Catherine Wicker-Planquart

Effects of dietary proteins on some pancreatic mRNAs encoding digestive enzymes in the pig

Abstract The response of pancreatic protease syntehsis to fish meal-enriched diets was investigated in the pig, which is generally held to be a suitable model for human digestive physiology. In three sets of experiments, pigs were fed either 7, 17, 48, or 68% protein diets for 8 days (1st set), or 17 or 48% protein diets for 3, 6, or 7 days (2nd set), or 7 or 48% protein diets for 4 days (3rd set). At the end of each experiment, the pancreata were removed for biochemical and gene expression assays. The specific activities of amylase, lipase, chymotrypsin, trypsin, and elastase decreased as the result of the 7% protein diet after an 8-day feeding period (1st set). In the same experiment, all the enzyme-specific activities (those of amylase, lipase, chymotrypsin, elastase as well as carboxypeptidases A and B) increased in response to the 48% protein diet, the most strongly affected enzyme being chymotrypsin. Only chymotrypsin and carboxy-peptidase A-specific activities were further increased after feeding with the 68% protein diet for 8 days as compared with the 48% protein diet. The amylase, lipase, and trypsinogen mRNA levels remained unchanged throughout the experiments, but the mRNA encoding procarboxypeptidase A2 decreased, and that coding for chymotrypsinogen was enhanced after the animals had been fed the experimental diets for 3 days, but showed no change thereafter. Procarboxypeptidase B mRNA increased slightly only after a 6-day feeding period. When pigs were fed the 7 and 48% protein diets for 4 days, the enzymes synthesized in vitro in pancreatic lobules were correlated with the relative levels of the corresponding mRNAs, as measured by means of an in vitro cell-free reticulocyte-lysate translation system: both amylase and carboxypeptidases specific activities and their mRNA levels decreased slightly, while those of serine proteases increased. It was concluded that the biosynthesis of each serine protease was regulated separately and transiently at the pre-translational level. On the other hand, it seems very likely that amylase may be regulated at the translational level, while a multiple-level control process may take place in the case of procarboxypeptidase A2.

Primary structure of rat pancreatic lipase mRNA

The sequence of a rat pancreatic lipase mRNA was determined. The data have been assigned the following accession number, X61925, in the EMBL data library. The total length of the messenger is 1531 nucleotides, plus a poly(A) stretch of about 60 nucleotides. A 72‐nucleotides 5′‐noncoding region is followed by a 1419‐nucleotides open reading frame which encodes a protein of 473 amino acids, including the 17 amino acid signal peptide. The mature enzyme (456 residues) has 6 additional C‐terminal amino acids, as compared with the amino acid sequence of pig (direct amino acid sequence), dog, man and rat isoenzyme from Genbank, M58369 (all deduced from the nucleotide sequence). A higher degree of homology exists between the amino acid sequence of rat mature enzyme with those of dog (88%), pig (75%) and man (75%) than with that of rat isolipase (74%).

Bovine Pancreatic Preproelastases I and II: Comparison of Nucleotide and Amino Acid Sequences and Tissue Specific Expression

Clones encoding bovine preproelastases I and II were isolated from a pancreatic cDNA library and were sequenced in order to define the structural characteristics of these enzymes. The bovine 947- and 884-nucleotide preproelastase I and II cDNAs encode proteins containing a signal peptide of the same length (16 amino acids), but with a slightly different number of amino acids for the activation peptide (10 and 12, respectively) and the mature enzyme (240 and 241, respectively). Considering amino acid sequences, each enzyme shares a high degree of identity (76-86%) within species. In contrast, only 55.3% identity is found between bovine elastases I and II. This difference could explain partly their own specificity. Analysis of the expression of the elastases in various bovine tissues demonstrated that they are specifically expressed in high levels in the pancreatic gland. These two approaches (structure and expression) allowed us to characterize the bovine pancreatic elastases I and II.

Interaction between Bacillus subtilis YsxC and ribosomes (or rRNAs)

The stoichiometry of YsxC ribosome subunit complex was evaluated. We showed that YsxC binding to the 50S ribosomal subunit is not affected by GTP, but in the presence of GDP the stoichiometry of YsxC‐ribosome is decreased. YsxC GTPase activity was stimulated upon 50S ribosomal subunit binding. In addition, it is shown for the first time that YsxC binds both 16S and 23S ribosomal RNAs.

The C-terminal α-helix of YsxC is essential for its binding to 50S ribosome and rRNAs.

YsxC is an essential P‐loop GTPase that interacts with the 50S subunit of the ribosome. The putative implication in ribosome binding of two basic clusters of YsxC, a conserved positively charged patch including R31, R116, H117 and K146 lying adjacent to the nucleotide‐binding site, and the C‐terminal alpha helix, was investigated. C‐terminal truncation variants of YsxC were unable to bind to both ribosome and rRNAs, whereas mutations in the other cluster did not affect YsxC binding. Our results indicate that the basic C‐terminal region of YsxC is required for its binding to the 50S ribosomal subunit.

Preduodenal Lipases and their Role in Lipid Digestion

Under normal physiological conditions the digestion and absorption of dietary lipids are highly efficiently processed. In humans, the diet generally contains 90 to 120 g of lipids (mostly triacylglycerols), more than 95% of which are absorbed, due to the interplay between the stomach, the small intestine, the liver, and the pancreas (Carey et al., 1983). Several steps can be distinguished in the processing of dietary lipids, including their emulsification, hydrolysis and solubilization, and, last, their uptake into the enterocyte. The emulsification of lipids starts in the stomach and is mediated by physical forces and is facilitated by the partial lipolysis of the dietary lipids (Carey et al., 1983). For a long time, the hydrolysis of dietary triglycerides was thought to begin in the intestinal lumen and to be catalyzed entirely by pancreatic lipase. The stomach was thought to be a transient storage organ, the role of which was limited to mixing lipids with the other nutriments and dispersing them as required. Although many authors observed the occurrence of lipolysis at the preduodenal level in humans and in several other species, the gastric phase of lipolysis was assumed to be negligible and to be of little or no significance in comparison with the intestinal step. Gastric lipolysis was even attributed to pancreatic contamination resulting from a duodeno-gastric reflux. At the beginning of the twentieth century, however, it was observed that gastric juice could hydrolyze fat. In 1901, Volhard stated that gastric lipase was the “ferment” present in gastric juice that was responsible for fat hydrolysis. Finally, the gastric origin of the lipase present in dog gastric juice was established by Hull and Keaton (1917) in dogs with Pavlov stomach under conditions precluding the possibility of any pancreatic contamination.