New concepts in understanding the pathophysiology of chronic pancreatitis.
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Calcium-Binding Proteins↗
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Neurodegeneration changes in primary central nervous system neurons transfected with the Alzheimer-associated neuronal thread protein gene.
The AD7c-NTP gene is over-expressed in brains with Alzheimer's disease (AD), and increased levels of the corresponding protein are detectable in cortical neurons, brain tissue extracts, cerebrospinal fluid, and urine beginning early in the course of AD neurodegeneration. In the present study, we utilized a novel method to transfect post-mitotic primary neuronal cell cultures, and demonstrated that over-expression of the AD7c-NTP gene causes cell death and neuritic sprouting, two prominent abnormalities associated with AD. These results provide further evidence that aberrantly increased AD7c-NTP expression may have a role in AD-type neurodegeneration. In addition, we demonstrate that primary post-mitotic neurons can be efficiently transfected with conventional recombinant plasmid DNA to evaluate the effects of gene over-expression in relevant in vitro models.
Alzheimer Disease↗
Neuronal thread protein regulation and interaction with microtubule-associated proteins in SH-Sy5y neuronal cells.
In Alzheimer's disease (AD), neuronal thread protein (NTP) accumulates in cortical neurons and colocalizes with phospho- tau-immunoreactive cytoskeletal lesions that correlate with dementia. To generate additional information about the potential role of NTP in AD, we characterized its expression and regulation in human SH-Sy5y neuronal cells. Quantitative real-time reverse transcription-polymerase chain reactin and Western blot analysis demonstrated prominent insulin, moderate insulin-like growth factor, type 1 (IGF-1) and minimal nerve growth factor stimulation of NTP expression. In addition, NTP protein was more stable and it progressively accumulated in cells that were stimulated with insulin for 24 or 48 h. Metabolic labeling and phospho-amino acid analysis demonstrated phosphorylation of NTP on Serine residues, 30-60 min after insulin or IGF-1 stimulation, when glycogen synthase kinase 3beta (GSK-3beta) activity would no longer have been suppressed. Kinase inhibitor and in vitro phosphorylation studies demonstrated a role for GSK-3beta in the positive regulation of NTP expression and phosphorylation. Coimmunoprecipitation studies demonstrated physical interactions between NTP and tau or microtubule-associated protein 1b (MAP-1b), and ubiquitin immunoreactivity in NTP immunoprecipitates. In summary, these studies showed that (i) NTP expression is regulated at the level of transcription by insulin and IGF-1 stimulation; (ii) NTP is phosphorylated by GSK-3beta; (iii) NTP can physically interact with tau and MAP-1b and (iv) NTP-MAP complexes are ubiquitinated. The results suggest a functional role for NTP in relation to the turnover or processing of neuronal cytoskeletal proteins, attributes that may be modulated by insulin/IGF-1-mediated signaling.
Antibodies, Monoclonal↗
Cyclic ADP-ribose-mediated insulin secretion and Reg, regenerating gene.
Glucose is the primary stimulus of insulin secretion in pancreatic beta-cells of the islets of Langerhans. CD38 has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose from NAD+, and cyclic ADP-ribose hydrolase, which converts cyclic ADP-ribose to ADP-ribose. ATP, produced by glucose metabolism, inhibits the cyclic ADP-ribose hydrolase of CD38 and therefore causes cyclic ADP-ribose accumulation in beta-cells. Then, cyclic ADP-ribose acts as a second messenger for Ca2+ mobilization from the endoplasmic reticulum to secrete insulin. The mechanism of insulin secretion as described above is completely different from the conventional hypothesis in which Ca2+ influx from extracellular sources was assumed to play a role in insulin secretion by glucose. On the other hand, strategies for influencing the replication of islet beta-cells and the growth of the beta-cell mass may be more important for ameliorating diabetes. Reg, regenerating gene, is involved in the growth of the beta-cell mass, and Reg protein has been shown to increase the beta-cell mass in a 90% depancreatized diabetic rat model, thereby ameliorating the diabetes. CD38 is involved in the formation of cyclic ADP-ribose and is essential for the glucose sensitivity of beta-cells for insulin secretion. Therefore, CD38 gene and Reg gene will become targets for genetic engineering for diabetic beta-cells.
ADP-ribosyl Cyclase↗
Reg (regenerating) gene overexpression in islets from non-obese diabetic mice with accelerated diabetes: role of IFNbeta.
AIMS/HYPOTHESIS: The expression of IFNbeta in beta cells results in accelerated type 1 diabetes. The REG family of beta cell proliferation factors have been described as autoantigens in autoimmune diabetes. The aim of this study was to determine the effect of IFNbeta on Reg expression, and the implications of this in terms of autoimmunity. METHODS: Reg gene expression was determined in islets from non-obese diabetic (NOD) RIP-HuIFNbeta mice by cDNA microarray, quantitative real-time PCR and immunohistochemistry. The effect of IFNbeta on Reg1 and Reg2 expression was assessed in the NOD insulinoma cell line NIT-1. IL-6, known to induce Reg expression, was measured in the insulitis microenvironment. Morphological studies were carried out to determine islet enlargement in this model. RESULTS: Reg2 was upregulated in islets from the NOD RIP-HuIFNbeta mice at the onset of the autoimmune attack. IFNbeta upregulates Reg1 and Reg2 genes in NIT-1 cells. The expression of Il6 was increased in islets from transgenic mice and in NIT-1 cells exposed to HuIFNbeta. Moreover, islets from transgenic mice were enlarged compared with those from wild-type mice. CONCLUSIONS/INTERPRETATION: Reg overexpression correlates well with the acceleration of diabetes in this model. The upregulation of Reg suggests that islets try to improve hyperglycaemia by regenerating the cells lost in the autoimmune attack. Reg expression is regulated by several factors such as inflammation. Therefore, the overexpression of an IFNbeta-induced autoantigen (REG) in the islets during inflammation might contribute to the premature onset of diabetes.
Animals↗
The Reg gene family and Reg proteins: with special attention to the regeneration of pancreatic beta-cells.
Pancreatic beta-cells of the islets of Langerhans are the only cells that produce insulin in humans, as well as in most animals, but they have been thought to have a limited capacity for regeneration, which is a predisposing factor for the development of diabetes mellitus. Strategies for influencing the replication and growth of the beta-cell mass are therefore important for the prevention and/or treatment of diabetes. We have established a model for islet regeneration in 90% depancreatized rats by the administration of poly(ADP-ribose) synthetase inhibitors such as nicotinamide. The regenerating islets in the remaining pancreas of poly(ADP-ribose) synthetase inhibitor-treated rats were markedly enlarged and consisted largely of insulin-producing beta-cells, preventing the development of diabetes mellitus that would otherwise be caused by the 90% pancreatectomy. In screening the regenerating islet-derived cDNA library, we found a novel gene and named it Reg (i.e., regenerating gene). The rate Reg cDNA had a single open reading frame that encoded a 165-amino acid protein with a 21-amino acid signal peptide. We also isolated the human REG cDNA which encoded a 166-amino acid protein with a 22-amino acid signal peptide. The amino acid sequence of human REG protein has 68% homology to that of rat Reg protein. Recombinant rat Reg protein without the signal peptide, produced in yeast, stimulated beta-cell replication and increased the beta-cell mass in the residual pancreas of 90% depancreatized rats, ameliorating the surgical diabetes. Recombinant human REG protein also induced an expansion of the beta-cell mass in non-obese diabetic (NOD) mice, resulting in the amelioration of diabetes. These results, as well as several other lines of evidence, indicate that Reg protein is a growth factor for pancreatic beta-cells and also suggest that the administration of Reg protein and/or activation of the Reg gene can be used as a potential therapeutic approach for diabetes mellitus. We have further isolated several Reg and Reg-related genes from human, rat and mouse, and revealed that they constitute a multigene family, the Reg gene family. Based on the primary structures of proteins encoded by the Reg gene family, we have grouped the members of the family into three subclasses, type I, II, and III. Type I Reg (Reg I) encodes a beta-cell growth factor, Reg I protein, as mentioned above. Some of the type III Reg (Reg III) have recently been suggested to play roles in the regeneration of cells other than pancreatic beta-cells, such as neuronal cells and epithelial cells in the alimentary tract.
Amino Acid Sequence↗
Activated pancreatic enzyme and pancreatic stone protein (PSP/reg) in bile of patients with pancreaticobiliary maljunction/ choledochal cysts.
Symptoms of pancreaticobiliary maljunction/choledochal cysts are caused by the obstruction of bile and pancreatic ducts due to protein plugs compacted in the common channel. However, the mechanism of protein plug formation remains unknown. Pancreatic stone protein (PSP) is reported to be a key protein to form protein plugs in chronic pancreatitis. Bile from 13 patients with pancreaticobiliary maljunction and bile from two normal controls were analyzed. Activity of pancreatic enzymes and the concentration of PSP were measured. The mean concentrations of PSP were 76.9+/-30.9 ng/mL in the bile-duct bile, and 76.9+/-29.8 ng/mL in the gallbladder bile. PSP was not detected in the controls (P < 0.05). In the bile-duct bile of the patients, trypsin(ogen) was detectable in nine patients, of which seven patients had activated trypsin. In the gallbladder bile, trypsin(ogen) was detectable in 12 patients, of which 9 patients had activated trypsin. Neither activated trypsin nor trypsinogen was detected in the controls. Bile in pancreaticobiliary maljunction patients contained both activated trypsin and PSP. Activated trypsin cleaves soluble PSP and creates insoluble PSP. Protein plugs in pancreaticobiliary maljunction may be formed by assembled insoluble PSP.
Amylases↗
Pancreatic stone protein, a phosphoprotein which inhibits calcium carbonate precipitation from human pancreatic juice.
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Calcium Carbonate↗
Cleavage of the Arg-Ile bond in the native polypeptide chain of human pancreatic stone protein.
The pancreatic stone protein (PSP) isolated from calculi (Mr 14,000) and the 5 protein forms (PSP S1-5) detected in pancreatic juice (Mr 14,000-19,000) derive from the same source differing seemingly in their carbohydrate contents or/and in their polypeptidic chain lengths. This kind of protein would inhibit in vivo CaCO3-crystal growth in pancreatic juice. PSP and PSP S1 N-terminal sequences are identical (NH2Ile-). This report demonstrates that: in PSP S2-5 the amino-terminal is blocked; the C-terminus is alike in every form; the single polypeptide chain of PSP S2-5 is converted into that of PSP S1 or PSP by the specific trypsin cleavage of the Arg-Ile bond.
Amino Acid Sequence↗
The amino-terminal domain of thrombomodulin and pancreatic stone protein are homologous with lectins.
Amino acid sequence alignment of the amino-terminal of thrombomodulin and pancreatic stone protein with the hepatic asialoglycoprotein receptor shows that these proteins are homologous. From the known disulfide bridge pattern of other proteins belonging to the same family two disulfide bonds can be predicted. The homology raises the question whether the amino-terminal part of thrombomodulin and the pancreatic protein binds carbohydrate or perhaps like tetranectin have a specific affinity for other proteins.
Amino Acid Sequence↗
The disulfide bridges of the immunoreactive forms of human pancreatic stone protein isolated from pancreatic juice.
Following the complete sequence elucidation of human pancreatic stone protein (immunoreactive form PSP S1 isolated from pancreatic juice) [(1987) Eur. J. Biochem. 168, 201-207], the location of the three S-S bridges of the protein was investigated. The cystine-containing peptides, detected after the separation of the peptic or chymotryptic digests on SP-Sephadex or Sephadex G-50, were submitted to Edman degradation and/or to oxidation. The cysteic peptides after separation on SP-Sephadex or Sephadex G-50 were characterized by their amino acid compositions. The pairing of the half-cystines: Cys 3-Cys 14, Cys 31-Cys 129 and Cys 104-Cys 121 was determined. The same experiments carried out with PSP S2-5 (other immunoreactive forms) gave an identical characterization.
Amino Acid Sequence↗
Remarks on the article 'Cleavage of the Arg-Ile bond in the polypeptide chain of human pancreatic stone protein' by P. Rouimi, J. Bonicel, M. Rovery and A. De Caro.
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Arginine↗
Isolation and characterization of human reg protein produced in Saccharomyces cerevisiae.
reg was originally identified as a gene expressed during the regeneration of insulin-producing pancreatic beta-cells of the rat. We built an expression vector containing human reg cDNA to drive Saccharomyces cerevisiae to synthesize the reg protein, and purified it from the culture medium. The 144-amino acid sequence of the recombinant protein was consistent with that deduced from the cDNA and genomic DNA sequence except that the signal sequence of 22 amino acids was eliminated, and the amino-terminal residue of the protein was pyroglutamic acid. The secondary structure of the reg protein was predicted by determination of the intramolecular cystine linkage and of alpha-helix and beta-sheet contents.
Amino Acid Sequence↗
A gene homologous to the reg gene is expressed in the human pancreas.
We have determined the nucleotide sequence of regl a human genomic DNA fragment homologous to the reg gene which is expressed in the exocrine pancreas and regenerating islets. Sequence comparisons of reg and regl suggested similar exon-intron organisation. Based on this assumption, specific oligonucleotides for regl exons were used to demonstrate expression of the regl gene in pancreas and liver. The proteins encoded by reg and regl comprise 166 amino acids and differ by 22 amino acids only.
Amino Acid Sequence↗
Human REG family genes are tandemly ordered in a 95-kilobase region of chromosome 2p12.
Reg, first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet beta-cells. Recently, it has been revealed that Reg and Reg-related genes constitute a multigene family, the Reg family. In human, the four REG family genes, i.e., REG 1 alpha, REG 1 beta, REG-related sequence (RS) and HIP/PAP, have so far been isolated. In this study, we analyzed YAC clones containing the four genes and performed two-color FISH to determine the map order of the genes. The human REG family genes are tandemly ordered in the 95-kbp DNA region of chromosome 2p12 as follows: 2cen-HIP/PAP-RS-REG I alpha-REG I beta-ptel.
Acute-Phase Proteins↗
Pancreatic stone protein. I. Evidence that it is encoded by a pancreatic messenger ribonucleic acid.
We have previously shown that the pancreatic stone protein (PSP) is an inhibitor of calcium carbonate crystal growth and may participate in the stabilization of the normally supersaturated pancreatic juice. Our aim in this study was to determine if PSP is a normal secretory product of the human pancreas by determining if the normal human pancreas contains a messenger RNA coding for PSP. Human pancreatic messenger RNAs were used to direct protein synthesis in a cell-free translation system. Immunoprecipitation of translation products with a monospecific antibody directed against the PSP yielded a product migrating as a single homogeneous band on sodium dodecyl sulfate-polyacrylamide gels. This product has a molecular weight of 16,000, the value expected for pre-PSP. Products selected by immunoprecipitation with antitrypsin-1 antibodies also migrated as a single band, with a molecular weight of 27,000. It is concluded that a messenger RNA coding for pre-PSP, distinct from the messenger RNA coding for pretrypsinogen, is present in human pancreas. These results support the hypothesis that PSP is a molecular entity, and not a degradation product of trypsinogen 1 or another pancreatic protein.
Animals↗
Pancreatic stone protein. II. Implication in stone formation during the course of chronic calcifying pancreatitis.
Pancreatic stone protein, a novel protein isolated from pancreatic stones of patients suffering from chronic calcifying pancreatitis and secreted in normal human pancreatic juice, was measured by radial immunodiffusion in pure pancreatic juice. Patients with chronic calcifying pancreatitis of different etiologies had significantly lower levels of pancreatic stone protein when compared with other pancreatic diseases and controls. Pancreatic stone protein suppresses in vitro calcium carbonate precipitation and therefore stabilizes normally supersaturated pancreatic juice. The decreased pancreatic stone protein levels observed could be a key factor in the growth of calcium carbonate crystals and stone development during the course of chronic calcifying pancreatitis.
Adolescent↗
Immunochemical characterization and quantitative distribution of pancreatic stone protein in sera and pancreatic secretions in pancreatic disorders.
A fluorometric immunoassay has been established to quantitate pancreatic stone protein providing a sensitivity for concentrations from 0.015 to 0.5 micrograms/mL. When concentrations of pancreatic stone protein were determined from pancreatic secretions obtained either from patients suffering from chronic pancreatitis (n = 31) [including the calcifying forms (n = 10)], pancreatic cancer (n = 22), or nonpancreatic diseases (n = 17), no significant differences were found. In contrast, increased concentrations were found in serum samples from patients with chronic (39/66) and acute pancreatitis (16/20) compared with control patients. The differences between these diagnostic groups and controls were highly significant (P less than 0.0001) and independent of pancreatic enzyme activity. Immunochemical analyses of serum pancreatic stone protein showed an isoelectric point (pH 9) similar to that reported for the pancreatic thread protein. With respect to recent communications, these data do not support the etiopathogenic role postulated for pancreatic stone protein in chronic pancreatitis and chronic calcifying pancreatitis by other investigators.
Adult↗