Martine Fouchereau-Peron

Impact of ultrafiltration and nanofiltration of an industrial fish protein hydrolysate on its bioactive properties

BACKGROUND Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides. RESULTS The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin-like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene-related peptide (CGRP)-like peptides demonstrated an increase in CGRP-like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin-converting enzyme (ACE)-1 inhibitory activity, which were not increased by UF and NF fractionation. CONCLUSION Fractionation of an FPH using membrane separation, with a molecular weight cut-off adapted to the peptide composition, may provide an effective means to concentrate CGRP-like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut-off of the membrane only, as is currently done in the literature.

TEM-89 β-Lactamase Produced by a Proteus mirabilis Clinical Isolate: New Complex Mutant (CMT 3) with Mutations in both TEM-59 (IRT-17) and TEM-3

ABSTRACT TEM-89 (CMT-3) is the first complex mutant β-lactamase produced by a clinical strain of Proteus mirabilis (strain Pm 631). This new enzyme, which has a pI of 6.28, is derived from TEM-3 and has a single amino acid substitution also encountered in TEM-59 (inhibitor-resistant TEM β-lactamase IRT-17): Ser-130 to Gly. TEM-89 hydrolyzed penicillins to the same extent that TEM-3 did but lost almost all hydrolytic activity for cephalosporins and, like TEM-59, was highly resistant to inhibitors.

Purification of a functional competitive antagonist for calcitonin gene related peptide action from sardine hydrolysates

Calcitonin gene related peptide (CGRP) related molecules were purified from sardine hydrolysates prepared using 0.1% alcalase and two hours of hydrolysis. Gel exclusion chromatography and HPLC performed purification of these molecules. The purified molecules were characterised using specific CGRP radioimmunoassays and radioreceptoraasays. From 22 mg of crude extract, we obtained 14 µg of CGRP related molecules, the molecular weight determined by mass spectrophotometry was 6000 daltons. The biological activity of these molecules was analysed using the ability of CGRP to stimulate the adenylate cyclase activity in rat liver membranes. The purified molecules induced an inhibition of the CGRP stimulated adenylate cyclase activity, this effect was specific as no such effect was observed on the glucagon stimulated adenylate cyclase activity measured in the same rat liver membrane preparation. These results suggest that the purified molecules may act as antagonists for peptides that bind to CGRP receptors in rat liver membranes. These new antagonists may be of particular importance in various aspects of CGRP action in vertebrates.

Cortisol mobilizes mineral stores from vertebral skeleton in the European eel: an ancestral origin for glucocorticoid-induced osteoporosis?

Endogenous excess cortisol and glucocorticoid (GC) therapy are a major cause of secondary osteoporosis in humans. Intense bone resorption can also be observed in other vertebrates such as migratory teleost fish at the time of reproductive migration and during fasting when large amounts of calcium and phosphate are required. Using a primitive teleost, the European eel, as a model, we investigated whether cortisol could play an ancestral role in the induction of vertebral skeleton demineralization. Different histological and histomorphometric methods were performed on vertebral samples of control and cortisol-treated eels. We demonstrated that cortisol induced a significant bone demineralization of eel vertebrae, as shown by significant decreases of the mineral ratio measured by incineration, and the degree of mineralization measured by quantitative microradiography of vertebral sections. Histology and image analysis of ultrathin microradiographs showed the induction by cortisol of different mechanisms of bone resorption, including periosteocytic osteolysis and osteoclastic resorption. Specificity of cortisol action was investigated by comparison with the effects of sex steroids. Whereas, testosterone had no effect, estradiol induced vertebral skeleton demineralization, an effect related to the stimulated synthesis of vitellogenin (Vg), an oviparous specific phospho-calcio-lipoprotein. By contrast, the cortisol demineralization effect was not related to any stimulation of Vg. This study demonstrates GC-induced bone demineralization in an adult non-mammalian vertebrate, which undergoes natural bone resorption during its life cycle. Our data suggest that the stimulatory action of cortisol on bone loss may represent an ancestral and conserved endocrine regulation in vertebrates.

Possible role of calcitonin gene-related peptide in osmoregulation via the endocrine control of the gill in a teleost, the eel, Anguilla anguilla

Osmoregulation is a major challenge in aquatic animals involving a complex endocrine control. We investigated the potential role of calcitonin gene-related peptide (CGRP, a neuromediator in mammals) in the endocrine control of the gill in a teleost, the eel. Transfer from freshwater to seawater induced an hyperosmolality and a concomitant large increase in plasma CGRP levels. Specific CGRP binding sites were characterized in the gill and their number was up-regulated after seawater transfer. This study suggests that the endocrine control of gill function during osmoregulation may represent an ancient role of CGRP in vertebrates.

Postneurosurgical Meningitis Due to Proteus penneri with Selection of a Ceftriaxone-Resistant Isolate: Analysis of Chromosomal Class A β-Lactamase HugA and its LysR-Type Regulatory Protein HugR

ABSTRACT We report on a case of a postneurosurgical meningitis due to ceftriaxone-susceptible Proteus penneri, with selection of a ceftriaxone-resistant isolate following treatment with ceftriaxone. The isolates presented identical patterns by pulsed-field gel electrophoresis and produced a single β-lactamase named HugA with an isoelectric point of 6.7. The ceftriaxone-resistant isolate hyperproduced the β-lactamase (increase in the level of production, about 90-fold). The sequences of the hugA β-lactamase gene and its regulator, hugR, were identical in both P. penneri strains and had 85.96% homology with those of Proteus vulgaris. The HugA β-lactamase belongs to molecular class A, and the transcriptional regulator HugR belongs to the LysR family.

Characterisation and distribution of calcitonin gene-related peptide in a primitive teleost, the eel, Anguilla anguilla and comparison with calcitonin

Radioimmunoassay (RIA), radioreceptor assay and chromatography were used to study the occurrence of calcitonin gene-related peptide (CGRP) in a primitive teleost, the eel, Anguilla anguilla. Immunologically and biologically active CGRP-like molecules were found in brain, heart, kidney, liver, spleen and ultimobranchial body with the higher concentrations in brain, spleen and heart. Gel exclusion chromatography of heart and spleen extracts followed by SDS-PAGE showed that the eel CGRP-like molecules presented a molecular weight between 3.30 and 3.95 kDa similar to that of human CGRP. The wide distribution of CGRP reflects its multiple role as brain neuromediator and peripheral paracrine effector as described in mammals. In comparison, the distribution of calcitonin (CT) was much more restricted, immunologically and biologically active CT-like molecules being localised in the ultimobranchial bodies (UBB) that is the site of CT synthesis in non-mammalian vertebrates. In plasma, CGRP-like concentrations were 10 to 100 higher than those of CT. These high concentrations in a primitive teleost strengthen the possible endocrine role of CGRP in early vertebrates and emphasise the important role of this hormone in evolution.

Use of a Commercial Protease and Yeasts To Obtain CGRP-like Molecules from Saithe Protein

Different bioactive molecules, such as CGRP-like peptides, can be found in fish protein hydrolysates. Calcitonin gene-related peptide (CGRP) is a neuropeptide known to act as a potent arterial and venous vasodilator in humans. This study focuses on the industrial obtaining of CGRP-like molecules from saithe (Pollachius virens) byproduct, derived from the filleting process. Protein from P. virens was primarily hydrolyzed with Alcalase and later treated with Saccharomyces cerevisiae live cells. Treatment with Saccharomyces doubled the quantity of bioactive molecules obtained. The CGRP-like molecules were partially purified by chromatography, and the immunoreactive material was further analyzed for its CGRP-like bioactivity, using a specific radioreceptor assay. The concentration of CGRP-like molecules increased over 100-fold after purification. The bioactive molecules were able to induce cyclic AMP stimulation in rat liver membranes. Finally, partial sequencing of the bioactive peptide was performed, showing some homology with alpha-actin and myosin of several fish species.

CGRP stimulates gill carbonic anhydrase activity in molluscs via a common CT/CGRP receptor.

The physiological significance of calcitonin gene-related peptide (CGRP) during biomineralization was investigated by assessing the effect of human CGRP on the carbonic anhydrase activity in gill membranes of the pearl oyster, Pinctada margaritifera. Salmon CT and human CGRP were able to induce a 150% increase of the basal activity. No additive effect was observed suggesting that both activities are mediated by the same receptor. The CGRP-stimulated effect was specific as demonstrated by the inhibition produced by the CGRP antagonist, hCGRP8-37. So, CGRP by its specific action on gill carbonic anhydrase controls the calcification process, an ancient role both in invertebrates and non-mammalian vertebrates.

Evidence for the Presence of Molecules Related to the Neuropeptide CGRP in Two Cephalopods, Sepia officinalis and Nautilus macromphalus: Comparison with Its Target Organ Distribution

Calcitonin gene-related peptide (CGRP) is a neuropeptide mainly involved in brain and cardiovascular functions in mammals. We investigated its presence and potential roles in two cephalopods, Sepia officinalis and Nautilus macromphalus. CGRP-like, but not calcitonin (CT)-like, molecules were detected by specific radioimmuno- and radioreceptor assays in the brain, optic lobes, branchial heart or afferent branchial vein and kidney. Gel exclusion chromatography of cephalopod brain extracts, followed by SDS-PAGE, indicated that CGRP-like molecules had a molecular weight of around 3 kDa, close to that of human CGRP. The distribution of CGRP target organs was characterized by binding studies in cuttlefish. Specific CGRP binding sites were detected in the brain, optic lobes, and kidney, indicating potential autocrine/paracrine roles of CGRP. Specific CGRP binding sites were also detected in the gills and shell sac that do not contain the peptide itself, indicating potential endocrine roles of CGRP. Accordingly, high circulating levels of CGRP-like molecules were detected in hemolymph of both cuttlefish and nautilus, unlike the situation in mammals. CGRP binding sites were further characterized in the cuttlefish gills by the Scatchard method. Our study indicates that the brain neurotransmitter role of CGRP could represent an ancient role in metazoa, already present in cephalopods and conserved among vertebrates. In contrast, the endocrine role of CGRP, which was suggested in cephalopods and also present in teleosts, may have been lost during the evolution of the tetrapod lineage. Our data support the hypothesis that CGRP represents the ancestral molecule of the CT/CGRP family appeared in metazoa before the vertebrate emergence.