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The bifunctional enzyme acylpeptide hydrolase (APEH) is involved in important metabolic processes both as an exopeptidase and as an endopeptidase. Hence, the growing interest in the study of this protein and the need to set up in vitro assays for its characterization. This chapter describes two in vitro assays able to detect the activities of APEH, one for the exopeptidase activity and one for the endopeptidase activity. In particular, these assays have been set up on the two APEH isoforms from Antarctic fish, characterized by a distinct functionality and marked exo- and endopeptidase activities.

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48–64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives. The data suggest that the presence of sulphoxide-modified methionines is an important prerequisite for the substrates to be processed by APEH and that the residue is crucial for switching the enzyme activity from exo- to endoprotease. The cleavage occurs on residues placed on the C-terminal side of Met(O), with an efficiency depending on the methionine adjacent residues, which thereby may play a crucial role in driving and modulating APEH endoprotease activity.

An overproduction of free radicals or reactive oxygen species, often due to environmental factors, can alter the DNA structure and irreversibly modify proteins and lipids in the living cells. The superoxide anion (O2−) is one of the strongest oxidant molecules produced under oxidative stress conditions but it can be neutralized by the action of the enzymes SuperOxide Dismutases (SODs). In all the human tissues, SODs are essential for the prevention of serious diseases and the protection against oxidative stress damages. In the dermo-cosmetic sector, SODs have found promising applications, but their use is limited due to the loss of activity following the addition of the enzyme in the skin care formulas and the exposure of the skin to UV radiations and heat. Extremophile organisms, which proliferate in extreme physical and/or geochemical conditions, represent a potential source of stable SOD enzymes, able to function even in harsh conditions of high temperature, acid pH and long UV exposures. In the present study we investigated on a Mn-SOD deriving from the extremophilic bacterium Deinococcus radiodurans and, after its expression in E.coli, the Mn-SOD was characterized in terms of chemical and physical properties. Its extraordinary features in terms of UV resistance prompted us to investigate further about its potential applications in the dermo-cosmetic sector. It was expressed in Solanum lycopersicum (tomato) cell cultures with the main goal of developing a new ingredient, capable of keeping its ROS neutralizing activity once exposed to UV radiations and even when added to skin care formulas.

Bacteria isolated from different environments can be exploited for biocontrol purposes by the identification of the molecules involved in the antifungal activity. The present study was aimed at investigating antifungal protein compounds purified from a previously identified plant growth promoting bacterium, Pseudomonas protegens N isolated from agricultural land in northern Algeria. Therefore, a novel protein was purified by chromatographic and ultrafiltration steps and its antifungal activity together with growth-inhibition mechanism was evaluated against different fungi by plate-based assays. In addition, stereomicroscopy and transmission electron microscopy (TEM) was performed to explore the inhibition activity of the compound on spore germination processes. The protein, showing a molecular mass of about 100 kDa under native conditions, was revealed to be in the surface-membrane fraction and displayed an efficient activity against a variety of phytopathogenic fungi, being Alternaria the best target towards which it exhibited a marked fungicidal action and inhibition of spore germination. Moreover, the compound was able to significantly decrease fungal infection on tomato fruits producing also morphological aberrations on conidia. The obtained results suggested that the isolated compound could represent a promising agent for eco-friendly management of plant pathogens in agriculture.

Nuclear factor erythroid 2–related factor2 (Nrf2) is a redox-sensitive transcription factor. Its activation by low dietary intake of ligands leads to antioxidant effects (eustress), while pro-oxidant effects (oxidative distress) may be associated with high doses. NADPH oxidases (NOXs) and the mitochondrial electron transport chain are the main sources of intracellular ROS, but their involvement in the biphasic/hormetic activity elicited by Nrf2 ligands is not fully understood. In this study, we investigated the involvement of NOX expression and mitochondrial function in the hormetic properties of omega-3 typically present in fish oil (FO) and conjugated linoleic acid (CLA) in the mouse liver. Four-week administration of FO, at both low and high doses (L-FO and H-FO) improves Nrf2-activated cyto-protection (by phase 2 enzymes), while a significant increase in respiration efficiency occurs in the liver mitochondria of H-FO BALB/c mice. Eustress conditions elicited by low dose CLA (L-CLA) are associated with increased activity of phase 2 enzymes, and with higher NOX1-2, mitochondrial defences, mitochondrial uncoupling protein 2 (UCP2), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression, compared with controls. Steatogenic effects (lipid accumulation and alteration of lipid metabolism) elicited by high CLA (H-CLA) elicited that are associated with oxidative distress, increased mitochondrial complex I/III activity and reduced levels of phase 2 enzymes, in comparison with L-CLA-treated mice. Our results confirm the steatogenic activity of H-CLA and first demonstrate the role of NOX1 and NOX2 in the eustress conditions elicited by L-CLA. Notably, the negative association of the Nrf2/PGC-1α axis with the different CLA doses provides new insight into the mechanisms underlying the hormetic effect triggered by this Nrf2 ligand.

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

Loranthus europaeus is a well-known and important medicinal plant, with a long history of traditional medicine use. Several studies showed that it contains many bioactive compounds with a wide range of pharmacological effects. In light of these past researches, L. europaeus were chosen to consider its potential antimicrobial action. To this aim, different protocols were performed to selectively extract protein compounds, from L. europaeus yellow fruits, and evaluate the antimicrobial activity against four phytopathogenic fungi (Aspergillus niger, Alternaria spp., Penicillium spp., Botritis cinereus) and a number of foodborne bacterial pathogens (Listeria monocytogenes, Staphylococcus aureus strains, Salmonella Typhimurium and Escherichia coli) by using serial dilutions and colony formation assays. Results evidenced no antifungal activity but a notable bactericidal efficiency of a crude protein extract against two foodborne pathogens, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values between 0.2 and 0.5 mg/mL, being S. aureus strains the most susceptible bacteria. Moreover, a strong bactericidal activity against S. aureus M7 was observed by two partially purified protein fractions of about 600 and 60 kDa molecular mass in native conditions. Therefore, these plant protein extracts could be used as natural alternative preventives to control food poisoning diseases and preserve foodstuff avoiding health hazards of chemically antimicrobial applications.

Oxidative stress is a key mediator of autoimmune/neurodegenerative disorders. The antioxidant/anti-inflammatory effect of a synthetic conjugated linoleic acid (CLA) mixture in MRL/MpJ-Fas lpr mice (MRL/lpr), an animal model of neuropsychiatric lupus, was previously associated with the improvement of nuclear factor-E2-related factor 2 (Nrf2) defenses in the spleen and liver. However, little is known about the neuroprotective ability of a CLA mixture. This study investigated the age-dependent progression of oxidative stress and the hyperactivation of redox-sensitive compensatory pathways (macroautophagy, Nrf2) in old/diseased MRL/lpr mice brains and examines the effect produced by dietary CLA supplementation. Disrupted redox homeostasis was evidenced in the blood, liver, and brain of 21- to 22-week-old MRL/lpr (Old) mice compared with 8- to 10-week-old MRL/lpr (Young) animals. This alteration was associated with significant hyperactivation of compensatory mechanisms (macroautophagy, Nrf2, and astrocyte activation) in the brains of Old mice. Five-week daily supplementation with CLA (650 mg/kg-1 body weight) of 16-week-old (CLA+Old) mice diminished all the pathological hallmarks at a level comparable to Young mice or healthy controls (BALB/c). Such data demonstrated that MRL/lpr mice can serve as a valuable model for the evaluation of the effectiveness of neuroprotective drugs. Notably, the preventive effect provided by CLA supplementation against age-associated neuronal damage and hyperactivation of compensatory mechanisms suggests that the activation of an adaptive response is at least in part accountable for its neuroprotective ability.

Dietary supplementation with pure cis9, trans11 isomer of Conjugated Linoleic Acid –known as Rumenic Acid (RA)– improves cytoprotective defenses downstream through the activation of nuclear factor-E2-related factor-2(Nrf2). This capability, when Rumenic Acid is consumed in the form of foods, is still unknown. The ability of standard (St) or cow milk naturally-enriched in RA (En) to activate Nrf2 pathway and its impact on dextran sodium sulfate (DSS)-induced colitis was comparatively evaluated. Activity of Nrf2 pathway was investigated in colonic tissue of BALB/c mice, receiving 4-week supplement with skimmed milk (SK), St or St reinforced with pure RA (RSt) providing increasing RA dose (0, 124 or 404 mg RA/kg-1 b.w, respectively). Next, the anti-oxidant/ anti-inflammatory effect produced by St or En treatment (383 mg RA/kg-1 b.w.) was explored. Finally, macroscopic and histomorphologic features of colitis were evaluated in animals challenged with 5% (w/v) DSS, at the end of St or En treatment. Significant activation of Nrf2 pathway is associated with RSt and En intake (P

Inflammation and oxidative stress play an important role in the pathogenesis of depressive disorders and Nuclear erythroid related factor 2 (Nrf2), a regulator of RedOx homeostasis and inflammation, is a promising target for depression prevention/treatment. As fish oil (FO) and Conjugated Linoleic Acid (CLA) are known Nrf2 inducer, their protective ability was comparatively evaluated in a murine model of depression (MRL/MpJ-Faslpr ). Oxidative stress, fatty acids content and critical factors reflecting brain functioning - namely brain-derived neurotrophic factor (BDNF), synaptic markers and cholinergic signaling - were preliminarily evaluated in frontal cortex of 8-weeks (Young) and in 22-weeks old animals (Old), which were used as model of depression. These markers were measured in Old mice at the end of a 5-week pre-treatment with FO or CLA (728 or 650 mg kg-1, respectively). Old mice exhibited disrupted Redox homeostasis, compensatory Nrf2 hyperactivation, lower Docosaheaxaenoic acid (DHA), lower BDNF and synaptic function proteins compared to Young mice. FO and CLA treatment relieved almost all the pathophysiological hallmarks at a level comparable to Young mice. Presented data provide the first evidence for the comparable efficacy of FO or CLA supplementation in preventing depression signs in Old MRL/lpr mice, likely through their ability of improving Nrf2-mediated antioxidant defenses.

In this study, we investigated by in silico analysis the possible correlation between microRNAs (miRNAs) and Anamnia V-SINEs (a superfamily of short interspersed nuclear elements), which belong to those retroposon families that have been preserved in vertebrate genomes for millions of years and are actively transcribed because they are embedded in the 3' untranslated region (UTR) of several genes. We report the results of the analysis of the genomic distribution of these mobile elements in zebrafish (Danio rerio) and discuss their involvement in generating miRNA gene loci. The computational study showed that the genes predicted to bear V-SINEs can be targeted by miRNAs with a very high hybridization E-value. Gene ontology analysis indicates that these genes are mainly involved in metabolic, membrane, and cytoplasmic signaling pathways. Nearly all the miRNAs that were predicted to target the V-SINEs of these genes, i.e., miR-338, miR-9, miR-181, miR-724, miR-735, and miR-204, have been validated in similar regulatory roles in mammals. The large number of genes bearing a V-SINE involved in metabolic and cellular processes suggests that V-SINEs may play a role in modulating cell responses to different stimuli and in preserving the metabolic balance during cell proliferation and differentiation. Although they need experimental validation, these preliminary results suggest that in the genome of D. rerio, as in other TE families in vertebrates, the preservation of V-SINE retroposons may also have been favored by their putative role in gene network modulation.

Alzheimer's disease (AD) is a progressive, multifactorial neurodegenerative disorder that is the main cause of dementia. To date, there are no definitive diagnostic tests that can predict or assess onset and progression of the disease. Blood biomarkers for AD are being sought for many years but their identification remains a challenging task. In this study, we investigated the potential relationship between AD and levels of acyl-peptide hydrolase (APEH) and proteasome in erythrocyte samples of 52 participants (26 AD and 26 cognitively healthy controls). A statistically significant decrease in proteasome and exopeptidase/endopeptidase APEH activities was found in AD samples compared to those of healthy controls. Moreover, in contrast to what was observed for proteasome transcripts, APEH activities reduction in AD patients was unrelated to its gene expression levels, suggesting the occurrence of posttranslational modifications or the expression of endogenous inhibitors that might impair enzyme activity. These preliminary data further support a relationship between the APEH-proteasome system and AD molecular players, providing the first evidence of its potential use as a novel blood-based indicator for the routine detection of AD.

Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease), while the less abundant (named SsMTP-1) one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50-90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Oxidative challenge is an important factor affecting the adaptive strategies of Antarctic fish, but data on antioxidant defenses in these organisms remain scarce. In this context, a key role could be played by acylpeptide hydrolase (APEH), which was recently hypothesized to participate in the degradation of oxidized and cytotoxic proteins, although its physiological function is still not fully clarified. This study represents the first report on piscine members of this enzyme family, specifically from the Antarctic teleost Trematomus bernacchii. The cDNAs corresponding to two apeh genes were isolated, and the respective proteins were functionally and structurally characterized with the aim of understanding the biological significance of these proteases in Antarctic fish. Both APEH isoforms (APEH-1Tb and APEH-2Tb ) showed distinct temperature-kinetic behavior, with significant differences in the Km values. Moreover, beside the typical acylpeptide hydrolase activity, APEH-2Tb showed remarkable oxidized protein endohydrolase activity towards oxidized BSA, suggesting that this isoform could play a homeostatic role in removing oxidatively damaged proteins, sustaining the antioxidant defense systems. The 3D structures of both APEHs were predicted, and a possible relationship was found between the substrate specificity/affinity and the marked changes in the number of charged residues and hydrophobicity properties surrounding their catalytic sites. Our results demonstrated the occurrence of two APEH isoforms in T. bernacchii, belonging to different phylogenetic clusters, identified for the first time, and showing distinct molecular and temperature-kinetic behaviors. In addition, we suggest that the members of the new cluster 'APEH-2' could participate in reactive oxygen species detoxification as phase 3 antioxidant enzymes, enhancing the protein degradation machinery.

Transposable elements have accompanied the evolution of the eukaryotic genome for millions of 26 years. The recently discovered Helitron order (class II, subclass 2 single-strand DNA transposons) 27 is common in eukaryotes and seems to play a highly active role in genome reshuffling. This study 28 provides novel insights into the characteristics of Helinoto, a helitron isolated in the genome of the 29 Antarctic fish Chionodraco hamatus. In particular, investigation of the structure of its 5‘ and 3' 30 ends, which are involved in the transposition process, enabled identification of the characteristic 31 motifs of the Helitron2 group. Moreover, identification of a deubiquitinating protease domain in the 32 region upstream two consecutive OTU domains extended and strengthened the ―deubiquitinase‖ 33 character of the N-terminal portion of Helinoto. Finally, Helinoto transcriptional activity was 34 detected in several C. hamatus tissues. Taken together, these data are particularly intriguing because 35 they document high transcription levels for genes involved in ubiquitination, which ensures protein 36 homeostasis in the extreme Antarctic environment.

The antioxidant defense mechanisms have a great impact on the life of Antarctic organisms. The present study could represent the first evidence of a direct involvement of the 26S proteasome in the antioxidant defense systems of fish adapted to cold., Protein homoeostasis is a fundamental process allowing the preservation of functional proteins and it has a great impact on the life of the Antarctic organisms. However, the effect of low temperatures on protein turnover is poorly understood and the cold-adaptation of the degradation machinery remains an unresolved issue. As the 26S proteasome represents the main proteolytic system devoted to the controlled degradation of intracellular proteins, the purpose of the present study was to investigate the functions of this complex in the notothenioid Trematomus bernacchii, in order to better understand its role in the physiology of Antarctic fish. To this aim, we purified and characterized the 26S proteasome from T. bernacchii and isolated the cDNAs codifying seven of the 14 subunits belonging to the proteasome 20S core particle. Results provided evidences of the high resistance of the piscine 26S proteasome to oxidative agents and of its ‘uncommon’ ability to efficiently hydrolyse oxidized bovine serum albumin (BSA), suggesting that this enzymatic complex could play a key role in the antioxidant defense systems in fish inhabiting permanently cold marine environments. These unique properties were also reflected by the 3D model analysis, which revealed a higher structural stability of the piscine complex respect to the murine template. Finally, a comparative analysis, performed in a variety of tissues collected from T. bernacchii and the temperate fish Dicentrarchus labrax, showed a lower protein retention in the cold-adapted fish, possibly due to a better efficiency of its degradation machinery.

The Campania region in Italy is facing an environmental crisis due to the illegal disposal of toxic waste. Herein, a pilot study (EcoFoodFertility initiative) was conducted to investigate the use of human semen as an early biomarker of pollution on 110 healthy males living in various areas of Campania with either high or low environmental impact. The semen from the "high impact" group showed higher zinc, copper, chromium and reduced iron levels, as well as reduced sperm motility and higher sperm DNA Fragmentation Index (DFI). Redox biomarkers (total antioxidant capacity, TAC, and glutathione, GSH) and the activity of antioxidant enzymes in semen were lower in the "high impact" group. The percentage of immotile spermatozoa showed a significant inverse correlation with TAC and GSH. Overall, several semen parameters (reduced sperm quality and antioxidant defenses, altered chemical element pattern), which were associated with residence in a high polluted environment, could be used in a further larger scale study, as early biomarkers of environmental pollution.

Article history:Received 27 July 2011Received in revised form 6 September 2011Accepted 7 September 2011Keywords:Antarctic skateCartilaginous fish immunoglobulin MCμ gene diversityExtra-cysteines Immunoglobulins (Ig) of Chondroichthyes have been extensively studied in sharks; in contrast, in skatesinvestigations on Ig remain scarce and fragmentary despite the high occurrence of skates in all of themajor oceans of the world. To focus on Rajidae Igμ, the most abundant heavy chain isotype, we have chosenthe Antarctic species Bathyraja eatonii, Bathyraja albomaculata, Bathyraja brachyurops, and Amblyrajageorgiana which live at high latitudes in the Southern Ocean, and at very low temperatures. We preparedmRNA from the spleen of individuals of each species and performed RT-PCR experiments using twooligonucleotides designed on the alignment of various elasmobranch Igμ heavy chain sequences availablein GenBank. The PCR products, about 1400-nt long, were cloned and sequenced. Nucleotide sequence identi-ties calculated for the constant region domains ranged from 88.5% to 97.5% between species, and from 91.1%to 99.7% within species. In a distance tree, including also Raja erinacea sequences, two major branches wereobtained, one containing Arhynchobatinae sequences, the other one Rajinae sequences. Four presumptive Dgene segments were identified in the region of the VH/D/JH recombination; two different D segments wereoften found in the same sequence. Moreover, 5–15 genomic fragments of different lengths, carrying thegene locus encoding Igμ chain were revealed by Southern blotting analysis. B. eatonii amino acid sequenceswere analyzed for the positional diversity by Shannon entropy analysis, showing CH4 as the most conserveddomain, and CH3 as the most variable one. B. eatonii CDR3 region length varied between 11 and 15 aminoacid residues; the mean length (13.4 aa) was greater than that of Leucoraja eglanteria sequences (7.7 aa).An alignment of representative sequences of Antarctic species and R. erinacea showed that more cysteineresidues not involved in the intradomain disulfide bridges were present in Antarctic species.© 2011 Elsevier B.V. All rights reserved.

Rolling-circle (RC) eukaryotic transposons, known as helitrons, are found in a wide range of organisms, from protist to mammals. Autonomous helitrons have a distinctive open reading frame (ORF) encoding a polypeptide that contains typical domains for RC replication (RCR): the Rep (RCR initiator) and the DNA helicase domains. These elements are believed to have an important role in the host genome evolution, owing to their frequent capture of host genes, some of which can evolve into novel genes or become essential for helitron transposition. We conducted a molecular analysis of the suborder Notothenioidei, a group of Perciformes that currently dominate the Antarctic waters by virtue of their remarkable cold-adaptation ability. A novel helitron from the genome of the icefish species Chionodraco hamatus, belonging to the Channichthyidae, the most derived Notothenioids family, was isolated, characterized and designated as HeliNoto (8.9 kb). Its ORF was compared to homologous sequences from different species in a comprehensive phylogenetic analysis. For the first time the putative functional domains of a helitron were subjected to a well accurate structural analysis including chromosomal localization. Finally, the distribution of HeliNoto among Notothenioids was investigated.

An extensive investigation of the organisation of globin genes has greatly contributed to the understanding of universal mechanisms of gene evolution and expression. Cartilaginous fish are the first organisms that have evolved the tetrameric form of hemoglobin (Hb). So far, there has been absolute lack of data about globin genes in chondrichthyans. Bathyraja is the dominant rajid south of 60 degrees S. In the framework of the investigations on globin genes of Antarctic red-blooded and Hb-less fish we obtained the cloning of the alpha- and beta-globin cDNAs of the main Hb (Hb 1) of the skate Bathyraja eatonii. Then, a genomic fragment of 6.2 kb was isolated where the Hb 1 alpha and beta genes are linked in a tail-to-head (3' to 5') orientation. The beta-globin gene promoter region and the chromosomal organisation of the Hb 1 genes of B. eatonii have been compared to their homologues in other vertebrates. The finding of a tail-to-head linkage of the Hb 1 alpha- and beta-globin genes in B. eatonii is the first characterisation of the organisation of globin genes in chondrichthyes; such finding offers a novel contribution to the understanding of the evolution of this class of genes. Moreover, the characterisation of chondrichthyan genes is very important for gaining insight into the ancestral state of vertebrate genomes.

Acylpeptide hydrolase (APEH) is a ubiquitous cytosolic protease that plays an important role in the detoxification of oxidised proteins. In this work, to further explore the physiological role of this enzyme, two apeh cDNAs were isolated from the Chionodraco hamatus icefish, which lives in the highly oxygenated Antarctic marine environment. The encoded proteins (APEH-1Ch and APEH-2Ch) were characterised in comparison with the uniquely expressed isoform from the temperate fish Dicentrarchus labrax (APEH-1Dl) and the two APEHs from the red-blooded Antarctic fish Trematomus bernacchii (APEH-1Tb and APEH-2Tb). Homology modelling and kinetic characterisation of the APEH isoforms provided new insights into their structure/function properties. APEH-2 isoforms were the only ones capable of hydrolysing oxidised proteins, with APEH-2Ch being more efficient than APEH-2Tb at this specific function. Therefore, this ability of APEH-2 isoforms is the result of an evolutionary adaptation due to the pressure of a richly oxygenated environment. The lack of expression of APEH-2 in the tissues of the temperate fish used as the controls further supported this hypothesis. In addition, analysis of gene expression showed a significant discrepancy between the levels of transcripts and those of proteins, especially for apeh-2 genes, which suggests the presence of post-transcriptional regulation mechanisms, triggered by cold-induced oxidative stress, that produce high basal levels of APEH-2 mRNA as a reserve that is ready to use in case of the accumulation of oxidised proteins.

Cartilaginous fish are very ancient organisms. In the Antarctic sea, the modern chondrichthyan genera are poorly represented, with only three species of sharks and eight species of skates; the paucity of chondrichthyans is probably an ecological consequence of unusual trophic or habitat conditions in the Southern Ocean. In the Arctic, there are 26 species belonging to the class Chondrichthyes. Fish in the two polar regions have been subjected to different regional histories that have influenced the development of diversity: Antarctic marine organisms are highly stenothermal, in response to stable water temperatures, whereas the Arctic communities are exposed to seasonal temperature variations. The structure and function of the oxygen-transport haem protein from the Antarctic skate Bathyraja eatonii and from the Arctic skate Raja hyperborea (both of the subclass Elasmobranchii, order Rajiformes, family Rajidae) is reported in the present paper. These species have a single major haemoglobin (Hb 1; over 80% of the total). The Bohr-proton and the organophosphate-binding sites are absent. Thus the haemoglobins of northern and southern polar skates appear functionally similar, whereas differences were observed with several temperate elasmobranchs. Such evidence suggests that, in temperate and polar habitats, physiological adaptations have evolved along distinct pathways, whereas, in this case, the effect of the differences characterizing the two polar environments is negligible.

The climatic features of Antarctic waters are more extreme and constant than in the Arctic. The Antarctic has been isolated and cold longer than the Arctic. The polar ichthyofaunas differ in age, endemism, taxonomy, zoogeographic distinctiveness and physiological tolerance to environmental parameters. The Arctic is the connection between the Antarctic and the temperate¡Vtropical systems. Paradigmatic comparisons of the pathways of adaptive evolution of fish from both poles address the oxygen-transport system and the antifreezes of northern and southern species. (i) Haemoglobin evolution has included adaptations at the biochemical, physiological and molecular levels. Within the study of the molecular bases of fish cold adaptation, and taking advantage of the information on haemoglobin amino acid sequence, we analysed the evolutionary history of the ƒÑ and ƒÒ globins of Antarctic, Arctic and temperate haemoglobins as a basis for reconstructing phylogenetic relationships. In the trees, the constant physico-chemical conditions of the Antarctic waters are matched by clear grouping of globin sequences, whereas the variability typical of the Arctic ecosystem corresponds to high sequence variation, reflected by scattered intermediate positions between the Antarctic and non-Antarctic clades. (ii) Antifreeze (glyco)proteins and peptides allow polar fish to survive at sub-zero temperatures. In Antarctic Notothenioidei the antifreeze gene evolved from a trypsinogen-like serine protease gene. In the Arctic polar cod the genome contains genes which encode nearly identical proteins, but have evolved from a different genomic locus¡Xa case of convergent evolution.

The pathways and mechanisms of genomic change that have led to the peculiar haemoglobinless phenotype of the white-blooded Antarctic icefishes (16 species in the family Channichthyidae) constitute an important model for understanding the rapid diversification of the Antarctic notothenioid fish flock. To provide complementary structural information on genomic change at globin-gene loci in Antarctic fish species, cytogenetic studies and in-situ chromosomal mapping have been undertaken. Using a DNA probe containing one α- and one β-globin gene from the embryonic/juvenile globin gene cluster of the red-blooded species Notothenia coriiceps, we mapped the cluster on the chromosomes of Antarctic teleosts by fluorescence in-situ hybridization. As anticipated on the basis of its molecular organization, the cluster was located on a single chromosome pair in all of the red-blooded fish species probed (N. coriiceps, N. angustata, Trematomus hansoni, T. pennellii). In contrast, the α/β-globin probe did not recognize complementary sequences on the chromosomes of the white-blooded species Chionodraco hamatus and Channichthys rhinoceratus. These results represent the first example of chromosomal mapping of embryonic/juvenile globin genes in teleostean fishes. Beyond its relevance to the evolutionary history of Antarctic notothenioids, this work contributes to our understanding of the evolution of the chromosomal loci of globin genes in fishes and other vertebrates.

The oxidative challenge represents an important factor affecting the adaptive strategies in Antarctic fish, but their impact on the protein degradation machinery still remains unclear. The previous analysis of the first 26S proteasome from the Antarctic red-blooded fish Trematomus bernacchii, evidenced improved antioxidant functions necessary to counteract the environmental pro-oxidant conditions. The purpose of this work was to carry out a study on 26S proteasomes from the temperate red-blooded Dicenthrarcus labrax and the icefish Chionodraco hamatus in comparison with the isoform already described from T. bernacchii, to better elucidate the cold-adapted physiological functions of this complex. Therefore, the 26S isoforms were isolated and the complementary DNAs (cDNAs) codifying the catalytic subunits were cloned. The biochemical characterization of Antarctic 26S proteasomes revealed their significantly higher structural stability and resistance to H2O2 with respect to that of the temperate counterpart, as also suggested by a comparative modeling analysis of the catalytic subunits. Moreover, in contrast to that observed in T. bernacchii, the 26S systems from C. hamatus and D. labrax were incapable to hydrolyze oxidized proteins in a ubiquitin-independent manner. Therefore, the ‘uncommon’ properties displayed by the Antarctic 26S proteasomes can mirror the impact exercised by evolutionary pressure in response to richly oxygenated environments.

Purpose: The beneficial effects of conjugated linoleic acid (CLA) mixture (cis9, trans11, c9; trans10, cis12, t10) against gliadin-induced toxicity in HLA-DQ8-transgenic mice (DQ8) have been associated with improved duodenal cytoprotective mechanisms [nuclear factor-E2-related factor- 2, Nrf2; acylpeptide hydrolase (APEH)/proteasome]. The present study was aimed at investigating the ability of individual CLA isomers to improve the efficacy of these defensive mechanisms and to protect against duodenal injury caused by the combined administration of gliadin and indomethacin (GI). Methods: Gluten-mediated enteropathy was induced in DQ8 mice by three intra-gastric administration of gliadin (20 mg kg-1/bw) and indomethacin (15 mg L-1) in drinking water for 10 days (GI). C9 or t10 CLA (520 mg kg-1/bw/day) were orally administered for 2 weeks. Pro-oxidant and toxic effects associated with GI treatment, anti-oxidant/detoxifying ability of c9 or t10-CLA and the protective effect induced by c9 pre-treatment (c9 + GI) were evaluated in DQ8 mice duodenum by combining enzymatic, immunoblotting, histological evaluation and quantitative real-time PCR assays. Results: GI treatment produces the time-dependent decline of the considered detoxifying mechanisms thus leading to pro-apoptotic and pro-oxidant effects. APEH/proteasome pathway was not markedly affected by individual CLA isomers, but duodenal redox status and activity/mRNA levels of Nrf2-activated enzymes were significantly improved by c9 administration. c9 pre-treatment protects against GI-mediated accumulation of oxidative stress markers, and histological examination reveals the increase of goblet cells number in mouse duodenum but induces only a partial recovery of APEH/proteasome activity. Conclusions: The activation of and adaptive response by low doses of c9 supplementation prevents distinct signs of gliadin-induced enteropathy in DQ8 mice

Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis 9, trans 11 (9,11-CLA) and trans 10, cis 12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/ day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis.-Mollica, M. P., G. Trinchese, G. Cavaliere, C. De Filippo, E. Cocca, M. Gaita, A. Della-Gatta, A. Marano, G. Mazzarella, and P. Bergamo. c 9, t 11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway. J. Lipid Res . 2014. 55: 837-849. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Various hydroxy-, oxo-, and conjugated fatty acids are generated by gut microbes throughout the metabolism of dietary PUFAs. Specifically, 10-Hydroxy-cis-12-(HYA), 10-oxo-cis12-(KetoA) and 10-oxo-cis9,trans11-octadecenoic acid (KetoC) are intermediates free fatty acids (iFFA) produced during the isomerization of dietary linoleic acid to cis9,trans11 (c9,t11) isomer of conjugated linoleic acid (CLA). Herein, immunomodulatory and antioxidant activity of selected iFFA was studied in resting (iDCs), LPS-matured DCs (mDCs) and in murine enterocyte cells (MODE-K). Phenotypic maturation of iDCs was not influenced by iFFA, but the release of pro-inflammatory molecules from mDCs was reduced following HYA pre-treatment. HYA ability to improve antioxidant/detoxifying defenses associated with decreased expression of maturation markers in mDCs and in MODE-K cells. The pro-oxidant activity, via NADPH oxidase activation, was responsible for the pro-inflammatory effect of 10-hydroxy-octadecanoic acid. Presented data suggest that the immunomodulatory ability of HYA is determined, at least in part, by its ability to improve antioxidant/detoxifying defenses.

The blood of Channichthyidae, the most phyletically derived notothenioid family, is devoid of haemoglobin (Hb). Three of the 15 channichthyid species retain inactive genomic remnants of the major adult notothenioid a-globin gene, but have lost the gene encoding adult β-globin. In an attempt to understand the mutational events leading to loss of globin expression in channichthyds, the organisation of globin genes in red-blooded notothenioid species was examined. Globin genes in fish are often organised as alβ pairs linked in 5' to 5' orientation, and the major adult α- and β-globins of the Antarctic yellowbelly rockcod Notothenia coriiceps have this organisation. A second globin gene cluster from N. coriiceps contains two sets of linked α- and β-globin genes but the deduced amino acid sequences of the globins differ from those of the adult globins. To test the hypothesis that the globins may be expressed at different stages of ontogenesis. total RNA from head kidney and erythrocytes of adult and juvenile N. coriiceps were probed for transcripts corresponding to the four globin genes. The data suggest that the globin genes of this cluster are transcribed maximally in the early phase of development. Southern analysis of the genomes of red- and white-blooded notothenioids revealed that the genes of this complex are absent in Hb-less icefish.

This research dealing with fish, bird and mammal hemoglobins is in the framework of the study of the molecular basis of cold adaptation in Antarctic organisms. The study of hemoglobin structural and functional properties in Antarctic teleosts allowed a correlation of amino acid sequence, multiplicity, and oxygen‐binding features with ecological constraints. Amino acid sequences of α and β chains were analysed to build phylogenetic trees, while the organisation and expression of globin genes was studied. The respiratory proteins of Antarctic birds were investigated, in relation with the oxygen demands arising from their characteristic behaviour (diving or flight) and, in general, from the extreme conditions of the Antarctic habitat. The study of Weddell seal hemoglobins indicated that the combined effect of carbon dioxide, organic phosphates and temperature optimises oxygen delivery to all tissues in spite of their relative heterothermia. The crystallographic structure of the carbonmonoxy derivati...

The icefishes of the Southern Ocean (family Channichthyidae, suborder Notothenioidei) are unique among vertebrates in their inability to synthesize hemoglobin. We have shown previously (Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella, L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 1817–1821) that icefishes retain inactive genomic remnants of adult notothenioid α-globin genes but have lost the gene that encodes adult β-globin. Here we demonstrate that loss of expression of the major adult α-globin, α1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus) results from truncation of the 5′ end of the notothenioid α1-globin gene. The wild-type, functional α1-globin gene of the Antarctic yellowbelly rockcod, Notothenia coriiceps, contains three exons and two A + T-rich introns, and its expression may be controlled by two or three distinct promoters. Retained in both icefish genomes are a portion of intron 2, exon 3, and the 3′-untranslated region of the notothenioid α1-globin gene. The residual, nonfunctional α-globin gene, no longer under positive selection pressure for expression, has apparently undergone random mutational drift at an estimated rate of 0.12–0.33%/million years. We propose that abrogation of hemoglobin synthesis in icefishes most likely resulted from a single mutational event in the ancestral channichthyid that deleted the entire β-globin gene and the 5′ end of the linked α1-globin gene.

Among piscine taxa, the Antarctic icefishes (family Channichthyidae) prosper in the absence of erythrocytes and hemoglobin, a unique condition among adult vertebrates. The genomes of three icefish species and four red-blooded notothenioid species were probed using α- and β-globin cDNA from the red-blooded Antarctic fish Notothenia coriiceps . High-stringency hybridization signals with the α-globin probe, but none with the β-globin probe, on genomic DNAs of both hemoglobinless and red-blooded fishes suggest that icefishes retain remnants of α-globin genes in their genomes but have lost the gene that encodes for β-globin, either through deletion or through rapid mutation. Northern blot analysis of major hematopoietic and non-hematopoietic tissues shows that the α-globin-related sequences of icefishes are nonexpressed derivatives of the α-globin genes of their red-blooded relatives. Mechanisms leading to the hemoglobinless phenotype are discussed in relation with the expression of myoglobin in the Channichthyidae family.

The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH-proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-kappaB, p21(Waf1), and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies.

Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade.

A novel acylpeptide hydrolase, named APEH-3(Ss), was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a "true" aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3(Ss) and apeh(Ss) (the gene encoding the previously identified APEH(Ss) from S. solfataricus), which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.