processing occurred but that normal zymogen activation did not, and cell fractionation experiments indicated that both mutants accumulated the three different extracellular proteins assayed in a position external to the cytoplasmic membrane, presumably in the periplasm. The two mutants differed, however, in that one was lysed during the osmotic shock procedures and also contained severely reduced amounts of two of the major protein components of the outer membrane. The wild-type chromosomal regions into which the transposon had been inserted in the two mutants were cloned. In each case, transconjugants of the mutants containing the corresponding cloned fragment were complemented for the defects in secretion, and one of the mutants was complemented by the heterologous clone as well, suggesting the possibility of an interaction between these two genes or gene products. These results indicate that two separate functions which are required for extracellular secretion were interrupted in the insertion mutants and that one of these is also critically important in the biogenesis of the outer membrane.

Aeromonas↗
PubMed1994

Heterotrimeric G proteins, vesicle trafficking, and CFTR Cl- channels.

Previously (E.M. Schwiebert, N. Kizer, D. C. Gruenert, and B. A. Stanton, Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992), we showed that heterotrimeric G proteins regulate adenosine 3',5'-cyclic monophosphate (cAMP)-activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in human airway epithelial cells. The goal of the present study was to test the hypothesis that heterotrimeric G proteins regulate vesicle trafficking and exocytosis and that these events are critical for cAMP activation of CFTR-mediated Cl- secretion. We report that cAMP stimulates exocytosis and CFTR Cl- conductance (GCl) in normal but not in CF cells. Stimulation of the heterotrimeric G protein G alpha i-2 inhibited cAMP-activated CFTR GCl and exocytosis in normal cells. In contrast, inhibition of G alpha i-2 stimulated exocytosis and allowed cAMP to stimulate CFTR GCl in cells isolated from patients with cystic fibrosis (CF). Brefeldin A and nocodazol prevented cAMP-induced exocytosis and also blocked cAMP stimulation of CFTR GCl in normal airway epithelial cells. Our studies suggest that the heterotrimeric G protein G alpha i-2 regulates CFTR GCl in human airway epithelial cells by modulating vesicle trafficking and the delivery of CFTR Cl- channels from an intracellular vesicular pool to the plasma membrane. Inhibition of G alpha i-2 may be a useful therapeutic approach to target mutant delta F508 CFTR Cl- channels from an intracellular vesicular pool to the plasma membrane and thereby correct defective Cl- secretion in CF airway epithelial cells.

Calcium↗
PubMed1995

[Molecular endocrinology of thyroid diseases].

Over the last decade it has become possible to investigate the molecular basis of functional and neoplastic thyroid diseases, leading to the elucidation of various genetic defects at the level of the pituitary, thyroid and target organs. Mutations in either the pituitary-specific transcription factor Pit-1 or its target gene, TSH beta, lead to rare forms of hereditary congenital hypothyroidism. However, somatic mutations in thyroid epithelial cells causing an increase in hormone production and/or cellular proliferation are much more frequent. Nearly 50% of all toxic adenomas were shown to harbour activating mutations in either the TSH-receptor or certain G-proteins. In contrast, follicular and papillary thyroid malignancies are associated with mutations in the ras and ret genes respectively. Intriguingly, different mutations and rearrangements in the ret gene were shown to cause medullary thyroid cancer and MEN2 as well as to be specifically associated with papillary thyroid cancer. In contrast, mutations in thyroid-specific genes, such as thyroid peroxidase and thyroglobulin, causing congenital hypothyroidism are extremely rare. Besides the molecular abnormalities at the pituitary and thyroidal level leading to altered hormone secretion, genetic defects impairing thyroid hormone action at the target level also occur. Specifically, mutations in one of the thyroid hormone receptor genes (the proto-oncogene c-erbA beta) were shown to cause the autosomal dominant syndrome of resistance to thyroid hormone. The quest for a better understanding of the molecular defects in the pituitary-thyroid axis has led to the cloning of some of the key proteins, which can now be used for diagnostic purposes in vivo and in vitro. The use of recombinant thyroid peroxidase and TSH-receptor proteins has made possible the development of more sensitive and specific in vitro assays for autoantibodies. In addition, recombinant TSH was recently shown to be effective in stimulating radioiodine uptake in patients with residual differentiated thyroid cancer who remained on suppressive thyroid hormone therapy. Recombinant human TSH may therefore become a convenient diagnostic tool in the follow-up of patients with thyroid cancer by allowing for thyroglobulin measurements and radioiodine scanning without the need for the patient to become hypothyroid.

DNA-Binding Proteins↗
PubMed1994

A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis.

The causative agent of plague, Yersinia pestis, contains a 75-kb plasmid, pCD1, which carries a virulence-related stimulon called the low-Ca2+ response stimulon (LCRS). LCRS operons are regulated by the environmental signals of temperature and Ca2+. This study characterized a portion of the lcrB region of pCD1, known to contain at least one gene necessary for the regulation of LCRS operons by Ca2+. The sequence of a 2-kb region revealed three open reading frames, designated yscQ, yscR, and yscS, predicted to encode acidic proteins of 34.4, 24.4, and 8.5 kDa. All three proteins were homologous to proteins involved in flagellar function or virulence. An antipeptide antibody specific for YscR was used to localize YscR to the inner membrane of Y. pestis. Analysis of yscR-phoA fusions supported a model for yscR which predicts four transmembrane regions and a large, central hydrophilic domain. In-frame deletion mutations of yscQ and yscR were constructed and moved into Y. pestis. Both mutants failed to show the restriction of growth that normally accompanies maximal LCRS induction. Unlike the parent Y. pestis, the yscR mutant did not respond to the absence of Ca2+ by increasing the net transcription or translation of the LCRS-encoded V antigen, YopM, or LcrG. The yscR mutant also was defective for secretion of V antigen, YopM, and LcrG. These findings implicate a dual role for YscR in regulation of LCRS operons and secretion of LCRS proteins and add to the developing picture of how secretion of virulence proteins may be coupled to transcriptional regulation in yersiniae.

Amino Acid Sequence↗
PubMed2004

Both relative insulin resistance and defective islet beta-cell processing of proinsulin are responsible for transient hyperglycemia in extremely preterm infants.

OBJECTIVE: Many extremely preterm infants develop hyperglycemia in the first week of life during continuous glucose infusion. The objective of this study was to determine whether defective insulin secretion or resistance to insulin was the primary factor involved in transient hyperglycemia of extremely preterm infants. METHODS: A prospective comparative study was conducted in appropriate-for-gestational-age preterm infants <30 weeks of gestational age with the aim specifically to evaluate the serum levels of proinsulin, insulin, and C-peptide secreted during transient hyperglycemia by specific immunoassays. Three groups of infants were investigated hyperglycemic (n = 15) and normoglycemic preterm neonates (n = 12) and normal, term neonates (n = 21). In addition, the changes in beta-cell peptide levels were analyzed during and after intravenous insulin infusion in the hyperglycemic group. Data were analyzed using analysis of variance and analysis of variance for repeated measures. RESULTS: At inclusion, insulin and C-peptide levels did not differ in hyperglycemic subjects and in preterm controls. Proinsulin concentration was significantly higher in the hyperglycemic group (36.5 +/- 3.9 vs 23.2 +/- 0.9 pmol/L). Compared with term neonates, proinsulin and C-peptide levels were higher in normoglycemic preterm infants (23.2 +/- 0.9 vs 18.9 +/- 2.71 pmol/L and 1.67 +/- 0.3 vs 0.62 +/- 0.12 nmol/L, respectively). During and after insulin infusion in hyperglycemic neonates, plasma glucose concentration fell and proinsulin and C-peptide levels were lowered (18.4 +/- 7.6 and 20.7 +/- 4.5 pmol/L, respectively). CONCLUSION: These data suggest that 1) preterm neonates are sensitive to changes in plasma glucose concentration, but proinsulin processing to insulin is partially defective in hyperglycemic preterm neonates; 2) hyperglycemic neonates are relatively resistant to insulin because higher insulin levels are needed to achieve euglycemia in this group compared with normoglycemic neonates. These results also show that insulin infusion is beneficial in extremely preterm infants with transient hyperglycemia.

Blood Glucose↗
PubMed1991

Ion transport abnormalities in rectal suction biopsies from children with cystic fibrosis.

Abnormalities in transepithelial electrolyte transport in cystic fibrosis rectum were analyzed by short-circuit current measurements on 11 control subjects and 11 subjects with cystic fibrosis in a modified Ussing chamber. As judged by the amiloride-sensitive component of the short-circuit current, electrogenic sodium absorption appeared unmodified in cystic fibrosis. In contrast, the short-circuit current response to specific stimuli of both cyclic adenosine monophosphate (cAMP)- and calcium-mediated chloride secretion was drastically altered in all of the cystic fibrosis biopsy specimens examined. Stimulation of the cAMP pathway by 8-bromo cAMP or forskolin resulted in a sustained increase in short-circuit current in control tissues (+ 2.51 +/- 0.63 microA/cm2) but in a slight change in the opposite direction in cystic fibrosis (-0.56 +/- 0.49 microA/cm2; P less than 0.05). Carbachol, a calcium-linked secretagogue, provoked a transient increase in short-circuit current in all of the control tissues (peak response, + 26.69 +/- 3.63 microA/cm2) but a transient change in the opposite direction in 6 of 11 cystic fibrosis tissues (-12.46 +/- 4.64 microA/cm2; P less than 0.05). In 2 of 11 patients with cystic fibrosis, however, a significant but subnormal and transient increase in short-circuit current was observed (+ 2.62 +/- 0.04 microA/cm2; P less than 0.05), whereas in 3 of 11 patients with cystic fibrosis a transient change in the opposite direction (-9.83 +/- 2.20 microA/cm2; P less than 0.05) was followed by a small and transient increase (+ 2.89 +/- 0.83 microA/cm2; P less than 0.05). Using the calcium-mediated secretory response therefore, patients with cystic fibrosis could be divided into two categories: a major population showing defective anion secretion but active cation secretion and a subclass (including three siblings) showing residual but subnormal anion secretion. The easy accessibility of rectal samples and the inversed direction of the cAMP- or calcium-provoked short-circuit current is of considerable advantage in the diagnosis of cystic fibrosis.

Adolescent↗
PubMed1980

Persistent defect in regulation of prolactin secretion after successful pituitary tumor removal in women with the galactorrhea-amenorrhea syndrome.

TRH, metoclopramide (MCP), chlorpromazine (CPZ), and insulin (ITT) stimulation tests of PRL secretion were carried out in age-matched controls and before and after successful removal of pituitary prolactinomas in women with the galactorrhea-amenorrhea syndrome. In preoperative patients there was a blunted or absent PRL response to TRH in 87%, to MCP in 100%, to CPZ in 100%, and to ITT in 93%. Two to 6 months after successful tumor removal, serum PRL rose 2-fold (the usual criterion for a normal response) in 73% after TRH, in 100% after MCP, but in only 13% after CPZ and in only 14% on ITT. However, the PRL increment with all four tests was significantly lower than that in normal controls. One to 8 yr after successful surgery, the PRL increments after TRH and MCP were returning to normal, but the PRL responses to CPZ and ITT remained blunted. GH, ACTH, and TSH reserves were intact in all patients. The diminished PRL response to all stimulation tests observed up to 6 months postoperatively might be explained by the persistence of a negative feedback effect from high PRL levels associated with the tumor. The more persistent impairment of the PRL response to CPZ and ITT is unexplained but suggests a hypothalamic defect.

Amenorrhea↗
PubMed1988

The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process.

The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents. Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids. The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein. To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively. All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity. The most N-terminal fusion joint is at amino acid 19 of Tsr. Tsr-lacZ fusions were found throughout the tsr gene. The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint. Fusions with low activity have fusion joints within the periplasmic loop of Tsr. The expression of these fusions is most likely reduced at the level of translation. In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB. A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature. The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain. We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins. Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.

ATP-Binding Cassette Transporters↗
PubMed1990

The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2.

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.

Blotting, Northern↗
PubMed2005

Plasma leptin and adiposity during antipsychotic treatment of schizophrenia.

Alterations in plasma leptin have been reported in schizophrenia patients treated with antipsychotics, suggesting the hypothesis that impairments in leptin secretion or signaling might play a role in antipsychotic-induced weight gain. Plasma leptin was measured in 72 schizophrenia patients chronically treated with olanzapine (n=27), risperidone (n=24) or typical antipsychotics (n=21) and 124 healthy adult control subjects. ANCOVA was used to test effects of adiposity (body mass index kg/m2; BMI), subject group (treated patients vs untreated controls), and treatment group (specific medication groups and untreated controls) on plasma leptin concentrations. Additional analyses were performed in a subset of patients and controls individually matched for BMI to further assess group differences in plasma leptin independent of adiposity. BMI strongly predicted plasma leptin concentrations in the overall sample. In addition, a significant three-way interaction between BMI, subject group, and gender was observed. In the individually BMI-matched sample, modestly reduced plasma leptin levels (effect size 0.4 SD) were observed in treated patients in comparison to the BMI-matched healthy controls, with both groups including males and females. However, no differences in plasma leptin levels were observed in the matched sample when separately comparing male patients vs untreated male controls and female patients vs untreated female controls. Plasma leptin in chronically treated patients with schizophrenia is strongly predicted by adiposity, similar to untreated healthy individuals despite adequate power to detect a difference. The results argue against a role for defective leptin secretion or sensitivity in the weight gain induced by antipsychotic medications.

Adipose Tissue↗
PubMed1996

Lipid processing and lipoprotein synthesis by the developing human fetal colon.

Despite significant progress in the elucidation of the ontogeny of gastrointestinal function, little attention has been given to colonic lipid processing during development. The major purpose of this study was to explore the intracellular phase of fat absorption, lipid synthesis, and secretion in the human fetal colon compared with the jejunum originating from the same fetuses. The synthesis of lipids and major apolipoproteins was examined using cultured fetal colonic explants incubated with [14C]oleic acid and [36S]methionine, respectively. Fetal colonic explants demonstrated substantial ability to incorporate [14C]oleic acid (dpm/mg protein) into phospholipids (48,743 +/- 4,783), triglycerides (25,687 +/- 2,469), and cholesteryl esters (6,751 +/- 1,227). The total amount of radiolabeled lipids was much higher within the tissue (87,472 +/- 9,142) than in the medium (51,916 +/- 4,970), indicating a limited capacity of the fetal colon to export newly synthesized lipids. The limited colonic lipid secretory process was even more evident when compared with homologous fetal jejunal de novo synthesized lipids in tissue (133,975 +/- 13,836) and medium (279,858 +/- 1,610), respectively. Similar to the jejunum, the colon was able to elaborate all the phospholipid classes, with phosphatidylcholine accounting for > 70% of tissue phospholipids. However, their individual levels were present in lesser amounts in the colon (P < 0.001). Colonic explants elaborated most of the major lipoprotein classes but were less efficient than jejunal explants in exporting chylomicrons (33-fold), very low density lipoprotein (1.5-fold), and high-density lipoprotein (9-fold) into the medium. Apolipoprotein (apo) B synthesis and apo B mRNA editing were comparable in colonic and jejunal explants; thus they are not responsible for the defective lipoprotein secretion in the fetal large bowel. These results establish for the first time te capability of the human fetal colon to form, but not to efficiently transport, lipids, lipoproteins, and apoproteins.

Apolipoproteins B↗
PubMed1995

Mitochondrial deoxyribonucleic acid content is specifically decreased in adult, but not fetal, pancreatic islets of the Goto-Kakizaki rat, a genetic model of noninsulin-dependent diabetes.

Considerable interest has recently been focused on the putative role of mutations in the mitochondrial genome for the development of noninsulin-dependent diabetes. The Goto-Kakizaki (GK) rat, a genetic model of defective insulin secretion and hyperglycemia, is characterized by partial maternal inheritance. Because the mitochondrial genome is known to be maternally transmitted, the aim of this study was to investigate whether the GK syndrome can be explained in terms of alterations of the mitochondrial DNA (mtDNA). For this purpose, pancreatic islets were isolated from adult and fetal control Wistar and diabetic GK rats. Using electron microscopy, the ultrastructural morphology of beta-cell mitochondria was analyzed in control and GK islets. It was found that the beta-cells of adult GK rats had a significantly smaller mitochondrial volume and an increased number of mitochondria per unit tissue volume as compared with the beta-cells of corresponding control islets. Moreover, mtDNA and mtRNA were isolated from the islets and, as a control tissue, from liver, and subsequently analyzed using Southern and Northern blot techniques. No major deletions or restriction fragment polymorphism could be detected in mtDNA from both GK liver and GK islets. The mtDNA sequence of the transfer RNAleu(UUS) gene was identical in both strains of rats. mtDNA contents of fetal GK islets and fetal GK liver were not different from those of fetal Wistar rats. However, adult GK islets contained markedly less mtDNA than the corresponding control islets, contrary to the mtDNA contents of adult liver, which were similar in the two strains. The lower islet mtDNA contents were paralleled by a decreased content of islet mtRNA (12S ribosomal RNA and cytochrome b messenger RNA). Islet insulin messenger RNA contents were similar in GK and Wistar rats. In conclusion, our results do not support a role of a genetic defect in mtDNA as a cause of the GK syndrome. Instead, mtDNA damage may occur specifically in islet cells as a consequence of the disturbed metabolic environment of the adult GK rat. It is speculated that a long-lasting metabolic dysfunction may induce mtDNA damage and/or inhibition of mtDNA replication leading to a gradual and late decrease in the mitochondrial volume fraction and subsequently an impaired capacity for oxidative metabolism.

Animals↗
PubMed1999

The effect of topical benzamil and amiloride on nasal potential difference in cystic fibrosis.

The electrochemical defect in the bronchial epithelium in cystic fibrosis (CF) consists of defective chloride secretion and excessive sodium reabsorption. The sodium channel blocker, amiloride, has been shown to reversibly correct the sodium reabsorption in CF subjects, but long term studies of amiloride have been disappointing due to its short duration of action. Benzamil, a benzyl substituted amiloride analogue, has a longer duration of action than amiloride in cultured human nasal epithelium. The results of the first randomized, placebo controlled, double blind, crossover study are reported here comparing the effects of benzamil and amiloride on nasal potential difference (nasal PD) in CF. Ten adults with CF attended on three occasions. At each visit baseline nasal PD was recorded, the drug (amiloride 1 x 10(-3) M, benzamil 1.7 x 10(-3) M, or 0.9% sodium chloride) was administered topically via a nasal spray, and nasal PD was measured at 15, 30 min, 1, 2, 4 and 8 h. Results were expressed as maximum change in nasal PD from baseline (PDmax), time for PDmax to return to 50% of baseline (t0.5), and the area under the curve (AUC). PDmax values for benzamil (20.6+/-0.9 mV) and amiloride (20.3+/-1.6 mV), were similar. The duration of effect was much longer for benzamil as measured as either AUC or t0.5 AUC values were 11.8+/-1.6 mV for benzamil, 2.8+/-0.4 mV for amiloride and 0.6+/-0.4 mV for placebo. The AUC value for benzamil was significantly greater than amiloride (95% confidence interval (CI) for the difference 5.3-12.7 mV, p<0.0001). t0.5 values were 4.3+/-0.7 h for benzamil and 0.6+/-0.1 h for amiloride (95% CI for the difference 2.0-5.3 h, p<0.001). It is concluded that benzamil has a similar maximal effect to amiloride but a more prolonged duration of action on nasal potential difference in cystic fibrosis. Benzamil may be a useful sodium channel blocker for the long-term treatment of the biochemical defect in the lungs of patients with cystic fibrosis.

Administration, Intranasal↗
PubMed2002

Anx7 is required for nutritional control of gene expression in mouse pancreatic islets of Langerhans.

BACKGROUND: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. MATERIALS AND METHODS: To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. RESULTS: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control islets that are principally associated with cell division and DNA repair. The latter genes have not specifically been associated with islet physiology in the past. By contrast, Anx7(+/-) mouse islets exhibit a greatly reduced ability to discriminate genomically between fed and fasted states for all classes of identified genes. Many of the validated genes are specific to islets in comparison to liver tissue examined. Real-time quantitative RT-PCR analysis of islets from Anx7 heterozygous mice and littermate controls revealed remarkable down-regulation in PTEN, Glut-2, PDX-1, IGF-1, and Neuro D1 expression, but not in liver. CONCLUSIONS: We conclude that reduced gene dosage in the Anx7(+/-) islet, with concomitant loss of ITPR3 expression and consequent defects in Ca(2+) signaling, may substantially contribute to the mechanism of the loss of genomic discrimination, in vivo, between the fed and fasted states. We believe that the requirement for complete Anx7 gene dosage and IPTR3 expression in islets of Langerhans will prove to be of fundamental importance for understanding the mechanism of nutritional sensing in health and disease.

Algorithms↗
PubMed1977

The contribution of hyperglycemic hormones to the pathogenesis of diabetes mellitus.

Changes in cortisol, growth hormone, and glucagon levels observed in diabetes mellitus appear to be secondary to insulin deficiency, and can be related to the severity of insulinopenia with its attendant metabolic sequelae. Similarly, disturbances in plasma concentrations of catecholamines in diabetes also appear to be secondary to insulin deficiency, although a primary disturbance in adrenergic function or receptors at the cellular level cannot be excluded. As "inappropriate" compensatory responses, these hormones may aggravate the diabetic syndrome, but their dysfunction is not the cause of diabetes and cannot be used to identify prediabetes. To date, the primary hormonal disturbance in insulin-dependent diabetes remains defective insulin secretion.

Diabetes Mellitus↗
PubMed1993

Intestinal electrolyte secretion. History of a paradigm.

The contemporary paradigm for active chloride secretion by vertebrate epithelial cells evolved, at least in part, from experiments that began in the laboratory of Dr William Silen at Beth Israel Hospital in Boston, Mass. It was first shown there that cyclic adenosine monophosphate and cholera toxin stimulate active chloride secretion when added to intestinal mucosa in vitro. The paradigm, which evolved further from experiments on shark rectal gland and flounder intestine at the Mt Desert Island Biological Laboratory in Salsbury Cove, Maine, is as follows: Chloride enters some epithelial cells by sodium-potassium-chloride cotransport with a stoichiometry of 1:1:2 and accumulates intracellularly because of the sodium gradient maintained by sodium-potassium-adenosinetriphosphatase in the basolateral membrane; the chloride is then released from the cell through chloride channels in the membrane opposite that of the cotransporter. If the cotransporter is basolateral and the channel is apical, chloride is secreted; if it is the other way around, chloride is absorbed. In a number of secretory epithelial cells, cyclic adenosine monophosphate activates these channels, thereby initiating secretion. A defect in the activation of these channels by cyclic adenosine monophosphate is the root cause of cystic fibrosis.

Animals↗
PubMed2000

Monoclonal antibodies specific for human tumor-associated antigen 90K/Mac-2 binding protein: tools to examine protein conformation and function.

As part of our effort to identify glycoproteins that contribute to colon cancer progression, we have previously described a family of structurally related glycoproteins expressing beta1-6 branched asparagine(Asn)-linked oligosaccharides defined by monoclonal antibody (MAb 1H9), which are differentially expressed, processed, and glycosylated by human colon carcinoma cell lines (Laferté and Loh [1992]; Biochem J; 283:193-201). MAb 1H9 immunoprecipitates three glycoproteins having apparent sizes of 92-100, 66-70, and 25 kDa, the size heterogeneity attributable to cell-type specific glycosylation differences. We report on the basis of partial protein and cDNA sequence information, that the 100-kDa glycoprotein detected by MAb 1H9 is identical to the 90-kDa glycoprotein variably known as tumor-associated antigen 90K (TAA90K), Mac-2 binding protein, and cyclophilin C-associated protein. Using a PCR-based cloning strategy, the complete cDNA encoding TAA90K was cloned into the eukaryotic expression vector pCDNA-3 (pCD-TAA90K(wt)) and the protein expressed in COS-1 cells. A [(35)S]methionine-labeled 60-kDa polypeptide, processed to an endoglycosidase H-sensitive 74-kDa glycoprotein in the presence of dog pancreas microsomes, was detected in a coupled transcription/translation in vitro reaction. The in vitro-translated 60-kDa polypeptide and N-glycanase-treated TAA90K (60-kDa species) immunoprecipitated from HT29 cells were shown to be structurally identical by limited proteolytic peptide mapping. Using a new panel of 11 TAA90K-specific monoclonal antibodies, including five specific for human TAA90K and six cross-reactive with a 90-kDa species expressed by COS-1 cells, we have detected conformational differences between recombinant wild-type TAA90K, in vitro-synthesized TAA90K, and mutant forms of TAA90K containing point mutations at residues 189, 223, and 259. Furthermore, we have shown that these mutant forms of TAA90K, as well as a truncated form of TAA90K containing amino acid residues 1-383, are defective in secretion. These studies demonstrate the potential usefulness of TAA90K-specific monoclonal antibodies for examining the structure and function of TAA90K, and highlight the contribution of specific amino acid residues to its normal processing and secretion.

3T3 Cells↗