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PubMed1996

Calcium carbonate crystals promote calcium oxalate crystallization by heterogeneous or epitaxial nucleation: possible involvement in the control of urinary lithogenesis.

A large proportion of urinary stones have calcium oxalate (CaOx) as the major mineral phase. In these stones, CaOx is generally associated with minor amounts of other calcium salts. Several reports showing the presence of calcium carbonate (CaCO3) and calcium phosphate in renal stones suggested that crystals of those salts might be present in the early steps of stone formation. Such crystals might therefore promote CaOx crystallization from supersaturated urine by providing an appropriate substrate for heterogeneous nucleation. That possibility was investigated by seeding a metastable solution of 45Ca oxalate with vaterite or calcite crystallites. Accretion of CaOx was monitored by 45Ca incorporation. We showed that (1) seeds of vaterite (the hexagonal polymorph of CaCO3) and calcite (the rhomboedric form) could initiate calcium oxalate crystal growth; (2) in the presence of lithostathine, an inhibitor of CaCO3 crystal growth, such accretion was not observed. In addition, scanning electron microscopy demonstrated that growth occurred by epitaxy onto calcite seeds whereas no special orientation was observed onto vaterite. It was concluded that calcium carbonate crystals promote crystallization of calcium oxalate and that inhibitors controlling calcium carbonate crystal formation in Henle's loop might play an important role in the prevention of calcium oxalate stone formation.

Calcium Carbonate↗
PubMed1999

The amino acid sequence of ovocleidin 17, a major protein of the avian eggshell calcified layer.

The amino acid sequence of ovocleidin 17, a major protein of the chicken eggshell calcified layer, contains 142 amino acids including 2 phosphorylated serines. Data base searches show that ovocleidin belongs to a heterogeneous group of proteins consisting of a single C-type lectin domain (CTL). The most similar sequences with an average of 30% identical amino acids were those of pancreatic stone protein (lithostathine) and lectins and anticoagulant proteins from snake venom.

Amino Acid Sequence↗
PubMed1996

Pancreatic calculi superimposed upon slow growing pancreatic cancer.

We report on a 59 year old male patient with cancer of the head of the pancreas, upon which pancreatic calculi were superimposed during the 3 year clinical course. Pancreatic calculi were noted in the main pancreatic duct (MPD) on both computed tomographic scans and ultrasonographs of the abdomen approximately 10 months after the recognizable dilatation of the MPD. Existence of the calculi was confirmed by autopsy. Elemental analysis and infrared spectrophotometry of the calculi demonstrated that the main constituent of the calculi was calcium carbonate. Histopathological examination showed that the pancreatic cancer was moderately differentiated adenocarcinoma. Immunohistochemical studies revealed that pancreatic stone protein (lithostathine) was present in the cytoplasm of tumour cells. In this case, pancreatic cancer progressed to obstruct the MPD unusually slowly, resulting in stagnation of pancreatic secretion and subsequent formation of the calculi.

Adenocarcinoma↗
PubMed1993

Human regeneration protein/lithostathine genes map to chromosome 2p12.

The pancreatic stone protein (lithostathine) secreted by the exocrine pancreas is an inhibitor of CaCO3 crystal growth. This protein, which is also present in endocrine pancreas, has also been called the regeneration protein (reg). Here we report the mapping of the REG gene to chromosome 2 using the polymerase chain reaction for the specific amplification of human reg sequences in rodent/human somatic cell hybrid DNA. A regional assignment has been made by in situ hybridization to metaphase chromosomes using two different fluorescently labelled genomic probes corresponding to the REG gene and a related gene REGL. Both probes hybridized to chromosome 2p12 suggesting the tandem organization of these genes.

Animals↗
PubMed1995

A limited genomic region contains the human REG and REG-related genes.

A glycoprotein expressed in exocrine pancreas (where it has been called lithostathine) and endocrine pancreas (where it has been called the regeneration protein) is encoded by a gene (REG) which maps to 2p12. A REG-related sequence (REGL) is also located in 2p12 and expressed in the pancreas. Here we describe the physical mapping of these genes within a 100-kb genomic region. A YAC clone was converted into an ordered cosmid contig. We constructed a restriction map of the cosmid contig and localized the loci corresponding to the genes. A third REG-related sequence also maps to this region. Although this sequence was previously described as a pseudogene, we show here that it is also expressed in the pancreas.

Base Sequence↗
PubMed2005

Proteomic analysis of urine in patients with intestinal segments transposed into the urinary tract.

Intestinal segments are used to replace or reconstruct the urinary bladder when it has become dysfunctional or develops life-threatening disease such as cancer. The quality of life in patients with intestinal segments used to either enlarge or completely replace the native bladder is adversely affected by recurrent urinary tract infections, excessive mucus production and the occasional development of malignancy. At present, there is no reliable method of predicting or noninvasively monitoring these patients for the development of these complications. The characterisation of proteins secreted into urine from the transposed intestinal segments could serve as important indicators of these clinical complications. Urine is an ideal source of material in which to search for biomarkers, since it bathes the affected tissues and can be obtained relatively easily by noninvasive methods. The urinary proteome of patients with intestinal segments transposed into the urinary tract is unknown and we present the first global description of the urinary protein profile in these patients. Sample preparation is a critical step in achieving accurate and reliable data. We describe a method to prepare urinary proteins that was compatible with their subsequent analysis using two-dimensional polyacrylamide gel electrophoresis. This method helped to overcome some of the technical problems encountered in analysing urine from this patient cohort. The method was used to analyse urinary proteins recovered from five healthy controls and ten patients with intestinal segments transposed into the urinary tract. Four low molecular weight proteins were found to be present in nine out of ten for the patient group but for none of the healthy controls. The four proteins were identified as lithostathine-1 alpha precursor, pancreatitis associated protein-1 precursor, liver fatty acid binding protein and testis expressed protein-12. The role of these proteins as potential biomarkers of intestinal cell activity within the reconstructed bladder is discussed.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1995

Analysis of the soluble organic matrix of five morphologically different kidney stones. Evidence for a specific role of albumin in the constitution of the stone protein matrix.

Our aims were to analyze the protein composition of the organic matrix of urinary stones and to investigate the role of albumin in its constitution. Five different morphological types of stones were studied. Proteins extracted from the stone were submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting with antibodies to 13 urinary proteins. Nine of the 13 proteins were found in all types of stone: human serum albumin (HSA), alpha 1-acid glycoprotein (alpha 1-GP), alpha 1-microglobulin (alpha 1-M), immunoglobulins (Igs), apolipoprotein A1 (apo-A1), transferrin (Tr), alpha 1-antitrypsin (alpha 1-T), retinol-binding protein (RBP) and renal lithostathine (RL). The beta 2-microglobulin (beta 2-M) was present only in calcium oxalate and uric acid stones. In contrast, ceruloplasmin, haptoglobin and Tamm-Horsfall protein (THP) were detected in none of them. Because HSA appeared as the major protein component in all stones, we wondered whether it might play a specific role in the constitution of the stone matrix. Association of HSA with urinary proteins that were present in stones was demonstrated by showing that proteins present in the matrix comigrated with HSA on gel filtration, whereas proteins that were absent did not. Moreover, HSA induced the binding of stone matrix proteins to an albumin-specific affinity column. Finally, we evidenced HSA binding to calcium oxalate monohydrate (COM) crystals in a solution similar to urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1992

Separation of rat pancreatic secretory proteins by cation-exchange fast protein liquid chromatography.

Rat pancreatic secretory proteins were separated by an automated liquid chromatography system utilizing a Mono S cation-exchange column. Optimal resolution was obtained with a multistep salt and pH gradient (0.01-2 M LiCl, pH 5.3-63). A total of fourteen well-separated peaks, as well as several minor peaks, were detected by UV absorption. The main pancreatic enzymes were resolved (two amylases, two chymotrypsinogens, two trypsinogens, proelastase, lipase, prophospholipase A2, procarboxypeptidase A, procarboxypeptidase B, and ribonuclease). In addition, proteins without enzymic activity, such as lithostathine and pancreatitis-associated protein, were identified. Activation of proenzymes did not occur during the separation. At a flow-rate of 0.5 ml/min, ca. 250 micrograms to 5 mg of protein could be applied with equal resolution. The reproducibility of retention volumes and peak areas was high (less than 1% or 5% variation, respectively). When radiolabeled proteins were separated, a comparable pattern of peaks was obtained. The technique described is, therefore, not only useful for analytical and preparative separation of pancreatic proteins but can additionally serve for quantitative determination of the pancreatic isoenzyme pattern.

Animals↗
PubMedDate not supplied

Identification of proteins in slow continuous ultrafiltrate by reversed-phase chromatography and proteomics.

Continuous modes of renal replacement therapy (CRRT) are increasingly being utilized in the intensive care unit. The removal of cytokines and other inflammatory proteins during ultrafiltration may be responsible for some of the beneficial effects of CRRT. We used proteomic tools to identify proteins found in the ultrafiltrate from a patient with acute renal failure. Identification of these proteins could help elucidate the mechanism(s) of improved outcome with continuous renal replacement therapy. Protein was loaded on a reversed-phase C4 column and eluted with stepwise isocratic flows starting with 0%, 5%, 10%, 25%, and 50% of acetonitrile. Effluent was collected, pooled, desalted, and separated by two-dimensional gel electrophoresis (2DE). Reversed-phase separation improved the resolution and the number of spots seen on the gels. Protein spots were digested with trypsin and spotted onto MALDI plates. Proteins were identified by either peptide mass fingerprinting using a MALDI-TOF mass spectrometer or by peptide sequencing using a MALDI-TOF/TOF tandem mass spectrometer. From 196 spots cut, 47 were identified, representing multiple charge forms of 10 different proteins. Proteins identified were albumin, apolipoprotein A-IV, beta-2-microglobulin, lithostathine, mannose-binding lectin associated serine protease 2 associated protein, plasma retinol-binding protein, transferrin, transthyretin, vitamin D-binding protein and Zn alpha-2 glycoprotein. Continuous renal replacement therapy is frequently used in acutely ill patients with renal failure. Removal of proteins occurs during this process. The physiological significance of this protein removal is unclear. Identification of these proteins will lead to better understanding of the role of protein removal in continuous renal replacement therapy.

Chromatography, High Pressure Liquid↗
PubMed1996

Two forms of hereditary chronic pancreatitis.

We report 11 families of hereditary pancreatitis characterized by the presence of calculi in pancreatic ducts. These were classified as (1) calcic lithiasis (one family with five cases), in which the calculi are composed of >95% calcium salts; and (2) protein lithiasis in 10 families, in which the calculi are composed of degraded amorphous residues of lithostathine, the pancreatic secretory protein that inhibits calcium salt crystallization. In both forms, transmission appears to be dominant. The average age at clinical onset of symptoms is 15 years. The clinical progression seems to be less severe than in alcoholic chronic pancreatitis (alcoholic calcic lithiasis). This report shows for the first time that hereditary chronic pancreatitis is a group of at least two diseases having a similar clinical picture and pathological features but different chemical compositions of calculi. This leads us to propose a revised Marseille-Rome classification.

Adult↗
PubMed1998

A review: acute and chronic effects of ethanol and alcoholic beverages on the pancreatic exocrine secretion in vivo and in vitro.

Whereas oral or intraduodenal ethanol causes a moderate stimulation of pancreatic bicarbonate and enzyme output, intravenous ethanol inhibits basal and hormonally stimulated pancreatic exocrine secretion in humans, dogs, cats, pigs, rabbits, and rats. This inhibition could be mediated by inhibitory cholinergic mechanisms or be the result of a direct cellular effect of ethanol. In vitro investigations have specified several signaling molecules that may be involved in the action of ethanol on stimulus-secretion coupling in the exocrine pancreas, including cyclic adenosine monophosphate, intracellular calcium, and cholecystokinin and somatostatin receptors. In difference to pure ethanol solutions and distilled spirits, beer strongly stimulates pancreatic enzyme output, probably by nonalcoholic fermentation products. During chronic alcoholism, the ethanol-induced inhibition is replaced by an enhanced enzyme output that causes intraductal protein precipitation. In vitro investigations suggest that this increase is reversible after alcohol withdrawal. The occurrence of protein precipitates is considered to be a crucial step in the development of chronic alcoholic pancreatitis in humans. Other ethanol-induced secretory alterations that may contribute to the development of alcoholic pancreatitis are a decreased secretion of trypsin inhibitor, an increased cholinergic tone, and changes in the concentration of lithostathine.

Alcoholic Beverages↗
PubMed1991

Pancreatic gene expression is altered during acute experimental pancreatitis in the rat.

We investigated pancreatic gene expression in the rat in response to taurocholate-induced acute pancreatitis. Concentrations of transcripts encoding pancreatic protein showed noncoordinated alterations. Contents in amylase, trypsinogen I, chymotrypsinogen B, elastase 1, and procarboxypeptidase A mRNAs decreased by greater than 50% during the acute phase (days 0-2), whereas actin and lithostathine mRNAs increased 5 and 0.6 times, respectively, and pancreatitis-associated protein (PAP) mRNA increased greater than 200 times, indicating redirection of the pattern of gene expression. Synthesis of pancreatic proteins was also altered in a noncoordinated manner. During the acute phase, it decreased more for trypsinogen I and chymotrypsinogen B than for amylase and lipase, whereas synthesis of the PAP increased dramatically. For amylase and chymotrypsinogen B, we compared the patterns of changes in mRNA concentrations, rates of synthesis, and pancreatic contents. Changes in enzyme contents and synthetic rates were temporally correlated during the acute phase. On the contrary, changes in mRNA concentrations and enzyme synthesis were not coordinated, suggesting that control of synthesis partly occurred at the posttranscriptional level. It was concluded that induction of pancreatitis is accompanied by transcriptional and posttranscriptional modifications resulting in rapid and massive rearrangement of the pattern of pancreatic protein gene expression.

Acute Disease↗
PubMed2004

C-type lectin-like domains in Fugu rubripes.

BACKGROUND: Members of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. Three genome-level studies on human, C. elegans and D. melanogaster reported previously demonstrated almost complete divergence among invertebrate and mammalian families of CTLD-containing proteins (CTLDcps). RESULTS: We have performed an analysis of CTLD family composition in Fugu rubripes using the draft genome sequence. The results show that all but two groups of CTLDcps identified in mammals are also found in fish, and that most of the groups have the same members as in mammals. We failed to detect representatives for CTLD groups V (NK cell receptors) and VII (lithostathine), while the DC-SIGN subgroup of group II is overrepresented in Fugu. Several new CTLD-containing genes, highly conserved between Fugu and human, were discovered using the Fugu genome sequence as a reference, including a CSPG family member and an SCP-domain-containing soluble protein. A distinct group of soluble dual-CTLD proteins has been identified, which may be the first reported CTLDcp group shared by invertebrates and vertebrates. We show that CTLDcp-encoding genes are selectively duplicated in Fugu, in a manner that suggests an ancient large-scale duplication event. We have verified 32 gene structures and predicted 63 new ones, and make our annotations available through a distributed annotation system (DAS) server http://anz.anu.edu.au:8080/Fugu_rubripes/ and their sequences as additional files with this paper. CONCLUSIONS: The vertebrate CTLDcp family was essentially formed early in vertebrate evolution and is completely different from the invertebrate families. Comparison of fish and mammalian genomes revealed three groups of CTLDcps and several new members of the known groups, which are highly conserved between fish and mammals, but were not identified in the study using only mammalian genomes. Despite limitations of the draft sequence, the Fugu rubripes genome is a powerful instrument for gene discovery and vertebrate evolutionary analysis. The composition of the CTLDcp superfamily in fish and mammals suggests that large-scale duplication events played an important role in the evolution of vertebrates.

Amino Acid Sequence↗
PubMed1992

[Description of a new disease: pancreatic lithiasis with radiolucent calculi].

118 patients presenting with pancreatic lithiasis were consecutively observed in our service. They underwent both an endoscopic pancreatography and god plain films of the abdomen. Calculi were classified in 3 groups: 1) Radiolucent calculi (17 cases, 5 females; 4 hereditary cases) are build up of amorphous residues of lithostathine S. They are not related to either alcohol, diet or tobacco. 2) Target calculi (27 cases, 4 females; 3 hereditary cases) have a radiolucent core as in 1 and a peripheral calcification. They are a late evolutionary stage of radiolucent lithiasis. The frequency of females and of hereditary cases is significantly greater in form 1 + 2 than in form 3. These two forms are a newly described disease without relationship with nutrition, alcohol or tobacco but the peripheral calcification of radiolucent calculi is favoured by alcohol and tobacco. This disease could be hereditary. 3) Calcic lithiasis (74 cases, 8 females, 2 hereditary cases) is the most frequent form of pancreatic lithiasis. Its cause is nutritional.

Alcohol Drinking↗
PubMed1992

[Description of a new disease, pancreatic lithiasis with radiotransparent calculi].

118 patients presenting with pancreatic lithiasis were consecutively observed in our service. They underwent both an endoscopic pancreatography and good plain films of the abdomen. Calculi were classified in 3 groups: 1) Radiolucent calculi (17 cases, 5 females; 4 hereditary cases) are build up of amorphous residues of lithostathine S. This disease is not related to either alcohol, diet or tobacco. 2) Taget calculi (27 cases, 4 females; 3 hereditary cases) have a radiolucent core as in 1 and a peripheral calcification. They are a late evolutionary stage of radiolucent lithiasis. The frequency of females and of hereditary cases is significantly greater in form 1 + 2 than in form 3. These two forms are a newly described disease without relationship with nutrition, alcohol or tobacco but the peripheral calcification of radiolucent calculi is favoured by alcohol and tobacco. This disease could be hereditary. 3) Calcic lithiasis (74 cases, 8 females, 2 hereditary cases) is the most frequent form of pancreatic lithiasis. Its cause is nutritional.

Adult↗
PubMed1993

[Glycoprotein inhibitors of urinary calculi formation].

Human urine is supersaturated with respect to calcium salts especially calcium oxalate which is the major mineral phase of urinary stones. Inhibitors of crystallization prevent renal calcification in urine. Small molecular weight components account for 20% of total inhibitory activity of urine. Macromolecular inhibitors (molecular weight greater than 10 kDa) are more powerful. Recently, several macromolecules have been identified in urine, in urinary stones and in the kidney: Nephrocalcin, Tamm-Horsfall protein, Uropontin, Crystal Matrix Protein, renal Lithostathine. The role of these proteins is stressed.

Crystallization↗
PubMed1996

[Uronic-acid-rich protein: a new glycoprotein inhibiting the crystallization of calcium oxalate in vitro].

During about ten years, nephrocalcin was considered the main calcium oxalate crystal growth inhibitor. Today, it appears only a urinary protein inhibitor among other ones such as non polymerized Tamm-Horsfall protein, uropontin or crystal matrix protein (CMP), a protein derived from prothrombin. All these molecules are able to inhibit either crystal growth or aggregation of calcium oxalate in urine. Another protein, named renal lithostathine, was also reported to be a potent inhibitor of secondary nucleation and growth of calcium carbonate crystals. A new urinary inhibitor of calcium oxalate formation was isolated from the urine of healthy subjects using chromatographic procedures. It is a macromolecule with a molecular weight (MW) of approximately 35 kDa as estimated by polyacrylamide gel electrophoresis. Its carbohydrate content represents an average of 8.5% of its MW. Glutamic and aspartic acids represent 24% of total amino acids. This protein is called Uronic-Acid-rich Protein (UAP) because of its uronic acid content. The same protein isolated from the urine of stone formers showed less inhibitory activity than that purified from the urine of healthy subjects. Structural modifications may explain this diminution of its inhibitory activity. This protein was also purified from rat urine using same procedures. Human and rat UAP exhibit similar biochemical characteristics and strongly inhibit calcium oxalate crystallization in vitro. Partial amino acid sequence analysis showed a homology with inter-alpha-trypsin inhibitor (ITI), confirmed by the immunological results on Western blot. Nevertheless, various chemical and enzymic treatments revealed that UAP and ITI are not identical molecules. Consequently, urine contains several macromolecular substances belonging to ITI superfamily which are involved in the inhibition of calcium oxalate crystallization. UAP takes place among the most efficient macromolecular substances known as inhibitors in calcium oxalate nephrolithiasis.

Animals↗
PubMed1999

pap, reg Ialpha and reg Ibeta mRNAs are concomitantly up-regulated during human colorectal carcinogenesis.

We have established the phenotype of a colorectal tumor by partial sequencing of 2166 transcripts that were eventually arrayed on high-density filters. These filters were used for differential screening with mRNAs of colorectal cancer and normal adjacent mucosa to characterize genes whose expression is altered in colorectal carcinoma. Three genes encoding related proteins, PAP, reg Ialpha and reg Ibeta, were over-expressed in cancer. Northern-blot analysis confirmed that their expression was very low in normal colonic epithelial cells, but elevated in 75% of tumors. Western blotting with specific antibodies to pap and reg Ialpha revealed in tumors a single band of the expected size ( 15-16 kDa), demonstrating synthesis of the proteins. Pap was localized by immunohistochemistry to the cytoplasm of epithelial cells. In cancerous tissue, many cells showed a strong staining signal, but the proportion of stained cells was variable among patients. In normal mucosa, staining was light and restricted to a few cells scattered in the epithelium. Similar results were obtained with antibodies against reg Ialpha. No significant relationship was found between concentrations of pap, reg Ialpha or reg Ibeta and clinical outcome. We looked at potential effectors of pap/reg gene over-expression by testing, in 2 adenocarcinoma cell lines, the efficacy of the pap promoter at driving a reporter gene; strong induction was observed upon exposure to IFNgamma and IL-6. By analogy with observations in hepatocellular carcinoma, our results suggest that prevention of PAP/reg expression in normal colon cells by silencing their gene promoters is relieved during colon carcinogenesis, allowing their up-regulation by mediators such as cytokines.

Biomarkers, Tumor↗