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D Lombardo

Publications and source records attributed to D Lombardo.

At least 73 records · Page 4Linked to original sources
PubMed1993

The structure of N-linked oligosaccharides of human pancreatic bile-salt-dependent lipase.

This study describes the structure of the N-linked oligosaccharide chains of bile-salt-dependent lipase isolated from the pancreatic juice of a normal donor. After hydrazinolysis, neutral sugar chains were separated from acidic chains by paper electrophoresis and were fractionated using serial column chromatography with immobilized lectins and Bio-Gel P-4 filtration. Structural analysis was performed by means of sequential glycosidase digestion and revealed that the neutral sugar chains are mainly of the biantennary complex type. Fucose residues were identified for some trimannosyl core structures and were alpha(1-6) or alpha(1-2) linked to the innermost GlcNAc residue and a terminal Gal residue, respectively. Sialyl residues were also involved in the oligosaccharide structures. Most of these structures have no linear N-acetyllactosamine repeats. Evidence from several approaches suggests that the sugar chains of the human pancreatic bile-salt-dependent lipase possess a blood-group-related antigenic determinant.

Bile Acids and Salts↗
PubMed1993

Variation of the glycosylation of human pancreatic bile-salt-dependent lipase.

Glycoproteins of human pancreatic juice were characterized by means of lectins after electrophoresis and electrotransfer to nitrocellulose membranes. For the detected glycoproteins, only a 100-kDa glycoprotein varied in the pancreatic juice from a normal patient (i.e. without any pancreatic disorder) compared to the pancreatic juice from a patient suffering from chronic pancreatitis. This protein, which is the only protein in human pancreatic juice which is O-glycosylated and N-glycosylated, was identified as the bile-salt-dependent lipase. Among the glycosylated proteins present in human pancreatic juice, only the glycosylation of bile-salt-dependent lipase differs between individuals. The enzyme was isolated either from normal or pathological human pancreatic juices. The purified variants have an identical molecular mass and amino-acid composition. As suspected from lectin affinity studies, the oligosaccharide composition differs between the variants. The structure of the N-linked oligosaccharides of the variant from the pancreatic juice of a normal donor correlated with complete processing and maturation of a complex-type N-glycan. Alteration of the maturation process can be detected for a bile-salt-dependent-lipase variant from a patient suffering with chronic pancreatitis, since the carbohydrate composition is compatible with the predominance of hybrid or high-mannose-type structures. The amount of sugar involved in O-glycosylation associated with the peanut agglutinin reactivity suggests the presence of 12-14 minimal Gal beta 1-->3GalNac-->T/S O-glycan structures which are sialylated and fucosylated. The amount of sugar involved in the O-linked oligosaccharide structure appears to be unchanged in the variants isolated from the pathological pancreatic juice.

Bile Acids and Salts↗
PubMed1993

Human fetoacinar pancreatic protein: an oncofetal glycoform of the normally secreted pancreatic bile-salt-dependent lipase.

A fetoacinar pancreatic protein (FAP) associated with the ontogenesis, differentiation and oncogenic transformation of the human exocrine pancreas has been purified from pancreatic juices of patients suffering from pancreatitis or duodenal cancers invading the pancreas [Escribano and Imperial (1989) J. Biol. Chem. 264, 21865-21871]. This protein has striking similarities, i.e. M(r), amino acid composition and N-terminal sequence, to the bile-salt-dependent lipase (BSDL) of normal human pancreatic secretion. The aim of this study was to gain further insight into the nature of the two proteins. Reactivity with the mouse monoclonal antibody J28 (mAb J28), which characterizes FAP, and enzyme activity could not be dissociated during biochemical purification of BSDL. Furthermore, a polyclonal antiserum raised against purified human BSDL reacted completely with FAP in Western-blot analysis giving additional support to the idea of similar molecular structures for BSDL and FAP. However, by the same technique, mAb J28 reacted with a relatively restricted population of BSDL molecules. The classical BSDL preparation could be separated into molecules bearing the J28 epitope and those devoid of it by immunoaffinity on immobilized mAb J28. The two subpopulations had identical N-terminal sequences and some differences in their amino acid compositions. However, they had different carbohydrate compositions. J28-epitope-bearing molecules were active on BSDL substrates, although their specific activity was decreased. These results are consistent with the existence of two closely related polypeptide chains with different glycan counterparts. Therefore, if the name FAP is reserved for molecules bearing the J28 epitope, which is linked to a carbohydrate-dependent structure. FAP could represent an oncofetal-related variant of BSDL. Our result is the first demonstration of the existence of an oncofetal-type subpopulation of an otherwise normally secreted human pancreatic enzyme.

Amino Acid Sequence↗
PubMed1993

Is bile salt-dependent lipase concentration in serum of any help in pancreatic cancer diagnosis?

The diagnostic value of bile salt-dependent lipase for pancreatic diseases was tested in sera of 187 patients. Of these patients, 76 suffered from pancreatic carcinoma, 43 from nonmalignant liver diseases (cirrhosis and chronic hepatitis), 18 from acute pancreatitis, and 20 from chronic pancreatitis. The remaining subjects were controls without pancreatic pathology. Bile salt-dependent lipase was determined by a sandwich enzyme-linked immunosorbent assay using polyclonal antibodies. Amylase and CA 19-9 antigen were also determined. In sera from control patients, the mean level of bile salt-dependent lipase was 1.5 micrograms/L. This level is quite similar to that of patients with benign liver diseases (1.1 micrograms/L) and with chronic pancreatitis (1.4 micrograms/L), but it was raised to 3.5 micrograms/L in patients with acute pancreatitis and decreased to 0.5 microgram/L in subjects with pancreatic adenocarcinoma. Thirty percent of control subjects and 73% of cancer patients had a bile salt-dependent lipase serum level below the cutoff value of 0.5 microgram/L. In acute pancreatitis, 11 of 16 subjects had levels above 1.5 micrograms/L. Amylase level largely increased in acute pancreatitis but was normal in all other groups. Concerning CA 19-9 antigen, 65% of control patients and > 80% of patients with nonmalignant pancreatic or liver diseases had normal levels. In sera from cancer patients, 80% presented with high levels. Accordingly, 36 of 38 patients with pancreatic cancer had either low serum levels of bile salt-dependent lipase (< 0.5 microgram/L) or high values of CA 19-9 antigen (> 37 U/ml; sensitivity 95%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Pancreatic bile salt dependent lipase from cod (Gadus morhua): purification and properties.

The enzymatic basis for cod digestive lipolysis has been investigated. Lipase activity was found in aqueous extracts from pyloric caeca as well as in pancreatic tissue surrounding the caeca and the bile duct. A bile salt-dependent lipase (BSDL) was purified from either defatted powder of cod pyloric caeca or aqueous pancreatic extracts by combined affinity chromatography on cholate-Sepharose and gel filtration on Sephacryl S-200 HR. By SDS-PAGE analysis the molecular weight of purified cod BSDL was estimated to 60 kDa. The enzyme was totally dependent on bile salts for hydrolysis of insoluble fatty acid esters. Antiserum raised against purified cod BSDL reacted specifically with selected mammalian pancreatic BSDLs by Western blot analysis. Results presented in this paper strongly suggest that the bile salt-dependent lipase is the only pancreatic enzyme involved in lipid digestion in cod. The enzyme has been characterized and compared to human pancreatic BSDL with respect to substrate specificity, temperature- and pH-dependence and inhibitors. Both soluble and insoluble fatty acid esters were hydrolysed and the enzyme was 1,3-specific in hydrolysis of triolein. The enzyme was inhibited by di-isopropyl fluorophosphate and phenyl boronic acid, but not significantly by phenyl methyl sulfonyl fluoride. The cod BSDL is probably homologous to mammalian pancreatic BSDLs.

Amino Acids↗
PubMed1991

N-Butyl-N-methyl-4-nitrophenyl carbamate as a specific active site titrator of bile-salt-dependent lipases.

The effect of a series of synthetic carbamates on the human (milk or pancreatic) bile-salt-dependent lipase (cholesterol esterase) was examined. N-isopropyl-O-phenyl, N-methyl-O-phenyl, N-butyl-(4-nitrophenyl), N-phenyl-(4-nitrophenyl), N-butyl-N-methyl and N-pentyl-O-phenyl carbamates were inhibitors of the enzyme activity, while O-isopropyl-N-phenyl, O-methyl-N-phenyl, O-benzyl-N-isopropyl and O-cyclohexyl-N-phenyl carbamates were not even recognized by the enzyme. The N-alkyl chain length is essential for the enzyme inhibition and N-butyl-(4-nitrophenyl) or N-pentyl-O-phenyl carbamates are more potent inhibitors than N-methyl-O-phenyl or N-isopropyl carbamates. The inhibition by reactive carbamates fits the criteria for mechanism-based inhibition: the inhibition is first-order with time, shows saturation kinetics with increasing carbamate concentration and leads to an inactive stoichiometric enzyme-inhibitor complex; the enzyme activity can be protected by a competitive inhibitor. Evidence is shown that the enzymatic nucleophilic attack of carbamates is directed at the carbonyl carbon atom and not the nitrogen atom. The inhibition of bile-salt-dependent lipase does not occur consecutive to the formation of a reactive isocyanate derivative of carbamate but via a tetrahedral intermediate involving essential residues implicated in the enzyme catalytic site. This intermediate evolves by liberation of alcohol (or phenol) and formation of an inactive carbamyl enzyme. Among the carbamates tested, N-butyl-N-methyl-(4-nitrophenyl) carbamate specifically inhibits the bile-salt-dependent lipase; the release of 4-nitrophenol from this carbamate is directly proportional to the enzyme inhibition and it may be defined as a specific active-site titrator for bile-salt-dependent lipases.

Animals↗
PubMed1991

The length of pregnancy: an echographic reappraisal.

A study of 998 pregnant women was conducted to estimate the random errors in dating pregnancy by menstrual history and by ultrasound measurements of the fetus (crown-rump length or biparietal diameter before 20 weeks, menstrual age) as well as the biological variability of pregnancy length. The latter was found to be 7.74 days. The random errors made when dating pregnancy by menstrual history and by ultrasound measurements were 7.16 days and 4.26 days, respectively. (All variabilities are expressed as standard deviations). The differences between the two methods were mainly attributable to some large errors occurring when dating pregnancy on the basis of menstrual history. Differences between methods of more than 10 days were observed in about 10% of our population. When the two methods were used on the same patients to define a pregnancy as preterm, at term, or postterm, discrepancies were found in about 7% of cases.

Female↗
PubMed1991

Change in bile salt dependent lipase in human breast milk during extended lactation.

Two hundred eighty-one milk samples collected from Zaïrian nonprivileged, undernourished mothers, in series of nine groups from 1 month to 18 months after parturition, and 66 milk samples collected from French privileged mothers in series of four groups from 2 days to 16 months postpartum, were analyzed for their lactose, lipid and protein contents. In addition, the activity of bile salt-dependent lipase (esterase), which may play an important role in the newborn infant's lipids digestion, was measured. After the first month postpartum, independent of the nutritional state of the mother, sugar and protein concentrations were identical. Lipid content of French mothers' milk was lower in transitional milk, but appeared constant in mature milk with an average value of 29.1 +/- 5.8 mg/mL of milk. In Zaïrian mothers' milk, the lipid content of mature milk plateaued at around 50-55 mg/mL independent of the stage of lactation. Bile salt-dependent lipase showed constant esterase activity within the lactation stage in privileged mothers' milk, but decreased by almost 80-90% during the first four months of lactation in undernourished mothers. The data suggest that milk from nonprivileged mothers may lose some of its ability to hydrolyze milk lipid esters, which could also be of consequence to the infant's normal growth in view of its effect on the esters of the lipid-soluble vitamins A, E and D.

Adult↗
PubMed1990

Hydrolysis of fluorescent pyrene-acyl esters by human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase.

Fluorescent esters containing pyrenedecanoic acid (P10) or pyrenebutanoic (P4) acid (P4cholesterol, P10cholesterol, P4- and P10-containing triacylglycerols) were synthesized and used as substrates for human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase from human milk. Both enzymes were purified by immunoaffinity chromatography. All fluorescent pyrene derivatives were hydrolyzed by pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase, but at different rates. The hydrolytic rates of the "short" acyl esters (P4-containing esters) were higher than those of the "long" ones (P10-containing esters). Conditions were optimized for sensitivity of the assay using fluorescent cholesteryl esters. The pH optimum was 7.5-8.0. Sodium cholate exhibited a stronger activating effect than taurocholate or taurodeoxycholate (maximal activation was achieved with 5 mmol/L cholate and with a molar ratio cholesteryl ester/cholate around 1:10). Both pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase from milk were strongly inhibited by the other amphiphiles tested, namely phosphatidylcholine and Triton X-100, and were inactivated by low concentrations (10 mumol/L) of the serine-reactive diethyl-paranitrophenyl phosphate (E600). Both enzymes were strongly inhibited by relatively low concentrations of plasma low density lipoproteins. These studies indicate that the fluorescent esters containing pyrene fatty acids can be used as substrates for assaying and investigating the properties of pancreatic carboxylic ester hydrolase as well as bile salt-stimulated lipase from milk.

Animals↗
PubMed1989

A cross-linked complex between horse pancreatic lipase and colipase.

The water soluble carbodiimide N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-p-toluolsulfona te was found to effectively covalently cross-link pancreatic colipase to lipase as evidenced by Western blotting experiments using antibodies directed either against lipase or colipase. Moreover the resulting covalent complex has a Mr consistent with a stoichiometry of 1 mol colipase per mol lipase. Cross-linked lipase and colipase retain their activity implying a correct covalent binding between the two proteins. The specificity of the lipase-colipase binding was further supported by the very low amount of cross-linked products when lipase or colipase alone were incubated in the presence of carbodiimide. The formation of a covalent lipase-colipase complex in the presence of carbodiimide clearly demonstrates that the binding between both proteins involves ion pairing. Furthermore, the formation of an active covalent complex strongly suggests that the lipase-colipase binding site is distinct from the colipase interfacial recognition site as well as from the lipase catalytic site.

Animals↗
PubMed1989

Inhibition of human milk bile-salt-dependent lipase by boronic acids. Implication to the bile salts activator effect.

The bile-salt-dependent lipase from human milk, which catalyzes the hydrolysis of the water-soluble substrate 4-nitrophenyl acetate and the water-insoluble substrate tributyrin, is competitively inhibited by phenyl boronic acid. This inhibitor does not interfere with the interaction of lipase either with the siliconized glass beads/water interface or with the activator bile-salt binding site. The boronic acid binds near or at the active site serine, since modification of this residue by diisopropylphosphofluoridate (DFP) was prevented by phenyl boronic acid. Phenyl boronic acid binds 15-fold as tightly to bile-salt-dependent lipase as does 4-nitrophenyl acetate. Therefore, phenyl boronic acid bears analogy to a substrate rather than to a tetrahedral intermediate analog. Bile salts such as sodium taurocholate which are non-essential activators for the milk lipase activity on water-soluble substrates decrease the Km as well as the enzyme inhibitor dissociation constant (Ki). They have a slight effect on kcat. These results are interpreted in terms of an increase of the stability of the enzyme-substrate tetrahedral intermediate and in general of any transition states for the formation and for the decomposition of these intermediates upon the enzyme bile salts interaction.

Adsorption↗
PubMed1989

Human milk bile-salt stimulated lipase: further investigations on the amino-acids residues involved in the catalytic site.

The bile-salt-stimulated lipase purified from human skim milk was modified with diisopropyl phosphofluoridate (DFP), N-ethyl-5-phenylisoxazolium-3'-sulfonate and ethoxyformic anhydride. These chemical modifications lead to the following results: (1) the inhibition of the enzyme by DFP is due to the phosphorylation of a single residue, probably a serine residue, which may represent the acylable group of the enzyme; (2) carbethoxylation of histidine residues leads to inhibition of the enzyme activity. Among the nine modified histidine residues, only one is essential for enzyme activity; (3) a free carboxyl group with a pKa of 5.4 is also involved in catalysis. These three essential residues are involved in the enzymatic hydrolysis of substrates whatever their physical state (soluble or emulsified). Upon treatment with DFP as well as with ethoxyformic anhydride, the enzyme remains able to bind to the model interface formed by siliconized glass-beads with almost the same efficiency (Kd between 4.1 and 7.4.10(-8) M) than the native bile-salt-stimulated lipase (Kd = 6.3.10(-8) M). Moreover, the modified and native enzymes occupy the same interfacial area (4000-4600 A2/molecule). By contrast, the enzyme modified by N-ethyl-5-phenylisoxazolium-3'-sulfonate reagent presents an interfacial area close to that of a denatured protein of size (approximately 18,300 A2/molecule) and a Kd more than 20-fold higher than that of the native enzyme. Solvent isotope effects measured on kcat/Km and kcat indicate that the catalytic mechanism of bile-salt-stimulated lipase involves transition states that are stabilized by hydrogen bonds as described in the two-step acylation-deacylation mechanism of serine enzymes.

Acylation↗
PubMed1989

Crystallization and preliminary X-ray study of horse pancreatic lipase.

Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.

Animals↗
PubMed1988

Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase.

A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).

Amino Acids↗
PubMed1988

Human monoclonal antibodies that protect mice against challenge with Pseudomonas aeruginosa.

Lymphocytes from healthy volunteers and from cystic fibrosis patients were transformed with Epstein-Barr virus and cultured at a limiting dilution to generate lymphoblastoid cell lines that secreted human monoclonal antibodies specific for lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Three cell lines (RM5, FDD7, and 11F9) produced immunoglobulin M (IgM) antibody species that reacted specifically with P. aeruginosa Fisher immunotypes 2, 4, and 5, respectively, and with LPS extracted from these immunotypes. A fourth cell line (9H10) produced a single IgM antibody species that recognized P. aeruginosa immunotypes 3, 6, and 7 and LPS extracted from them. Monoclonal antibodies secreted by cell lines RM5, FDD7, and 11F9 protected neutropenic mice prophylactically against challenge with P. aeruginosa immunotypes 2, 4, and 5, and those secreted by 9H10 protected against P. aeruginosa immunotypes 3 and 6 but did not protect against immunotype 7. In vivo experiments indicated that antibodies protected mice against infection by increasing the rate of bacterial clearance.

Animals↗
PubMed1987

Subcellular localization of cholesterol ester hydrolase in the human intestine.

Immunocytochemistry and subcellular fractionation were used to localize the cholesterol ester hydrolase in the human small intestine. A positive immunoreaction, when using antibodies directed against pancreatic cholesterol ester hydrolase, was mainly found in endocytotic vesicles. Moreover, a label by gold particles was observed in intercellular spaces where lymphatic tissue merges. No specific immunoreactivity was obtained with the mucosa when sera directed against human pancreatic chymotrypsinogen and human pancreatic lipase were used. Conventional subcellular fractionation was performed after extensive washing of enterocytes to rule out any possible contamination by pancreatic enzymes. In these conditions a bile salt-dependent cholesterol ester hydrolase activity was detected in the soluble fraction of cells. Data agree with the concept that the intestinal cholesterol ester hydrolase may have a pancreatic origin. The absorption, if any, of this enzyme by enterocytes seems specific since other pancreatic (pro)enzymes tested (lipase, chymotrypsinogen) are not detected in these cells.

Adult↗