Neogenesis of islet cells.
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Antigens, Neoplasm↗
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Expression of human regenerating gene mRNA and its product in normal and neoplastic human pancreas.
BACKGROUND: Localization of human regenerating gene (reg) mRNA and its product was investigated in normal and neoplastic human pancreas with the in situ hybridization method and immunohistochemical studies. METHODS: Both reg mRNA and reg protein were observed in acinar cells of the pancreas, but neither was found in ductal or islet cells. Immunoreactive reg was observed in an acinar cell carcinoma, a pancreatoblastoma, a solid and cystic tumor, and 5 of 20 duct cell carcinomas, but it was not found in 15 endocrine tumors and 2 microcystic adenomas. RESULTS: For comparison with reg, alpha-1-antitrypsin (AAT), lysozyme, chromogranin A (CMG), CA 19-9, carcinoembryonic antigen (CEA), cytokeratin, vimentin, and alpha-fetoprotein (AFP) were assessed in those tumors. An acinar cell carcinoma and a pancreatoblastoma had positive results for AAT, lysozyme, cytokeratin, and AFP but negative results for vimentin. An acinar cell carcinoma showed cells focally immunoreactive for CMG and CEA. A solid and cystic tumor had strongly positive results for AAT and vimentin and focally positive results for CMG and pancreatic hormones. Microcystic adenomas had abundant glycogen and strong immunoreactivity for cytokeratin. Ductal cell carcinomas showed cells focally positive for AAT, lysozyme, CMG, CA 19-9, and CEA. CONCLUSIONS: The localization of reg protein was not consist with that of any other proteins examined in the current study. Thus, reg protein was considered a useful marker for acinar cell differentiation; however, ectopic expression of reg also was observed in ductal cell carcinomas. In ductal cell carcinomas, expression of reg immunoreactivity was considered as one of phenotypic heterogeneity, as seen in AAT, lysozyme, and CMG immunoreactivity.
Calcium-Binding Proteins↗
REG gene expression is associated with the infiltrating growth of gastric carcinoma.
BACKGROUND: The Reg gene is known to be involved in the growth of not only pancreatic B-cells, but also epithelial cells of the gastrointestinal tract and carcinoma of its lineage. METHODS: Because, to the authors' knowledge, no studies have been reported regarding REG expression in gastric carcinoma, the authors investigated REG mRNA and REG protein expression using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemical study and correlated the results with the clinical features of gastric carcinoma. RESULTS: Using RT-PCR and Western blot analyses, reg mRNA and 16-kilodalton REG proteins were detected in two of eight human gastric carcinoma cell lines. Cytoplasmic localization of REG proteins in the cell lines was confirmed by fluorescent immunocytochemistry. The RT-PCR analysis revealed the presence of REG mRNA in as many as 77% (87 of 112 tumors) of primary gastric carcinoma tumors. Screening of a total of 195 patients with primary gastric carcinoma using immunoperoxidase staining revealed positive REG immunoreactivity in 60 of the 195 primary tumors (31%). REG expression in infiltrating tumors was found to be significantly higher compared with localized tumors (P < 0.05). Strong REG expression was noted in the cytoplasm of signet ring cell carcinoma tumors at a significantly higher incidence than in nonsignet ring cell tumors. Moreover, patients with REG-negative differentiated adenocarcinoma were found to have a significantly better prognosis compared with patients with REG-positive tumors. The incidence of venous invasion of REG-positive tumors was significantly higher than that of REG-negative tumors. CONCLUSIONS: The results of the current study suggest that the expression of the REG gene is closely related to the infiltrating property of gastric carcinoma, and may be a prognostic indicator of differentiated adenocarcinoma of the stomach.
Adenocarcinoma↗
Expression of regenerating gene I in gastric adenocarcinomas: correlation with tumor differentiation status and patient survival.
BACKGROUND: Regenerating gene I (REG) was identified as a growth factor for pancreatic islet beta cells. Enhanced REG expression was observed during the healing of gastric mucosa. REG expression was observed in various tumors including gastric carcinoma, but to the authors' knowledge, the correlation between REG expression and clinicopathologic characteristics and patient prognosis have not been evaluated. METHODS: The REG messenger RNA level was analyzed by Northern blot analysis and localization was performed by in situ hybridization and immunohistochemistry in gastric adenocarcinoma specimens. The correlations between REG expression and clinicopathologic features and survival of the patients were analyzed. RESULTS: Of the 68 patients studied, 24 (35%) were positive for REG. There was a significant consistency in the intensity and localization of REG transcript and protein expressions. REG expression was enhanced in advanced T classification tumors and in tumors that were not well differentiated. A significant number of metastatic lymph nodes were present in REG-positive tumors. Overall and disease-free survival were found to be poor for patients with REG-positive tumors. REG expression was reported to be an independent predictor of overall patient survival. CONCLUSIONS: Determination of REG expression may help to identify aggressive gastric tumors and to tailor appropriate therapy for patients with REG-positive tumors.
Adenocarcinoma↗
Reg IV, a new member of the regenerating gene family, is overexpressed in colorectal carcinomas.
A better understanding of the mechanisms by which colon tumor cells are able to survive exposure to drugs would be valuable for the development of new therapeutic strategies. We used differential display-PCR to compare gene expression in the drug-sensitive HT-29 colon cancer cell line and 3 drug-resistant subpopulations derived from this parental cell line. One of the genes identified is a new gene, Regenerating IV gene (Reg IV), and was strongly overexpressed in HT-29 drug-resistant cells. Other drug-resistant cell lines expressed Reg IV at a high level, whereas a low expression was noted in sensitive cell lines. Northern blot and real-time PCR analysis showed that Reg IV is more strongly expressed in 71% of colorectal tumors (in particular in mucinous carcinomas) than in normal colon tissues. The comparison of Reg IV expression with that of other REG genes, Regenerating Ialpha or (Reg Ialpha), Regenerating Ibeta (Reg Ibeta) and Pancreatitis-associated protein (PAP), highlights its predominant expression in colorectal tumors. Reg IV mRNA-positive tumor cells display different phenotypes: mucus-secreting, enterocyte-like or undifferentiated. Interestingly, whereas Reg IV expression is low in normal colon, its level in normal small intestine is similar to that in some colorectal tumors. In normal tissue, Reg IV mRNA-positive cells are mostly enteroendocrine cells and goblet cells. Our results point out the potential role of Reg IV in colorectal tumors and its subsequent interest as a pronostic indicator of tumor survival.
Adenocarcinoma↗
Enzyme immunoassay and immunochemical characterization of pancreatic stone protein in human serum.
Monoclonal antibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5 was employed as the standard in the assay. The assay's measurable range was 25-1,500 ng/ml and within run coefficient of variation was 3.7-6.4%. Analytical recovery of the assay was 101.5 +/- 5.65% (mean +/- SD). The results of experiments in which serum was fractionated by Mono S (cation exchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5. The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis.
Antibodies, Monoclonal↗
Enzyme immunoassay for specific analysis of pancreatic stone proteins in human pancreatic juice.
In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form of PSP-S1 converted from PSP-S2-5 on PSP determination and the assay method for PSP-S1 precipitate after solubilizing PSP-S1. When bovine trypsin was added to pancreatic juice, PSP-S1 was converted from PSP-S2-5 and precipitated about 45-85% after 1 h. The precipitated PSP-S1 was dissolved in 0.1 M sodium acetate buffer, pH 4.0, and the concentration was measured by the enzyme immunoassay, with similar reactivity to PSP-S1 and PSP-S2-5. The proposed method can offer accurate and specific analysis of the PSP level in pancreatic juice. The results of the fractionation of pancreatic juice and duodenal juice on Mono S cation-exchange chromatography suggested that the major component of PSP was PSP-S2-5 in pancreatic juice and PSP-S1 in duodenal juice.
Calcium-Binding Proteins↗
Enzyme immunoassay and characterization of pancreatic stone proteins in human urine.
An enzyme immunoassay of pancreatic stone protein (PSP) in human urine was developed. Mean analytical recovery of pure PSP-S2-5 added to urine was 102.3% (SD 5.9%), and the precision of the assay was 2.0-2.7% within an assay and 2.5-2.9% between assays. In healthy volunteers (age 20-55 years), the mean value of the PSP concentration, expressed as ratios to urine creatinine, was 129 +/- 88 (mean +/- SD) micrograms/g without any differences for sex. Urine PSP correlated with urine N-acetylglucosaminidase (NAG) (r = 0.354). The molecular forms of immunoreactive PSP in urine were characterized by using cation exchange chromatography (Mono S), SDS-PAGE, N-terminal sequence, and enzyme immunoassay analysis. The urine PSP, eluted at the position corresponding to PSP-S2-5 on cation exchange chromatography, was converted to PSP-S1 by trypsin digestion. The difference in mobility on SDS-PAGE between urine PSP and PSP-S2-5 seems to be due to a glycosylated undecapeptide (N-terminal 1-11). The proposed method offers a sensitive, specific, and reproducible tool for laboratory analysis of human urine PSP levels.
Acetylglucosaminidase↗
Immunological characterization of pancreatic stone protein in human urine.
In order to study the mechanism and origin of urine pancreatic stone protein (PSP), PSP was analyzed in the urine and sera from healthy subjects, patients with renal disease, and intensive care patients by Mono S chromatography and Western blotting. The elution patterns could be classified into three types. In control urine, a single peak of immunoreactive PSP (peak I) was identified at the position of PSP-S2-5 (type A). In three of seven patients with renal disease, another peak of urine immunoreactive PSP (peak II) was recognized at the position slower than that corresponding to that of PSP-S1 (type B). In urine from one patient with diabetic nephropathy, a third peak of immunoreactive PSP (peak III) was eluted between peaks I and II (type C). In Western blotting, the bands in urine from patients with renal disease and of those in ICU mainly appeared at the positions of high-molecular-weight types of PSP and PSP-S2-5, respectively. These results suggest that the kidney can be another major source of urine PSP in addition to the pancreas.
Blotting, Western↗
NH2-terminal sequence of novel pancreatic stone protein-related protein in human urine.
In a previous report, we detected the novel PSP-related protein in urine from a patient with diabetic nephropathy. This protein was different from PSP S1 and PSP S2-5 on the elution points of Mono S chromatography. To investigate the NH2-terminal sequence, we purified this protein by reverse phase chromatography and performed amino acid analysis. The results showed that the NH2-terminal sequence of this protein was one residue (arginine) longer than that of PSP S1.
Amino Acid Sequence↗
Comparative modeling of the three-dimensional structure of type II antifreeze protein.
Type II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C-type lectins. The structure of this domain from rat mannose-binding protein (MBP-A) has been solved by X-ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 360:127-134) and provided the coordinates for constructing the three-dimensional model of the 129-amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP-A, and as many as 50 carbohydrate-recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two alpha-helices, two beta-sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP-A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117.(ABSTRACT TRUNCATED AT 250 WORDS)
Adaptation, Physiological↗
Induction of lithostathine/reg mRNA expression by serum from rats with acute pancreatitis and cytokines in pancreatic acinar AR-42J cells.
During the acute phase of pancreatitis, expression of most pancreatic enzymes decreases, whereas mRNAs of pancreatitis associated protein and lithostathine/reg increase dramatically. In the present study we have investigated the effect of serum from rats with acute pancreatitis (SAP) and cytokines on the lithostathine/reg mRNA expression in AR-42J cells. Lithostathine/reg mRNA was strongly induced by SAP in a dose-dependent manner. Induction was abolished by preheating the SAP or by treating the cells with cycloheximide. Treatment with interleukins (IL) IL-1 or IL-6 or dexamethasone alone was ineffective. Combination of IL-1 with IL-6 was also ineffective. Combination of IL-6 with dexamethasone resulted in strong induction of the lithostathine/reg gene, but the further addition of IL-1 to the mixture reduced induction. Treatment with tumor necrosis factor-alpha (TNFalpha) or interferon-gamma (IFNgamma) induced lithostathine/reg mRNA expression. Combination of dexamethasone with TNFalpha or IFNgamma showed an inhibitory effect on lithostathine/reg mRNA expression. These findings suggest that expression of the lithostathine/reg mRNA during acute pancreatitis could be mediated by specific combinations of cytokines and/or glucocorticoids.
Acute Disease↗
Human pancreatic thread protein, an exocrine thread protein with possible implications to Alzheimer's disease: secondary structure in solution at acid pH.
The secondary structure of human pancreatic thread protein (HPTP) in solution at acid pH was derived using Fourier transform infrared (FT-IR) and laser Raman spectroscopic studies. The experimentally derived secondary structure of HPTP was compared with the secondary structure obtained by the Chou-Fasman algorithm. Pancreatic thread protein is a major exocrine secretory protein that in vitro forms filamentous bundles reminiscent of the paired helical filaments of Alzheimer's disease (AD). PTP immunoreactivity in brains afflicted with AD has been demonstrated previously and high levels of its mRNA in the developing human brain have also been reported in the literature. The above studies suggest that AD is associated with enhanced expression of PTP-related transcripts with interneuronal accumulation of PTP-like proteins. The experimentally derived secondary structure of HPTP consists of a significant proportion of beta-sheets and beta-turns and lesser amounts of alpha-helical structures. The beta-sheet component presumably plays an important role in the pH-dependent globule-fibril transformation of HPTP leading to antiparallel beta-sheet structure in the aggregated state. The secondary structure of HPTP and its globule-fibril transformation lend credence to the belief that AD may be viewed as a conformational disease.
Algorithms↗
Preferential expression of reg I beta gene in human adult pancreas.
In human pancreas two genes, reg I alpha and reg I beta, have been characterized but only the reg I alpha protein has been isolated from human pancreatic secretion. To examine their respective physiological roles in fetal and adult pancreas we have compared the patterns of gene expression using a specific RT-PCR method. No progressive evolution in the two mRNAs levels was observed during fetal development (16--41 weeks). A discoordinate expression of the two genes was found with a higher level of reg I alpha mRNA in fetus and a higher level of regI beta in adult. In addition, if reg I alpha mRNA level was correlated with the expression of genes encoding exocrine proteins in adults, reg I beta mRNA level presented no correlation with any ductular, endocrine, or exocrine gene expression. In human pancreatic cell lines we showed the only expression of reg I beta gene and protein. All these data suggest that the two reg genes and proteins could play different roles in the pancreas.
Aged↗
Assignment of the human pancreatic regenerating (REG) gene to chromosome 2p12.
A cDNA termed reg (for regenerating gene) has been isolated and characterized from a rat pancreatic library. Expression of reg is markedly increased in regenerating islets and decreased when insulin gene expression is inhibited. These findings have led to the hypothesis that reg may be involved in the expansion of beta-cell mass during regeneration as well as in the maintenance of normal beta-cell function. The human reg gene has a high degree of similarity to the rat reg gene. To determine the chromosomal location of the human reg gene, we analyzed two panels of mouse- or hamster-human hybrid cell lines containing a single human chromosome or several different human chromosomes. DNA extracts from these cell lines were analyzed for the presence of the human reg gene by polymerase chain reaction. In addition, human metaphase chromosomes were used for fluorescence in situ hybridization to further confirm the chromosomal assignment and to determine the subchromosomal localization. With these approaches, we show that the human reg gene is located on the short arm of chromosome 2 near the centromere at band 2p12.
Animals↗
A recombinant rat regenerating protein is mitogenic to pancreatic derived cells.
Pancreatic regenerating protein (reg I) is expressed in acinar cells and is mitogenic to beta- and ductal cells. Isolation of large amounts of endogenous reg I for in vivo and in vitro experiments has been difficult. The aim of this study was to develop a recombinant protein and determine its bioactivity on rat pancreatic derived cells. cDNA of the rat reg I coding region was created with unique BamHI flanking sequences using reverse transcriptase PCR. The coding region was then cloned into a bacterial expression vector in which expression is controlled by a T7 promoter. After transformation into the Escherichia coli strain B21(DE3) and induction by isopropyl-beta-d-thiogalactopyranoside, a fusion protein of 24 kDa in size, named reg-PET, was noted in the bacterial lysate. This protein contained a polyhistidine and S-peptide sequence to facilitate isolation and identification, respectively. This protein was purified using affinity chromatography, and identity was confirmed with gel electrophoresis and Western analysis. The reg-PET protein was mitogenic to both ARIP and RIN cells, rat pancreatic ductal and beta-cell lines, respectively. Antibodies raised to the protein reacted against rat reg I in pancreas. The purified recombinant reg I fusion protein, like endogenous reg I, is mitogenic to pancreatic derived cells. It is more potent than reg I isolated from pancreatic tissue. This protein can be isolated rapidly, easily, and with a high amount of purity.
Amino Acid Sequence↗
Coordinate regulation of secretory stress proteins (PSP/reg, PAP I, PAP II, and PAP III) in the rat exocrine pancreas during experimental acute pancreatitis.
BACKGROUND: Pancreatic stone protein (PSP/reg) is a constitutively secreted protein in pancreatic juice. Pancreatitis-associated protein (PAP) belongs to the same family of proteins. PAP is highly increased during acute pancreatitis, while no exact data exist regarding PSP/reg protein synthesis and secretion. Recently, an attempt to determine PSP/reg and PAP levels in sera of rats with acute pancreatitis showed a significant increase in PAP but failed to demonstrate changes in PSP/reg. Others reported that surgical manipulation of the pancreas, including sham controls, affected mRNA levels of PSP/reg. Neither report determined protein levels of PSP/reg. METHODS: Rats were treated intraperitoneally with a supramaximal dose of caerulein to induce pancreatitis, a physiological dose of caerulein, or a saline injection. Pancreata were analyzed for PAP and PSP/reg using ELISAs. RNA was extracted for Northern blot analysis of PAP I, II, and III and PSP/reg mRNA. RESULTS: Experimental induction of acute pancreatitis caused a coordinate increase in both PSP/reg and PAP. PAP showed an acute response and returned to low levels within 48 h while PSP/reg exhibited a more sustained response. Intraperitoneal application of a physiological dose of caerulein and even a saline injection caused an increase in PSP/reg. CONCLUSION: PSP/reg and PAP levels are increased through similar mechanisms by physiological and supramaximal doses of caerulein. However, PSP/reg regulation appears to sustain high levels while PAP levels are more transient. Since the regulation of this protein family is affected even under mild stress, we define them as secretory stress proteins.
Acute Disease↗
PAP I interacts with itself, PAP II, PAP III, and lithostathine/regIalpha.
PAP I, PAP II, PAP III, and lithostathine/regIalpha are members of a multigenic family of proteins expressed in several tissues. PAP I was shown to be antiapoptotic, mitogenic, and anti-inflammatory and can promote cell adhesion to the extracellular matrix. Lithostathine/regIalpha can be mitogenic. Because polymerization might regulate activity, we examined the ability of rat PAP I to interact with itself (homodimerization), PAP II, PAP III, and lithostathine/regIalpha (heterodimerization) by the yeast two-hybrid system, affinity experiments, and crosslinking. PAP I interacted significantly with all members of the PAP protein family, homodimerization showing the strongest interaction as judged by the beta-galactosidase test. This was confirmed by showing specific affinity between a MBP-rPAP I fusion protein and the native rPAP I. Finally, crosslinking experiments showed that rPAP I formed dimers in solution. These findings should be taken into account in functional studies involving PAP I and PAP-related proteins.
Acute-Phase Proteins↗