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C Figarella

Publications and source records attributed to C Figarella.

At least 19 recordsLinked to original sources
PubMed1992

Monoclonal antibodies to human pancreatic trypsin 1 inhibit the activation of human trypsinogens 1 and 2.

Two monoclonal antibodies (Mab) raised against human pancreatic trypsin 1, Mab G6 and A8, were previously isolated and characterized. The two Mab which recognize trypsinogen 1 are found to inhibit the activation of trypsinogen 1 by enterokinase. The inhibition of activation by the two Mab is concentration-dependent, rapid and virtually complete with Mab G6. Activation of trypsinogen 2 is totally inhibited by Mab G6, while Mab A8 has no effect on the activation of trypsinogen 2. The two monoclonal antibodies have opposite effects on the proteolytic activity of trypsin 1; Mab G6 increases proteolytic activity while Mab A8 inhibits trypsin activity by as much as 40%. This inhibition is concentration dependent but cannot account for the complete inhibition of activation of trypsinogen 1. Neither monoclonal antibody significantly inhibits the esterolytic activity of either form of human trypsin. Western-blot analysis of the reactivity of the two monoclonal antibodies with trypsinogens of various species shows that only Mab G6 cross-reacts with dog trypsinogen.

Animals↗
PubMed1992

Levels and molecular forms of immunoreactive trypsin and chymotrypsin in amniotic fluids from normal and cystic fibrosis fetus: evidence for a lack of activation of proteolytic zymogens in cystic fibrosis fetus.

We previously suggested that an activation defect of pancreatic proteolytic zymogens in newborns suffering from cystic fibrosis (CF) might contribute (by an adaptative-like process) to the significant increase of the serum trypsin level observed in the disease at birth. To give support to this hypothesis we studied two pancreatic enzymes: trypsin 1 (IRT) and chymotrypsin A (IRChT) by noncompetitive enzyme immunoassays in amniotic fluids taken at 17-18 weeks of pregnancy. In normal fluids (102), the levels of the two enzymes were widely dispersed between 5 and 100 micrograms/L. A similar pattern was observed for the fluids with a 1 in 4 risk of CF with a normal outcome (24). In contrast, the levels of pancreatic enzymes in the fluids with affected fetus (40) were always below 45 micrograms/L for IRT and 55 micrograms/L for IRChT and most of them were under 20 micrograms/L for both enzymes. The molecular forms of IRT and IRChT in amniotic fluids were studied by gel filtration. In amniotic fluids with affected fetus, a major form of IRT was eluted in a position consistent with the elution of proteins around 25 kDa and two peaks of IRChT were eluted at 75 kDa and 25 kDa. These patterns are similar to those observed in normal serum when zymogens are present and are quite different from the patterns obtained by gel filtration of amniotic fluids with normal outcome.(ABSTRACT TRUNCATED AT 250 WORDS)

Amniocentesis↗
PubMed1992

Immunohistochemical study of secretory proteins in the developing human exocrine pancreas.

We have studied, by immunohistochemical methods using specific antisera, the development of three glycoproteins of human pancreatic secretion: lipase, carboxyl ester hydrolase (CEH) and the P19 protein (precursor of the non glycosylated protein X or "pancreatic thread/stone protein"). We have compared their development to that of trypsinogens (Tgs) and chymotrypsinogen A (ChTgA), as well as to that of FAP (feto acinar pancreatic protein), a glycoprotein associated with the differentiation of human pancreas. Our studies show the characteristic appearance and development of lipase, the immunoreactivity of which appears later (at the 21st week of pregnancy) than it does for Tgs and ChTg (at the 16th week of pregnancy). Moreover, the lipase labelling is first observed in a few acini dispersed in the pancreas and then spreads out progressively to be present in all the acini after the age of 15 days. By contrast, as soon as they appear, Tgs and ChTg are observed uniformly in all acinar cells. The intensities of the lipase, Tgs and ChTg labellings increase greatly at birth. The ontogenesis of CEH does not follow that of lipase but that of Tgs and ChTg. The ontogenesis of P19 is parallel to that of Tgs. As previously observed, FAP presents a maximal immunoreactivity at the 24th-27th weeks of pregnancy, which decreases slowly up until birth.

Aging↗
PubMed1992

Secretory proteins and glycoconjugates synthesized by human tracheal gland cells in culture.

To study the proteins and glycoconjugates synthesized by serous cells from human tracheal glands (HTG), isolated HTG cells were cultured in the presence of radiolabeled precursors 14C-proline, Na2(35)SO4, and 3H-fucose. The secretory 14C/35S/3H-radiolabeled proteins and glycoproteins, de novo synthesized by HTG cells, were analyzed by gel filtration chromatography. We observed the incorporation of 14C-proline into antileukoprotease and an unknown 30 kD protein, and the incorporation of 35SO4-- and 3H-fucose into high molecular weight glycoconjugates and sulfoconjugates (M(r) > 1,000,000) and into components with apparent M(r) of approximately 250 and 100 kD. After specific chemical and enzymatic treatment, the 35S- and 3H-glycoconjugates were shown to be -O-linked mucin-like glycoproteins and proteoglycans. These results show that cultured HTG cells synthesize some of the macromolecules identified in bronchial secretions.

Cells, Cultured↗
PubMed1991

Characterization of trypsinogens 1 and 2 in two human pancreatic adenocarcinoma cell lines; CFPAC-1 and CAPAN-1.

Proteins with trypsin-like immunoreactivity (first detected by a specific immunoenzymatic assay) were isolated from CAPAN-1 and CFPAC-1 cell culture-conditioned media by chromatography on an immunoadsorbent prepared with a polyclonal antibody directed against trypsin 1. The adsorbed proteins were devoid of free trypsin activity but trypsin activity was present after enterokinase activation demonstrating that the immunoreactive trypsin present in cell supernatants corresponds to trypsinogens. When characterised by Western blotting using a monoclonal antibody directed against human trypsin 1 two protein bands corresponding to trypsinogen 1 (23 kDa) and trypsinogen 2 (25 kDa) gave a positive reaction. These results demonstrate the presence of trypsinogens 1 and 2 in CAPAN-1 and CFPAC-1 cells and in their culture-conditioned media.

Adenocarcinoma↗
PubMed1991

Identification of 'cystic fibrosis protein' as a complex of two calcium-binding proteins present in human cells of myeloid origin.

Cystic fibrosis protein is a serum protein characterized by a pI close to 8.4 and present with a higher concentration in serum and plasma of cystic fibrosis carriers than in controls. This protein was found immunologically indistinguishable from the cystic fibrosis antigen isolated from granulocytes and presenting a sequence analogous to that of MRP-8, a calcium-binding protein expressed in the myeloid cell lineage. Using antibodies directed against MRP-8 and its closely associated calcium-binding protein, MRP-14, we demonstrate here that cystic fibrosis protein purified from serum is a complex of the two proteins MRP-8 and MRP-14.

Blood Proteins↗
PubMed1990

Evidence for a different mechanism of lactoferrin and transferrin translocation on HT 29-D4 cells.

The fate of [125I] lactoferrin after binding on its specific receptor on HT29-D4 cells, a clone of HT29 cells, was studied and compared with that of [125I]transferrin. Time courses of protein binding in relation with temperature clearly show that different mechanisms of ligand-transportation for the two glycoproteins exist on these cells: whereas transferrin would enter into the cell probably by a relatively classic pathway of receptor-mediated endocytosis, lactoferrin and its specific receptor would not be internalized. Thus the mechanism by which lactoferrin promotes HT29 cell growth seems quite different from that of transferrin on the same cells.

Biological Transport↗
PubMed1990

Production and characterization of two monoclonal antibodies to human pancreatic trypsin 1.

Two monoclonal antibodies (MAb) G6 and A8 directed against human trypsin 1 have been produced by hybridization of myeloma cells with spleen cells of OF1 immunized mice. Antibodies were screened by radioimmunoassay. Monoclonal antibodies were purified by affinity chromatography on Protein A-Sepharose, and we found that MAb G6 was of the IgG2b class and MAb A8 of the IgG2a class. Both MAbs had a high affinity for trypsin 1 with the respective affinity constants equal to 1.3 x 10(8) l/mol for G6 and 3.2 x 10(7) l/mol for A8. Epitope specificity was studied by western blotting, using human trypsinogens 1 and 2. Both MAbs gave a positive reaction with native trypsinogen 1 and no reaction with the same protein after reduction. Only MAb G6 reacted with trypsinogen 2 in the native form. Its affinity for trypsin 2 was found similar to that for trypsin 1 with a constant equal to 2.7 x 10(7) l/mol. Both antibodies appeared directed against conformational and not sequential epitopes.

Animals↗
PubMed1989

Behaviour of serum immunoreactive trypsin after serum activation by enterokinase in normal and cystic fibrosis patients. Evidence of a trypsin-alpha 1-proteinase inhibitor complex in some cystic fibrosis patients.

Serum immunoreactive trypsin (IRT) concentrations are elevated in newborn children with cystic fibrosis (CF) and subsequently fall, in most cases, to values below normal. To evaluate the molecular form(s) of IRT present in serum, we have performed serum activation by enterokinase and have measured serum IRT before and after activation. This approach is based on the postulate that enterokinase converts trypsinogen into trypsin, and this trypsin would then be mainly trapped by alpha 2-macroglobulin, thus escaping the assay. This assumption was confirmed in the 28 controls studied, where the mean percentage (+/- S.D.) of IRT recovery after serum activation was 13.7 +/- 2.9. Previous inhibition of alpha 2-macroglobulin by methylamine raised the recovery over 85%, confirming that most of the serum IRT present in controls was in the form of trypsinogen. Identical results were obtained in the serum of 10 obligate heterozygotes and in 57 out of 80 CF patients. In 23 CF patients the mean percentage of IRT recovery after serum activation was 41.6 +/- 17.6. Gel-filtration studies were performed on the sera of the CF patients showing an abnormal increase in the IRT recovery after serum activation. We could demonstrate that IRT was distributed in two fractions: one eluted with the Mr 25,000 protein as usually found in controls and other CF sera, and the other eluted with the Mr 75,000 protein corresponding to a complex of trypsin with alpha 1-proteinase inhibitor. These results show that, in these sera, active trypsin has been directly released in blood. These findings suggest that in some patients with CF, subclinical attacks of acute pancreatitis may occur.

Chromatography, Gel↗
PubMed1989

Lactoferrin-binding sites at the surface of HT29-D4 cells. Comparison with transferrin.

The binding of 125I-lactoferrin to HT29-D4 cells, a clone of HT29 cells, was studied and compared to the binding of 125I-transferrin to the same cells. The binding of the two iron-transport proteins is saturable and reversible suggesting the presence of specific receptors for each protein. Scatchard analysis suggests the existence of binding sites for lactoferrin with the relatively high equilibrium dissociation constant, Kd1 of 408 nM. Additionally, the cell is capable of binding large amounts of lactoferrin with very low affinity, probably in a non-receptor intermediate fashion. The dissociation constant of transferrin and its receptor was calculated 9.29 nM which corresponds well to values found in the literature. In contrast to lactoferrin, the cell was capable of binding only low amounts of transferrin in a non-receptor intermediate fashion. After chemical crosslinking of lactoferrin to the cell surface, the radiolabeled lactoferrin was found in a complex of molecular mass 300 kDa. Crosslinking of transferrin resulted in a complex of much higher molecular mass. These data clearly show a binding site for lactoferrin different from the transferrin receptor. Only if competition experiments were performed with a high molar excess of both ligand proteins did a small percentage of either of the two ligands crossreact with the receptor for the other, possibly due to a structural similarity of the two glycoproteins.

Adenocarcinoma↗
PubMed1989

Plasma and serum lactoferrin levels in cystic fibrosis. Relationship with the presence of cystic fibrosis protein.

Plasma lactoferrin concentrations were measured in blood of cystic fibrosis patients, heterozygotes and controls using a specific and sensitive enzyme immunoassay. 67 plasmas were studied (26 controls, 23 heterozygotes, 18 cystic fibrosis patients) and the results showed a statistically significant increase (p less than 0.05) of the level of plasma lactoferrin in cystic fibrosis patients (265 +/- 224 micrograms/l) compared to controls (168 +/- 100 micrograms/l) and heterozygotes (150 +/- 72 micrograms/l). Since it is well established that plasma lactoferrin level could be influenced by the number of neutrophils, a second set of experiments was performed on 20 cystic fibrosis patients on whom leukocyte counts were also made. When the 15 plasmas with normal neutrophils (in the range 2 to 6 giga/l) were considered, the mean lactoferrin level was 318 +/- 116 micrograms/l, still far above the normal values. For serum, a similar significant increase of lactoferrin concentration was observed in 33 cystic fibrosis patients (610 +/- 551 micrograms/l) compared to the values observed for 25 controls (237 +/- 155 micrograms/l) and 37 heterozygotes (272 +/- 231 micrograms/l). Cystic fibrosis protein (CFP) was identified in the same sera by isoelectric focusing and the intensity of the band was closely related to the increase of lactoferrin concentration in cystic fibrosis patients. In contrast, no difference in serum lactoferrin concentrations was observed between heterozygotes with or without CFP, indicating that the increased CFP concentration cannot be due only to altered granulocyte function.

Blood Proteins↗
PubMed1989

Human cationic and anionic trypsins: differences of interaction with alpha 1-proteinase inhibitor.

Human cationic (trypsin 1) and anionic (trypsin 2) trypsins were obtained by controlled activation of purified trypsinogens 1 and 2, respectively. The interactions of trypsin 1 and trypsin 2 with human alpha 1-proteinase inhibitor (alpha 1PI) were analysed and compared by studies in vitro. The enzymatic activity and inhibitory capacity measurements were assessed using Glp-Gly-Arg-Nan as substrate. The association rate constants showed that the inhibition of trypsin 2 occurred more than 10 times faster than that of trypsin 1. The equimolar complexes obtained between either trypsin and alpha 1PI were visualized by electrophoresis followed by immunoblotting. The inhibition of the two trypsins was temporary i.e. the complexes trypsin 1-alpha 1PI and trypsin 2-alpha 1PI broke down with time yielding inactive alpha 1PI (Mr 50,000) and active enzyme. But the stability time for trypsin 1-alpha 1PI was much larger than that of trypsin 2-alpha 1PI. In vivo, alpha 1PI is not able to control the activity of trypsin 1 except when alpha 2-macroglobulin (alpha 2M) is already saturated. According to the delay times of inhibition calculated from normal concentrations in serum, alpha 1PI inhibits trypsin 2 as fast as alpha 2M inhibits trypsin 1. These results suggest that a significant role can be assigned to alpha 1PI in the inhibition of trypsin 2 in physiological conditions and of trypsin 1 in pathological ones.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Isolation of the "cystic fibrosis protein" from serum.

"Cystic fibrosis protein" (CFP), a minor serum protein marker of the cystic fibrosis allele, was isolated from serum from patients with cystic fibrosis by use of the "FPLC" high-resolution chromatography system and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CFP currently is characterized by its isoelectric point (8.4) on isoelectric focusing. However, after the first purification steps, we identified the protein, which was present in a very low concentration, by the immunosorbent assay of Hayward et al. (J Immunol Methods 1986;91:117-22), by virtue of its immunological relationship with the "CF antigen," a protein characterized in granulocytes by these same authors [Nature (London) 1985;315:513-5]. CFP, a protein of low molecular mass, about 14 kDa, appears to be strongly associated with IgG in serum. Using the same procedure with control serum permits us to assume that CFP normally is present in serum in trace amounts.

Antibodies, Monoclonal↗
PubMed1988

Characterization of two glycoproteins of human pancreatic juice: P35, a truncated protease E and P19, precursor of protein X.

Four glycoproteins were separated by SDS-polyacrylamide gel electrophoresis of proteins of human pancreatic juice devoid of free proteolytic activity. The two low molecular weight glycoproteins were isolated and characterized. Protein P19, the precursor family of protein X, was analyzed by its carbohydrate content which seemed to play an important role in protein solubility at pH 8.0. Protein P35 was found to be a Con A-binding protein rich in mannose. Its N-terminal amino acid sequence covering 33 residues revealed a strong homology with human protease E without the dipeptide Val-Val. Is P35 a protein homologous to the subunit III of bovine procarboxypeptidase A?

Amino Acid Sequence↗