The early ontogeny of digestive and metabolic enzyme activities in two commercial strains of arctic charr (Salvelinus alpinus L.)

The extent to which growth performance is linked to digestive or energetic capacities in the early life stages of a salmonid species was investigated. We compared two strains of Arctic charr known to have different growth potentials during their early development (Fraser and Yukon gold). Trypsin, lipase, and amylase activities of whole alevins were measured at regular intervals from hatching through 65 days of development. To assess catabolic ability, we also measured five enzymes representing the following metabolic pathways: amino acid oxidation (amino aspartate transferase), fatty acid oxidation (beta-hydroxy acyl CoA-dehydrogenase), tricarboxylic acid cycle (citrate synthase), glycolysis (pyruvate kinase), and anaerobic glycolysis (lactate dehydrogenase). The measurement of these enzyme activities in individual fish allowed a clear evaluation of digestive capacity in relation to energetic demand. We also compared triploid and diploid individuals within the Yukon gold strain. For the whole experimental period, diploid Yukon gold fish exhibited the highest growth rate (1.08+/-0.18% length/day) followed by triploid Yukon gold fish (1.00+/-0.28% length/day) and finally Fraser strain fish (0.84+/-0.28% length/day). When differences in enzyme activities were observed, the Fraser strain showed higher enzyme activities at a given length than the Yukon gold strain (diploid and triploid). Higher growth performance appears to be linked to lower metabolic capacity. Our results suggest that fish may have to reach an important increase in the ratio of digestive to catabolic enzyme activities or a leveling off of metabolic enzyme activities before the onset of large increases in mass.     

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.     

Epithelial and stromal uterine cells cultured in vitro protect bovine sperm from hydrogen peroxide

It is known that large amounts of leukocytes colonize the uterus, and that these leukocytes can produce considerable quantities of hydrogen peroxide (H2O2) and other reactive oxygen species that are toxic to sperm. It has been shown recently that oviductal fluid has a catalase that helps to maintain sperm motility. Therefore, the current experiment was performed to determine if a similar mechanism of protection exists against peroxides within uterine cells. Sperm motility and velocity were recorded after a 6h incubation in 1) conditioned media in the presence of endometrial cells, 2) conditioned media without endometrial cells, 3) control media (48h without cells) over endometrial cells, or 4) control media alone. All these treatments were performed in the presence or absence of added catalase. Conditioned media, endometrial cells and catalase had a significant positive effect on the maintenance of sperm motility and velocity. Addition of anti-catalase antibodies did not neutralize the beneficial effect of the conditioned media. However, the concentrations of aromatic amino acids, known substrates for sperm amino acid oxidase, were significantly lower in uterine conditioned media as compared to control medium. This reduction of aromatic amino acids was in correlation with reduced H2O2 production by sperm as estimated by chemiluminescence. These results suggest that epithelial and stromal uterine cells do not maintain sperm motility by secreting catalase in the conditioned media, but rather by reducing the levels of aromatic amino acids and thus of peroxides generated in the presence of spermatozoa.     

Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins

Endonuclease assays of the H-N-H proteins encoded by two group I introns in the Chlamydomonas moewusii chloroplast psbA gene revealed that the CmpsbA·1 intron specifies a site-specific DNA endonuclease, designated I-CmoeI. Like most previously reported intron-encoded endonucleases, I-CmoeI generates a double-strand break near the insertion site of its encoding intron, leaving 3′ extensions of 4 nt. This enzyme was purified from Escherichia coli as a fusion protein with a His tag at its N-terminus. The recombinant protein (rI-CmoeI) requires a divalent alkaline earth cation for DNA cleavage (Mg²⁺ > Ca²⁺ > Sr²⁺ > Ba²⁺). It also requires a metal cofactor for DNA binding, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date. rI-CmoeI binds its recognition sequence as a monomer, as revealed by gel retardation assays. K_m and k_cat values of 100 ± 40 pM and 0.26 ± 0.04 min^–1, respectively, were determined. Replacement of the first histidine of the H-N-H motif by an alanine residue abolishes both rI-CmoeI activity and binding to its substrate. We propose that this conserved histidine residue plays a role in binding the metal cofactor and that such binding induces a structural modification of the enzyme which is required for DNA recognition.     

Conformationally constrained opioid peptide analogs with novel activity profiles

Novel conformationally constrained opioid peptide analogs with antagonist, mixed agonist/ antagonist or agonist properties were developed. TIP(P)-related antagonists showed unprecedented antagonist potency and receptor selectivity, and may have potential for use in analgesia in combination with agonists. A definitive model of their receptor-bound conformation was developed. Three prototype mixed agonist/ antagonists were discovered. They represent the only known compounds with this pharmacological profile and, as expected, one of them was shown to be a potent analgesic and to produce no dependence and less tolerance than morphine. Novel dipeptide derivatives turned out to be potent and selective agonists. Because of their low molecular weight and lipophilic character, these compounds may cross the blood-brain barrier and, thus, may have potential as centrally acting analgesics.     

Biochemical characterization and structure–function relationship of two plant NCS2 proteins,the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent K_M values were determined with 10.12 μM, 4.85 μM and 19.95 μM, respectively for NAT3 and 1.74 μM, 2.44 μM and 29.83 μM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton–nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1–7 and TM8–14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure–function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.     

Functional calcitonin gene-related peptide receptors are formed by the asymmetric assembly of a calcitonin receptor-like receptor homo-oligomer and a monomer of receptor activity-modifying protein-1

In addition to their interactions with hetero-trimeric G proteins, seven-transmembrane domain receptors are now known to form multimeric complexes that can include receptor homo- or hetero-oligomers and/or accessory proteins that modulate their activity. The calcitonin gene-related peptide (CGRP) receptor requires the assembly of the seven-transmembrane domain calcitonin receptor-like receptor with the single-transmembrane domain receptor activity-modifying protein-1 to reach the cell surface and be active. However, the relative stoichiometric arrangement of these two proteins within a receptor complex remains unknown. Despite recent advances in the development of protein-protein interactions assays, determining the composition and stoichiometric arrangements of such signaling complexes in living cells remains a challenging task. In the present study, we combined bimolecular fluorescence complementation (BiFC) with bioluminescence resonance energy transfer (BRET) to probe the stoichiometric arrangement of the CGRP receptor complex. Together with BRET competition assays, co-immunoprecipitation experiments, and BiFC imaging, dual BRET/BiFC revealed that functional CGRP receptors result from the association of a homo-oligomer of the calcitonin receptor-like receptor with a monomer of the accessory protein receptor activity-modifying protein-1. In addition to revealing the existence of an unexpected asymmetric oligomeric organization for a G protein-coupled receptor, our study illustrates the usefulness of dual BRET/BiFC as a powerful tool for analyzing constitutive and dynamically regulated multiprotein complexes.     

Phenotypic flexibility of digestive system in Atlantic cod (Gadus morhua)

This study examined the restoration of the digestive capacity of Atlantic cod (Gadus morhua Linnaeus) following a long period of food deprivation. Fifty cod (48 cm, 1 kg) were food-deprived for 68 days and then fed in excess with capelin (Mallotus villosus Müller) on alternate days. Ten fish were sampled after 0, 2, 6, 14 and 28 days and the mass of the pyloric caeca, intestine and carcass determined. Two metabolic enzymes (cytochrome c oxidase and citrate synthase) were assayed in white muscle, pyloric caeca and intestine, and trypsin activity was measured in the pyloric caeca. A delay of 14 days was required before body mass started to increase markedly, whereas most of the increase in mass of both the pyloric caeca and intestine relative to fish length occurred earlier in the experiment. By day 14, the activities of trypsin and citrate synthase in the pyloric caeca as well as citrate synthase in the intestine had reached maxima. The growth of the digestive tissues and restoration of their metabolic capacities thus occur early upon refeeding and are likely required for recovery growth to take place. The phenotypic flexibility of the cod digestive system is therefore remarkable: increases in trypsin activity and size of pyloric caeca resulted in a combined 29-fold increase in digestive capacity of the fish during the refeeding period. Our study suggests that Atlantic cod are able to cope with marked fluctuations in food availability in their environment by making a rapid adjustment of their digestive capacity as soon as food availability increases.