Human pancreatic cancer is characterized by an alteration in fucose-containing surface blood group antigens such as H antigen, Lewis b, Lewis y, and sialyl-Lewis. These carbohydrate determinants can be synthesized by sequential action of alpha(2,3) sialyltransferases or alpha(1,2) fucosyltransferases (Fuc-T) and alpha(1,3/1,4) fucosyltransferases on (poly)N-acetyllactosamine chains. Therefore, the expression and the function of seven fucosyltransferases were investigated in normal and cancer pancreatic tissues and in four pancreatic carcinoma cell lines. Transcripts of FUT1, FUT2, FUT3, FUT4, FUT5, and FUT7 were detected by RT-PCR in carcinoma cell lines as well as in normal and tumoral tissues. Interestingly, the FUT6 message was only detected in tumoral tissues. Analysis of the acceptor substrate specificity for fucosyltransferases indicated that alpha(1,2) Fuc-T, alpha(1,3) Fuc-T, and alpha(1,4) Fuc-T were expressed in microsome preparations of all tissues as demonstrated by fucose incorporation into phenyl beta-d-galactoside, 2'-fucosyllactose, N-acetyllactosamine, 3'-sialyl-N-acetyllactosamine, and lacto-N-biose. However, these fucosyltransferase activities varied between tissues. A substantial decrease of alpha(1,2) Fuc-T activity was observed in tumoral tissues and cell lines compared to normal tissues. Conversely, the activity of alpha(1,4) Fuc-T, which generates Lewis a and sialyl-Lewis a structures, and that of alpha(1,3) Fuc-T, able to generate a lactodifucotetraose structure, were very important in SOJ-6 and BxPC-3 cell lines. These increases correlated with an enhanced expression of Lewis a, sialyl-Lewis a, and Lewis y on the cell surface. The activity of alpha(1,3) Fuc-T, which participates in the synthesis of the sialyl-Lewis x structure, was not significantly modified in cell lines compared to normal tissues. However, the sialyl-Lewis x antigen was expressed preferentially on the surface of SOJ-6 and BxPC-3 cell lines but was not detected on Panc-1 and MiaPaca-2 cell lines suggesting that several alpha(1,3) Fuc-T might be involved in sialyl-Lewis x synthesis.

The effect of ethanol metabolism on the energetic parameters and intracellular pH of the isolated perfused rat liver from fed rats was studied by phosphorus-31 nuclear magnetic resonance spectroscopy. This technique allowed us to analyze nondestructively and in real time the role of low oxygen tension on the possible injurious effect of ethanol on the liver cells. A quantitative analysis of nuclear magnetic resonance data recorded on a perfused rat liver within a 30 mm diameter probe has been performed at 80.9 MHz. Under normoxic and normothermic conditions, the levels of phosphorylated metabolites detected by nuclear magnetic resonance were 2.8, 0.3 and 2 mumoles per gm liver wet weight for ATP, ADP and inorganic orthophosphate, respectively. The cytosolic pH was 7.25 +/- 0.05. During a period of 4 min of hypoxia induced by reducing the perfusion flow rate to 25% of its initial value (i.e., from 12 ml to 3 ml per min per 100 gm body weight), the level of ATP dropped to 2.2 mumoles per gm liver wet weight. Concomitantly, ADP and inorganic orthophosphate increased to 0.6 and 3.3 mumoles per gm liver wet weight. Cytosolic pH fell to 7.02 +/- 0.05. Perfusion of the liver with a Krebs medium containing 70 mM (0.4%) ethanol induced a sharp decrease in intracellular inorganic orthophosphate to reach 1.3 mumole per gm liver wet weight and after a lag time of 4 to 6 min, a decrease in ATP level (2.15 mumoles per gm liver wet weight). A large increase in phosphomonoesters (mainly sn-glycerol 3-phosphate) up to 6 mumoles per gm liver wet weight was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of our study was to define the mechanism by which cholesterol uptake is inhibited by lecithin but not by lysolecithin. The work compared the cholesterol uptake by everted rat jejunal sacs from bile salt-lecithin-cholesterol or bile salt-lysolecithin-cholesterol micelles. The micellar size and the cholesterol saturation were measured. The size or molecular weight increases when the lecithin concentration rises, and the cholesterol uptake decreases and leads to zero when the micelles contain more than 30% lecithin. The size of bile salt-lysolecithin-cholesterol micelles is smaller than that of lecithin micelles in comparable molar ratios. Consistent with this result is the fact that, for a given phospholipid concentration, cholesterol uptake is greater in the presence of lysolecithin than in the presence of lecithin. The diffusion rate of the micelles through the unstirred water layer decreases when micellar size increases. However, the comparison of uptakes from lecithin or lysolecithin micelles similar in size and in cholesterol saturation showed that the cholesterol uptake is still lower for lecithin micelles. This shows that with larger micelles some factor other than micellar size and cholesterol content of the micelles is important. We observe that lysolecithin absorption is 15-fold greater than lecithin absorption. We suggest that lysolecithin absorption results in a rapid supersaturation with cholesterol leading to cholesterol absorption.

Feto-acinar pancreatic protein (FAPP) characterized by mAbJ28 reactivity is a specific component associated with ontogenesis and behaves as an oncodevelopment-associated antigen. We attempted to determine whether pancreatic tumoral SOJ-6 cells are expressed at their surface FAPP antigens and to examine if specific antibodies directed against these FAPP epitopes could decrease the growth of pancreatic tumors in a mice model. For this purpose, we used specific antibodies against either the whole FAPP, the O-glycosylated C-terminal domain, or the N-terminal domain of the protein. Our results indicate that SOJ-6 cells expressed at their surface a 32-kDa peptide corresponding to the C-terminal domain of the FAPP. Furthermore, we show, by using endoproteinase Lys-C or geldanamycin, a drug able to impair the FAPP secretion, that this 32-kDa peptide expressed on the SOJ-6 cell surface comes from the degradation of the FAPP. Finally, an in vivo prospective study using a preventative tumor model in nude mice indicates that targeting this peptide by the use of mAb16D10 inhibits the growth of SOJ-6 xenografts. The specificity of mAb16D10 for pancreatic tumors and the possibility to obtain recombinant structures of mucin-like peptides recognized by mAb16D10 and mAbJ28 are promising tools in immunologic approaches to cure pancreatic cancers.

The fetoacinar pancreatic protein (FAP), characterized by the mAb J28, is an oncofetal form of bile salt dependent lipase (BSDL), the expression of which is related to pancreatic differentiation and neoplastic processes. Because the J28 epitope, recognized by mAb J28, is suggested to be dependent upon carbohydrates, we have attempted to gain information about the structure of this epitope. Indeed, treatment of FAP with sodium periodate abolished the reactivity of the protein to mAb J28, which demonstrates the implication of oligosaccharides in the structure of the J28 epitope. FAP offers both O-linked and N-linked carbohydrate structures, of which, as we have determined, one is involved. Peptides obtained after cyanogen bromide cleavage were desialylated then separated by affinity chromatography on an immobilized peanut agglutinin agarose column. The peptide retained on this column carried out the reactivity with the mAb J28. Although some differences in amino acid analysis were observed, the N-terminal sequence of this peptide correlates with that of the C-terminal part of the enzyme. Carbohydrate analysis of the peptide bearing the J28 epitope revealed fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, and N-acetylneuraminic acid. The competition observed between mAb J28 and Ulex europaeus I lectin for binding to the J28 epitope suggested that fucose residue alpha (1-2) linked to a galactose residue was implicated in the structure of the J28 epitope. Alternatively, the loss of the mAb J28 reactivity upon treatment of FAP either with bovine kidney or bovine epididymis fucosidase was observed indicating that fucose residues linked at the alpha (1-2) and alpha (1-6) positions may be involved in the establishment of the structure of the J28 epitope. These observations suggest that mAb J28 recognized a particular fucosylated O-linked oligosaccharide structure located at the mucin-like extended C-terminal part of FAP.

Several α(1,3/1,4) fucosyltransferases expressed in human pancreatic cancer cells can participate in the biosynthesis of cell surface sialyl‐Lewis a and sialyl‐Lewis x antigens that contribute to hematogenous metastatis. Previously, we observed a significant increase of the α(1,4) fucosyltransferase activity in tumoral pancreatic cell lines, suggesting that FUT3 could be involved in the sialyl‐Lewis antigen expression. Therefore, we invalidated the expression of FUT3 by expressing FUT3 antisense sequence in the human pancreatic tumor BxPC‐3 cell line, which expresses the α(1,4) fucosyltransferase activity and harbors the cell surface sialyl‐Lewis antigens. The decrease of FUT3 transcript after transfection of antisense cDNA of FUT3 in these cells results in a substantial reduction of sialyl‐Lewis antigen expression on cell surface. This decreased antigen expression was associated with an inhibition of adhesive properties to E‐selectin and a decrease of metastatic power of FUT3 antisense‐transfected BxPC‐3 cells as tested in nude mice. Our study provides evidence that the expression level of FUT3 may regulate the expression of sialyl‐Lewis a and sialyl‐Lewis x surface antigens and consequently could play an important role in metastatic properties of human pancreatic cancer cells. Int. J. Cancer 88:558–565, 2000. © 2000 Wiley‐Liss, Inc.

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We assayed the lipid content of bile from rats that had been fed either a standard diet (5% fat) or a high fat diet (25% fat, 1.2% cholesterol) in the presence or in the absence of various dietary fibers (namely, wheat bran, pectin and cellulose). The cholesterol concentration in bile from rats fed the high fat diet plus wheat bran or pectin was lower than that of the rats fed the high fat, high cholesterol diet without fiber. Bile phospholipids did not vary significantly from one group to another. In comparison to the standard diet, the high fat, high cholesterol diet led to a greater ratio of primary to secondary bile salts and a higher level of glycoconjugates. The observed differences may be explained by a variation in the metabolism of bile salts brought about by the difference in diet.