CENTA as a chromogenic substrate for studying beta-lactamases.

CENTA, a chromogenic cephalosporin, is readily hydrolyzed by beta-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of beta-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.

Cloning, transcription and chromosomal localization of the porcine whey acidic protein gene and its expression in HC11 cell line.

The whey acidic protein (WAP) is the major whey protein of rodent, rabbit and camel. Recently, it was identified in the milk of swine (Simpson et al., 1998. J. Mol. Endocrinol. 20, 27-35). In this paper, the cloning of the pig WAP cDNA and of bacterial artificial chromosome (BAC) construct containing the entire porcine WAP gene is reported. The comparison of the coding sequence of the pig WAP gene to rodent or lagomorph WAP sequence already published demonstrated that only exon sequences are partially conserved. The porcine WAP gene was localized on the subtelomeric region of the chromosome 18. The estimation of the expression of the swine WAP gene in the mammary gland from lactating animals revealed a high level of expression. In order to compare the expression level of the porcine WAP gene from the large genomic fragment which contained 70 kb downstream and 50 kb upstream the pig WAP gene or the smaller one (1 kb downstream and 2.4 kb upstream), these two genomic fragments were transfected in HC11 cell line. The BAC construct was expressed 15 times higher than the plasmid when reported to the integrated copy number. This report suggests that the HC11 cell line is a useful tool to identify the regulatory sequences of milk protein genes.

Caseins and casein hydrolysates. 1. Lipoxygenase inhibitory properties.

Whole casein from bovine origin, the different casein subtypes alpha, beta, and kappa, and the related dephosphorylated proteins were assayed as modulators of soybean lipoxygenase 1 activity and were found to inhibit it. To define the lipoxygenase inhibitory domain, whole casein and beta-casein were digested by proteases (trypsin, clostripain, and subtilisin). The beta-casein tryptic digest and the tryptic and subtilisin digests of whole casein retained their inhibitory properties. The tryptic beta-casein digest was the most potent inhibitor of lipoxygenase activity and was further fractionated by FPLC or HPLC. The collected peptides inhibited the lipoxygenase-catalyzed reaction to different extents. The active fractions were analyzed by ESI-MS, and the sequences of several lipoxygenase inhibitory peptides, corresponding mainly to the C-terminal moiety of beta-casein, were identified.

Caseins and casein hydrolysates. 2. Antioxidative properties and relevance to lipoxygenase inhibition.

The antioxidant activity of caseins and casein-derived peptides was evaluated by using three free radical producing reactions-the lipoxygenase- and AAPH-catalyzed oxidation of linoleic acid and the hemoglobin-catalyzed oxidation of linoleic acid hydroperoxide. Caseins and casein-derived peptides were able to inhibit enzymatic and nonenzymatic lipid peroxidation, suggesting they were preferred targets for the free radical intermediates. The antioxidative feature was not lost with the dephosphorylation or the proteolysis of the proteins. The fractionation of the tryptic beta-casein digest yielded peptides with antioxidant activity. A structure-function relationship between the amino acid sequence and the antioxidant capacity and effectiveness is proposed. In addition, indirect evidence suggested that the trapping of free radicals by the proteins/peptides was accompanied by the oxidation of proteins/peptides, according to a sequence-specific mechanism.

The efficiency of different IRESs (internal ribosomes entry site) in monocistronic mRNAS.

The IRES from poliovirus and from encephalomyocarditis virus (EMCV) added between the cap and the AUG initiator codon were strong inhibitors of chloramphenicol acetyltransferase gene expression in three different cell types. The poliovirus IRES also inhibited bGH (bovine growth hormone) cDNA expression in the HC11 mammary cell line when added between the rabbit whey acidic gene promoter and the cDNA whereas the HTLV-1 IRES showed a stimulatory effect in the same situation. RNA stem loops were added before HTLV-1 (SUR) and the BiP (Immunoglobulin heavy-chain Binding Protein) IRESs followed by the firefly luciferase gene under the control of Rous sarcoma virus (RSV) promoter. The RNA loops abolished the expression of the reporter gene almost completely. These data suggest that the different IRESs may favour or inhibit translation of monocistronic mRNA.

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.