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.

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.

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.

Occurrence of a CGRP-like molecule in siki (Centroscymnus coelolepsis) hydrolysate of industrial origin.

Fish protein hydrolysates (FPH) may have potential as bioactive components in functional foods as nutraceuticals. This study focused on the identification of calcitonin gene-related peptide (CGRP) molecules in FPH. CGRP is a neuropeptide belonging to the calcitonin/CGRP family and is known as potent arterial and venous vasodilator in humans. Hydrolysates of industrial origin were prepared from siki (Centroscymnus coelolepsis) heads and were analyzed for the presence of CGRP-like molecules using specific radioimmunoassays and radioreceptorassays. The biological activity of the CGRP-related molecules was assessed by their ability to stimulate the adenylate cyclase activity in rat liver membranes. They were finally purified using gel exclusion chromatography and high-performance liquid chromatography (HPLC). These molecules presented a molecular weight around 1500-2500 Da and were obtained with a purification factor of 79. The incorporation of FPH with CGRP-like molecules in functional foods could lead to the development of new useful products for health and nutrition markets.

Evolution of the CT/CGRP family: comparative study with new data from models of teleosts, the eel, and cephalopod molluscs, the cuttlefish and the nautilus.

In mammals, alternative splicing of the calcitonin gene generates two distinct peptides: calcitonin (CT), synthesised in the thyroid C cells and involved in the regulation of calcium metabolism, and calcitonin gene-related peptide (CGRP), brain neuromediator synthesised in the peripheral and central nerves. CGRP is well represented and molecularly conserved during evolution whereas CT has not been detected in any of the invertebrates analysed so far. In order to better understand the evolution of this CT/CGRP peptide family we reviewed the major data concerning its evolution from the literature and our recent data obtained in models of teleosts and cephalopod molluscs. The presence of both CGRP-like molecules and its specific bindings sites in the central nervous system of eel, cuttlefish and nautilus, suggests that the brain neurotransmitter role of CGRP could represent an ancient role in metazoa, already present in cephalopods and conserved among vertebrates, as still observed in mammals. In contrast, the presence of CGRP specific binding sites, and not the peptide itself, in the gills suggests an endocrine role for CGRP, in cephalopods and teleosts, that may have been lost during the evolution of the tetrapod lineage. These data, and the absence of CT-like molecules that we observed in cephalopods, support the hypothesis that CGRP represents the ancestral molecule of the CT/CGRP family, appeared in metazoa before the vertebrate emergence. The distinction between CT and CGRP receptors appears to be an event posterior to the emergence of ecdysozoan and lophotrochozoan protostomes, probably in relation to the CT appearance. The evolution of the CT/CGRP peptide family is probably similar to the evolution of the CT/CGRP receptor family. In fact, the genic duplication that induced the appearance of the two separate molecules, CT and CGRP, may constitute an event close to that, which induced the appearance of the two specific receptors. These events remain to be further studied in order to better understand the peptide and receptor evolution of the CT/CGRP family.

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.

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.

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.

Substitution of Met-69 by Ala or Gly in TEM-1 beta-lactamase confer an increased susceptibility to clavulanic acid and other inhibitors.

In some inhibitor-resistant TEM-derived beta-lactamases, Met-69 is substituted by Leu, Ile or Val. Residue 69 is located in a region of strong structural constraints, at the beginning of H2 alpha-helix, and in the vicinity of B3 and B4 beta-strands. Analysis of the three-dimensional structure of TEM-1 beta-lactamase suggests that alteration of the substrate-binding site can be produced by changes of the size of residue 69 side chain. Met-69 was substituted by alanine or glycine in TEM-Bs beta-lactamase (a TEM-1-related enzyme) using site-directed mutagenesis. The minimum inhibitory concentrations of the mutants compared with the wild-type revealed an increased susceptibility to beta-lactamase inhibitor-beta-lactam combinations and to first-generation cephalosporins. Comparing the Met69Ala and Met69Gly beta-lactamases with TEM-Bs, K(m) constants of the mutants showed an increased affinity for most beta-lactams but the kcat for most substrates did not change substantially. Mutants also demonstrated lower IC50 for the three inhibitors (clavulanic acid, tazobactam and sulbactam). The two substitutions of the residue 69 by alanine and glycine had a noticeable effect on K(m) values of TEM-Bs beta-lactamase, and on affinity for beta-lactamase inhibitors.

Postneurosurgical meningitis due to Proteus penneri with selection of a ceftriaxone-resistant isolate: analysis of chromosomal class A beta-lactamase HugA and its LysR-type regulatory protein HugR.

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 beta-lactamase named HugA with an isoelectric point of 6.7. The ceftriaxone-resistant isolate hyperproduced the beta-lactamase (increase in the level of production, about 90-fold). The sequences of the hugA beta-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 beta-lactamase belongs to molecular class A, and the transcriptional regulator HugR belongs to the LysR family.