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Apolipoprotein E (APOE) ε, catechol-O-methytranferase (COMT) Val108/158Met and brain-derived neurotrophic factor (BDNF) Val66Met single nucleotide polymorphisms (SNPs) were shown to affect stress perception and response. The present study explored possible associations between these SNPs and changes in subclinical anxiety- and depressive symptoms, sense of coherence (SOC) and vital exhaustion (VE) during compulsory basic military training. The study encompassed 179 conscripts of a training base in Greece. The neuropsychiatric assessment was based on the Beck Depression Inventory, the State-Trait Anxiety Inventory, the Antonovsky SOC scale and the Maastricht Questionnaire. It was conducted at three time points of the 19-day basic military training: on day one (baseline), day six (follow-up I) and day 13 (follow-up II). Statistical analyses included Mann-Whitney test, Chi-square test and cross-sectional time series regression models based on the Skillings-Mack statistic. APOE ε4 non-carriers encountered significant changes in anxiety- and depressive symptoms and SOC (in all cases P < 0.001) over the observation period, whilst ε4 carriers did not. The changes in anxiety, depressive symptoms and SOC attained statistical significance in both BDNF Met66 carriers (in all cases P < 0.001) and non-carriers (P = 0.036; < 0.001; < 0.001, respectively) as well as in COMT Met108/158 carriers (P = 0.004; < 0.001; < 0.001, respectively) and non-carriers (P = 0.02; 0.01; 0.021, respectively. Changes over time in VE were not significant (P > 0.05). The observed resistance of APOE ε4 carriers vs non-carriers to changes in anxiety- and depressive symptoms and SOC when exposed to a stressful environment may point to superior coping capacities of healthy young men carrying the ε4 allele.

The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression, and metastasis. RANK-c is a RANK receptor isoform produced through alternative splicing of the TNFRSF11A (RANK) gene and a dominant-negative regulator of RANK-induced nuclear factor-κB (NF-κB) activation. Here we report that RANK-c transcript is expressed in 3.2% of cases in The Cancer Genome Atlas breast cancer cohort evenly between ER-positive and ER-negative cases. Nevertheless, the ratio of RANK to RANK-c (RANK/RANK-c) is increased in ER-negative breast cancer cell lines compared to ER-positive breast cancer cell lines. In addition, forced expression of RANK-c in ER-negative breast cancer cell lines inhibited stimuli-induced NF-κB activation and attenuated migration, invasion, colony formation, and adhesion of cancer cells. Further, RANK-c expression in MDA-MB-231 cells inhibited lung metastasis and colonization in vivo. The RANK-c-mediated inhibition of cancer cell aggressiveness and nuclear factor-κB (NF-κB) activation in breast cancer cells seems to rely on a RANK-c/TNF receptor-associated factor-2 (TRAF2) protein interaction. This was further confirmed by a mutated RANK-c that is unable to interact with TRAF2 and abolishes the ability to attenuate NF-κB activation, migration, and invasion. Additional protein interaction characterization revealed epidermal growth factor receptor (EGFR) as a novel interacting partner for RANK-c in breast cancer cells with a negative effect on EGFR phosphorylation and EGF-dependent downstream signaling pathway activation. Our findings further elucidate the complex molecular biology of the RANKL/RANK system in breast cancer and provide preliminary data for RANK-c as a possible marker for disease progression and aggressiveness.

Breast cancer is the most common malignancy, affecting one in eight women in North America and Europe. The human epidermal growth factor receptor (EGFR) protein comprises a major determinant of normal development but also cancer. RANK receptor (Receptor Activator of Nuclear factor-κB) is a tumor necrosis superfamily member and a binding partner for RANKL, which was recently implicated in breast cancer initiation, progression and metastasis. Here we provide preliminary evidence of a possible interplay between RANK and EGFR signaling in breast cancer. TCGA (cancergenome.nih.gov) publicly available data for EGFR and TNFRSF11A (RANK) genes from breast cancer patients and breast cancer cell lines were retrieved and analyzed. RANK mRNA showed a statistically significant positive correlation ( p 0.001) with the mRNA and protein expression of EGFR , but not with ERBB2 /3/4 . Further analyses of survival data of a group of breast cancer patients (n = 248) from TCGA, revealed an EGFR hi / RANK hi subpopulation that showed a statistically significant ( p = 0.001) reduced overall survival when compared to EGFR low / RANK low group of patients. Finally, EGFR and RANK combinatorial in vitro analyses revealed a significant upregulation of AKT and ERK signaling after EGF stimulation in cell lines and also an increase of breast cancer cell invasiveness.

Alternative splicing (AS) is a major post-transcriptional modification taking place in all cells. Many members of the TNF receptor superfamily modulate their function through protein isoforms produced by alternative splicing. TNFRSF11A (RANK) gene, through alternative splicing produces multiple isoforms truncated in their intracellular domain, with distinct functions. Here, we report the identification and characterization of a novel human TNFRSF11A (RANK) variant from human normal brain, named RANK-e5a (TNFRSF11A_e5a). The novel variant lacks 42 nucleotides from exon 5, giving rise to a novel shorter form of exon 5, named exon 5a. The incorporation of the novel exon 5a in RANK mRNA does not affect the open reading frame, producing a truncation of thirteen amino acids of the third and fourth TNFR motifs of the extracellular part of the receptor. By western blot analysis and immunofluorescence staining we were able to further characterize the RANK-e5a isoform at the protein level. In addition, we performed an ELISA assay to characterize RANK/RANKL and RANK-e5a/RANKL binding capacities, and we identified a reduced affinity of RANK-e5a to bind RANKL. Finally, when RANK-e5a is stimulated by RANK ligand, its capability to activate NF-κB is reduced compared to the wild type RANK receptor. Overall, our data provide a novel regulatory mechanism for the RANK/RANKL system, at the RANK receptor level.

Aven was originally identified as a protein that regulates apoptosis by binding to apoptotic regulators, Bcl-xL and Apaf-1. Recently was found that Aven protein is a potent activator of ATM, critical for its DNA damage-induced activation. An Aven cDNA clone was isolated from chicken (Gallus gallus) after screening of a cerebellum cDNA library. The full-length cDNA is 1,430 nt in size, encoding for a polypeptide of 352 amino acid residues. The predicted amino acid sequence of the chicken Aven is 69, 46, 45 and 37% identical to those of zebra finch, human, xenopus and zebrafish orthologs, respectively. Expression analysis reveals that the chicken Aven gene is expressed in the adult brain, heart, intestine, kidney, lung, stomach and spleen, as well as in the whole embryos of 4- and 6-days old. Phylogenetic analysis of the Aven ortholog proteins from various organisms clusters the chicken Aven in the same group with other bird Avens.

Humoral cytotoxicity results from the assembly of terminal components of complement, called membrane attack complex (MAC), which lead to the formation of pores on pathogen membranes. The complement components involved in MAC formation are C5b, C6, C7, C8alpha, C8beta, C8gamma and C9. Among them, C6 protein interacts with C5b through a metastable binding site to form a soluble C5b-6 dimer in the vicinity of the activating cell. Formation of the MAC is controlled by complement regulatory molecules, such as CD59, vitronectin and clusterin. Here, we report the molecular characterization of the C6 complement component, as well as the spatial and temporal expression profile of MAC structural (C6, C7, C8alpha, C8beta, C8gamma) and regulatory (CD59, vitronectin and clusterin) genes in chicken (Gallus gallus). The deduced polypeptide sequence of chicken C6 consists of 935 amino acid residues and exhibits 81%, 58%, 56% and 44% identity with zebra finch, human, frog and trout orthologs, respectively. The 'domain' architecture of chicken C6 resembles that of mammalian counterparts and the cysteine backbone is also conserved. MAC structural and regulatory genes are expressed in a wide range of adult chicken tissues, with the liver being the major source of their produced transcripts. The developmental expression profile of chicken MAC structural genes shows that their transcripts initially appear in the 12th embryonic day in the liver, exhibiting a pick in the 17th, while no expression was detected in the early whole embryo (day 4 and 6), as well as in the 2-day old neonate chicken liver. On the other hand, MAC regulatory genes are expressed in all the developmental stages investigated.

Postmortem phenobarbital redistribution was studied in a rat model. A total of 54 rats were sacrificed in a series of six experiments. The cadavers were left in an olive grove for 0, 4, 5, 6, 7 or 8 days before autopsy was performed. Tissues, blood samples and insect larvae collected at autopsy were then homogenized and the phenobarbital was extracted. Phenobarbital concentration in the extracts was determined using a fluorescence polarization immunoassay (FPIA). Postmortem redistribution of phenobarbital occurred in all the samples analyzed: blood, heart, lungs, kidneys, liver, and the insect larvae that had colonized the cadavers during their exposure. In kidneys and liver, the concentration of phenobarbital peaked on Day 5 and in larvae on Day 6. In kidneys and larvae, phenobarbital concentration reached near Day 0 values by Day 8. In blood, heart and lungs, the concentration of phenobarbital kept increasing throughout the study period. We conclude that the procedures taking place in the cadave...

Mechanical loading factors at the bone-implant interface are critical for the osseointegration and clinical success of the implant. The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti-6Al-4V/osteoblast interface, using an in vitro model. Homogeneous strain was applied to human bone marrow derived osteoblasts (HBMDOs) cultured on Ti-6Al-4V, at physiological levels (strain magnitudes 500 microstrain (μe) and 1000 μe, at frequencies of load application 0.5 Hz and 1 Hz), by a mechanostimulatory system, based on the principle of four-point bending. Semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) was used to determine the mRNA expression of Cbfa1 and osteocalcin at different loading conditions. The release of growth factors as a response to stretch was also investigated by transferring stretch-conditioned media to nonstretched cells and by measuring their effect on the regulation of DNA synthesis. Mechanical loading was found to contribute to the regulation of osteoblast differentiation by influencing the level of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the level of osteocalcin, which is regarded as the most osteoblast-specific gene. Both genes were differentially expressed shortly after the application of different mechanical stimuli, in terms of strain frequency, magnitude, and time interval. Media conditioned from mechanically stressed HBMDOs stimulate DNA synthesis more intensely compared to media conditioned from unstressed control cultures, indicating that mechanical strain induces the release of a mitogenic potential that regulates cell proliferation. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009

The scaffold/matrix attachment regions (S/MARs) are chromosomal elements that participate in the formation of chromatin domains and have origin of replication support functions. Because of all these functions, in recent years, they have been used as part of episomal vectors for gene transfer. The S/MAR of the human beta-interferon gene has been shown to support efficient episome retention and transgene expression in various mammalian cells. In Jurkat and other cells, DNA plasmid vectors containing Epstein-Barr virus origin of replication (EBV OriP) and the EBV nuclear antigen-1 gene mediate prolonged episome retention in the host cell nucleus, which, however, diminishes over time. In order to enhance retention, we combined this system with an S/MAR element. Unexpectedly, this completely eliminated the capacity of episomes to replicate. Calculation of the stress-induced DNA duplex destabilization profile of the vectors suggested that the S/MAR element had created an increase in molecular stability at the OriP site that may have disturbed replicative potential. In contrast, introduction of an alternative initiation of replication region from the beta-globin gene locus, instead of EBV OriP and the EBV nuclear antigen-1 gene, restored replicative capacity and enhanced episome retention mediated by the S/MAR. These effects were associated with a destabilization profile at the initiation of replication region. These data demonstrate a correlation between S/MAR-mediated vector retention and the presence of an unstable duplex at a replication origin, in this particular setting. We consider that the calculation of stress-induced duplex destabilization may be an informative first step in the design of units that replicate extrachromosomally, particularly as the latter present a safer and, therefore, attractive alternative to integrating viral vectors for gene therapy applications.

The beta 2 integrin CR3 is a leukocyte adhesion heterodimeric glycoprotein which functions both as receptor for iC3b and in several cell-cell and cell-substrate adhesion interactions. In order to elucidate the molecular evolution of the CR3 receptor, here we report the cloning and characterization of its beta2 (CD18) and aM (CD11b) subunits in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout CD18 and CD11b-like exhibit 50, 49, or 61% and 25, 25, or 30% identity with human, mouse, and zebrafish orthologs, respectively. The 'domain' architecture of trout CD18 and CD11b-like subunits retains several characteristics of the mammalian ortholog proteins, such as cysteine-rich regions, N-linked glycosylation sites and several proposed domains and signal sequences (von Willebrand factor type A, Integrin alpha, Integrin B tail, EGF, and Transmembrane domain). The tissue expression profiles of trout CR3 subunits diverge from those of mammalian counterparts, showing the kidney as the main source of the trout CD18 and CD11b-like mRNA transcripts. This is the first report of cloning and characterization of the CR3 receptor in low vertebrates.

The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti-6Al-4V-osteoblast interface, using an in vitro model. Homogeneous strain was applied to Human Bone Marrow derived Osteoblasts (HBMDOs) cultured on Ti-6Al-4V, at levels which are considered physiological, by a four-point bending mechanostimulatory system. A simple model for the estimation of maximum hydrodynamic shear stresses developed on cell culture layer and induced by nutrient medium flow during mechanical loading, as a function of the geometry of the culture plate and the load characteristics, is proposed. Shear stresses were lower than those which can elicit cell response. Mechanical loading was found that contributes to the regulation of osteoblast differentiation by influencing the expression of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the osteocalcin expression, whereas osteopontin gene expression was unaffected by mechanical loading at all experimental conditions.

Properdin is a plasma glycoprotein and the only known naturally occurring positive regulator of the complement system, stabilizing the alternative pathway convertase (C3bBb). In order to elucidate the molecular evolution of properdin factor P (pfc), here we report the cloning and characterization of three gene isoforms of properdin in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout properdins pfc1, pfc2 and pfc3 (447, 449 and 447 amino acids, respectively) share 78-90% identity to each other, showing the highest identity score (47%) with their zebrafish ortholog protein. The overall identity with human, mouse and xenopus properdin polypeptides is 44%, 42% and 45%, respectively. The 'domain' architecture of trout properdins resembles that of the mammalian counterpart proteins, composed of six thrombospondin repeat type 1 domains (TSR-1-TSR-6). TSR domains of the three trout properdin isoforms seem to adopt the folding pattern of human thrombospondin 1 TSP-1 domains, where each TSP-1 domain forms an antiparallel three-stranded structure that consists of alternative stacked layers of Trp and Arg residues from respective strands capped by disulfide bonds on each end. The trout pfc2 and pfc3 genes are arranged in nine and ten exons, respectively, which span approximately 3.5kb of the genome. In contrast to the expression profile of the properdin gene in mammals, liver is the main source of the trout properdin mRNA transcripts. In a phylogenetic analysis, trout pfc1, pfc2 and pfc3 genes are clustered with their orthologs from other teleost species. This is the first report of three separate genes coding for properdin factor P in a vertebrate species.

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types and has been shown to be associated with several physiological and pathological functions. In order to study the molecular evolution of clusterin, here we report the cloning and characterization of two clusterin genes in rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequences of clusterin-1 and a partial clusterin-2 clone are 89% identical to each other, showing 45, 42 and 38% identity with chicken, frog and human orthologs, respectively. Most of the putative N-glycosylation sites, as well as all 10 cysteine residues which are involved in disulfide bond formation in the mature trout clusterin-1 protein, are fully conserved when aligned with its orthologs from various species. Although trout clusterin genes exhibit the same exon-intron organization, in line with that of human clusterin, they show a totally different mRNA expression profile among various trout tissues. Phylogenetic analysis indicates an early segregation of the clusterin ancestral gene within the taxon of fish leading to the formation of a separate subgroup.

Sevoflurane has been shown to improve ischemia/reperfusion injury (IRI) through several mechanisms, including amelioration of inflammatory response. However, there haven't been any studies considering the potential role of the complement system in sevoflurane-mediated amelioration of ischemia/reperfusion injury. Our purpose was to investigate the molecular mechanisms involved in sevoflurane preconditioning in liver and lung injury induced by liver ischemia-reperfusion (LIR), giving emphasis to the immunological mechanisms. In order to do that, fifty male Wistar rats were randomly allocated in five groups (n=10 each): Animals in group LIR received ketamine and xylazine and were then subjected to ischemia of the right and median hepatic lobe for 45 min and reperfusion for 6h. Group SEVO/LIR received sevoflurane and then LIR was induced, as in group LIR. Animals in group SHAM/LIR were anesthetized with ketamine and xylazine and then laparotomy followed. Group SHAM/SEVO received sevoflurane for 30 min and then laparotomy followed. Finally, in group VEN, animals only received ketamine and xylazine. Our results showed that sevoflurane preconditioning significantly improved liver-biochemical tests (decreased Alanine transaminase (ALT), Alkaline phosphatase (ALP), Aspartate transaminase (AST) and Alkaline phosphatase (ALP) levels) and limited inflammatory cell infiltration in BALF. Additionally, compared with the LIR group, the reduction in plasma C3 was significantly reduced in the SEVO/LIR group. No significant differences were observed in histological examination in the liver and lung. Immunostaining of the liver for Intracellular Adhesion Molecule 1 (ICAM1) however, showed a decrease in ICAM1 levels in the SEVO/LIR group. In the lung, sevoflurane seemed to exert no effect in ICAM1 levels. Caspase 3 (CASP3) levels in the liver and the lung also appeared unaffected by sevoflurane preconditioning. In the SEVO/LIR group, ICAM1 mRNA expression was significantly reduced in the liver. No statistical significantly differences were observed in Complement component 3 (C3), Complement component 5 (C5) and Clusterin (CLU) mRNA levels in the liver or the lung tissue. Summarizing, sevoflurane preconditioning seems to ameliorate LIR-induced injury in the rats, mediated by mechanisms that include ICAM1 and complement C3 down regulation.

The ‘three-finger’ protein family consists of structurally related proteins which display a variety of biological functions. Snake venom α-neurotoxins, Ly-6 alloantigens, urokinase-type plasminogen activator receptor (uPAR) and the complement regulatory protein CD59 are members of this protein family. Their characteristic feature is the disposition of four disulfide bridges forming a hydrophobic core. Here it is reported the cloning and characterization of a toxin-like gene (toxin-1) in the rainbow trout (Oncorhynchus mykiss). Phylogenetic analysis showed that trout toxin-1 classified into the short-chain neurotoxins (60–65 residues), confining to a distinct group. Trout toxin-1 gene contains three exons interrupted by two introns, in line with the other toxin members of the ‘three-finger’ family. Trout liver, brain, and spleen express the toxin-1 gene and its encoded protein is identified in trout plasma. This is the first report of a toxin-like gene which has been described so far in teleost.

RNase P is an essential and ubiquitous endonuclease that mediates the maturation of the 5' ends of all precursor tRNA molecules. The holoenzyme from Dictyostelium discoideum possesses RNA and protein subunits essential for activity, but the exact composition of the ribonucleoprotein complex is still under investigation. Bioinformatic analysis of D. discoideum genome identified seven open reading frames encoding candidate RNase P protein subunits. The gene named drpp30 encodes a protein with a predicted molecular mass of 40.7 kDa that clusters with Rpp1 and Rpp30 RNase P protein subunits from Saccharomyces cerevisiae and human respectively, which have significantly lower molecular masses. Cloning and heterologous expression of DRpp30 followed by immunochemical analysis of RNase P active fractions demonstrates its association with RNase P holoenzyme. Furthermore, we show that DRpp30 can bind D. discoideum RNase P RNA and tRNA transcripts in vitro, giving a first insight of its possible role in D. discoideum RNase P function. Homology modeling using as a template the archaeal Ph1887p, and molecular dynamics simulations of the modeled structure suggest that DRpp30 adopts a TIM-barrel fold.

CD59, the major inhibitor of the complement membrane attack complex, is an 18-20 kDa glycoprotein, linked to the membrane via a glycosylphosphatidylinositol (GPI)-anchor. It restricts binding of C9 to the C5b-8 complex, preventing the formation of the complement membrane attack complex C5b-9. In this study we report the cloning of a second CD59-like gene in the rainbow trout, Oncorhynchus mykiss (referred to as CD59-2 and the previously deposited trout CD59-like gene as CD59-1). Trout CD59-2 is 56% identical to CD59-1 at the amino acid level. Both of trout CD59s show the highest identity score (54%) with putative CD59-like molecules from other teleost, and the overall identity with their mammalian orthologs is less than 30%. Trout CD59s are expressed in brain, heart, intestine, kidney, liver and spleen. Particularly, CD59-2 is abundant in trout brain, while CD59-1 seems to be absent in the trout spleen. Moreover, both of trout CD59 genes seems to be present as a single copy in trout genome.

The alpha subunit of the eighth complement component (C8) is a single-chain plasma glycoprotein which functions in the cytolytic process mediated by the complement system through a sequence of polymerization reactions with other terminal components. We have previously isolated and characterized the C8beta and C8gamma subunits of the eighth complement component in rainbow trout (Oncorhynchus mykiss). Here, we report the primary sequence, the tissue expression profile, the domain architecture and the phylogenetic analysis of the trout C8alpha gene. The deduced amino acid sequence of the trout C8alpha gene exhibits 44 and 43% identity with human and frog orthologs, respectively. The domain architecture of the trout C8alpha resembles that of mammalian orthologs, and the cysteine backbone shows a high degree of conservation. The trout C8alpha shows a similar expression profile with that of trout C8beta and C8gamma, pointing to the liver as the main source of the C8 genes expression. Although the presence of a fully developed lytic pathway of complement system is expected in teleost, this is the first report of the C8alpha gene in an organism other than mammalian.

The sixth complement protein (C6) is an essential component of the membrane attack complex (MAC); the end product of the lytic pathway of complement activation. The MAC complex constitutes a supramolecular assembly containing the five precursor proteins C5b, C6, C7, C8, and C9. Once assembled on the target surface it forms transmembrane channels that cause membrane damage and cytolysis of complement-opsonized pathogens. Besides mediating direct pathogen elimination, exposure of cells to sublytic doses of MAC can trigger diverse cellular responses such as, cell activation, induction of apoptosis, cell cycle re-entry and proliferation in various biological settings. The terminal complement components (C6-C9) are structurally related proteins, differing in size and complexity. In order to study their evolution, we report here the cloning and molecular characterization of C6 component in rainbow trout. The deduced amino acid sequence of trout C6 exhibits 55 and 44% identity with zebra fish and human orthologs, respectively. The 'domain' architecture of trout C6 resembles that of mammalian counterparts, and the cysteine backbone is also conserved. Finally, trout C6 gene appears to exist as a single copy in the trout genome, and is expressed in a wide range of trout tissues.

The seventh component of complement is a single-chain plasma glycoprotein that is involved in the cytolytic phase of complement activation through a sequence of polymerization reactions with other terminal components. We have previously isolated and characterized a C7 gene in rainbow trout (Oncorhynchus mykiss). Here, we report the cloning of a second trout C7 gene (C7-2). The deduced amino acid sequence of the C7-2 gene exhibits 43 and 50% identity with human C7 and trout C7-1, respectively. The structural motifs of trout C7-2 resemble those of mammalian C7 more than trout C7-1, and the cysteine backbone shows a high degree of conservation. C7-2 presents a different tissue expression profile from trout C7-1, which correlates with that of mammalian counterparts. Although duplication of complement genes is a common observation in teleost fish, this is the first report of two gene isotypes of a terminal membrane attack complex/perforin complement component in any organism.

In humans, leukocyte cell-derived chemotaxin 2 (LECT2) is a 16kDa chemotactic protein that consists of 133 amino acids and three intramolecular disulphide bonds. Although it was originally demonstrated to have a chemotactic function in vitro, recent data sustain a further multifunctional role of LECT2 that extends from cell growth, differentiation, damage/repair process and carcinogenesis to autoimmune diseases. The in vivo function of LECT2 protein still remains obscure. In order to study the phylogeny of LECT2, a full-length cDNA clone of LECT2 gene, 720 bp in size, was isolated in rainbow trout (Oncorhynchus mykiss). Its deduced amino acid sequence of 156 residues, presents 40, 45 and 61% overall identity to human, mouse and carp LECT2 proteins, respectively. In contrast to mammalian LECT2 protein, trout LECT2 protein reveals two potential N-glycosylation sites. Phylogenetic analysis shows that trout LECT2 is clustered with the known homologous proteins. Trout LECT2 mRNA is predominately expressed in liver and spleen, showing lower expression in kidney, intestine, heart and brain.

C7 is an integral component of the lytic pathway of complement, which interacts in a sequence of polymerization reactions with other terminal components to form the membrane attack complex. C7 plays a central role in the terminal complement cascade, since its incorporation into the nascent complex allows its hydrophilic-amphiphilic transition, which subsequently leads to the direct binding of the complex to the target membrane. To date, only human and porcine C7 have been cloned and characterized. Here we report the isolation of a C7 molecule from the rainbow trout (Oncorhynchus mykiss). The full-length trout C7 cDNA was isolated, and the predicted amino acid sequence exhibits 44 and 65% identity with human and Japanese flounder C7, respectively. The cysteine backbone and two putative N-linked glycosylation sites are conserved in trout C7. It also contains the same structural motifs as those found in mammalian C7. Trout C7 mRNA expression was detected in all tissues investigated, except kidney and spleen.

Complement-mediated killing of pathogens through the lytic pathway is an important effector mechanism of the innate immune response. C8 is one of the components of the lytic pathway and is composed of an alpha, beta, and gamma subunit. In the present study we report the cloning and characterization of the primary structure of the C8beta subunit in the rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout C8beta shows 72 and 47% identity with that of Japanese flounder and human, respectively. It also contains many of the same structural motifs as those found in mammalian lytic components. The C8beta gene appears to exists as a single copy in the trout genome and is expressed primarily in the liver. The protein encoded by the gene was identified by Western blotting using an anti-peptide antibody and was approximately 65kDa.

The complement component C5 plays important roles in inflammatory responses and complement-mediated cytolysis. In bony fish, although C5 has been identified at the DNA or the protein level in trout, carp and gilthead seabream, only partial C5 sequences are available. The present study was designed to obtain the complete primary structure of C5 from the common carp ( Cyprinus carpio) and to examine its possible structural diversity. Reverse-transcribed polymerase chain reaction amplification from carp hepatopancreatic RNA resulted in isolation of six distinct C5-like cDNA segments, which were grouped into two divergent types (type I and type II). Using two sequences representative of the two types as probes, two distinct full-length cDNA clones (C5-1 and C5-2) were isolated, in addition to a truncated isoform of C5-1 (C5-1'). The deduced amino acid sequences of C5-1 and C5-2 share 83% identity and predict a typical two-chain structure of the mature protein that lacks the thioester bond, as in C5 from other animals. Southern hybridization of genomic DNA suggested the presence of multiple genes encoding C5-type I and a single gene encoding C5-type II. Interestingly, carp C5-type I contains novel subtypes like C5-1 that have a histidine instead of the well-conserved arginine at the cleavage site for the C5 convertase, both in the complete and truncated forms. Northern blotting analysis suggested that C5-type I and C5-type II are mainly expressed in hepatopancreas, and the expression levels are significantly increased by stimulating carp with lipopolysaccharide or beta-1,3-glucan. Possible functional divergence among the C5 isotypes in carp is discussed.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.

Unlike mammalian species, several cold-blooded species have been shown to possess multiple forms of complement components. The multiple forms of C3 characterized in several fish species can bind with different specificities to various complement-activating surfaces. Here, Oriol Sunyer, Ioannis Zarkadis and John Lambris explore the possible advantages conferred by having multiple forms of individual complement proteins in a single organism.

Purpose To elucidate whether polymorphisms of C2, C3, and CFB genes are major genetic determinants of age-related macular degeneration (AMD) in a Greek population. Methods This was a case-control association study comprising 120 Greek patients with early and late-stage AMD and 140 independent controls of Caucasian origin. All participants were genotyped for rs547154, rs2230199, rs641153, and rs12614 polymorphisms by a combination of PCR and direct DNA sequencing assays. Results The frequency of the rs2230199 G allele (minor allele) was significantly higher in patients with AMD in comparison with controls (0.34 vs 0.22, p = 0.0031) and similar to the frequency of other reported populations. There was a significant difference in the frequencies of the rs2230199 genotypes among cases and controls (p = 0.0055). rs2230199 was found to be a significant predictor of advanced AMD status (odds ratio 6.41, confidence interval [CI] 2.72-15.09, pConclusions The rs547154, rs641153, and rs12614 SNPs were not associated with AMD development in Greek patients. However, this finding should be viewed with caution as the particular polymorphisms presented with very low frequencies in the Greek population. Finally, the replication of the reported associations of C3 with AMD suggests that the presence of the C3 G allele could serve as a high-risk genetic marker for the development of AMD and the progression of the disease to the advanced clinical stage.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is implicated in pericellular proteolysis, and, together with tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), in the activation of pro-matrix metalloproteinase-2 on the cell surface. It is expressed on the cell surface either activated or as a proenzyme. A soluble form of MT1-MMP (sMT1-MMP) has been previously identified in periprosthetic tissues and fluid of patients with loose arthroplasty endoprostheses. The aim of this study was to examine periprosthetic tissues and fluids from patients with loose arthroplasty endoprostheses, as well as tissues and fluids from patients with other disorders, for the presence of sMT1-MMP, and to investigate its activation state and possible role. With antibody against MT1-MMP, a protein with molecular mass of ~ 57 kDa was detected by western blotting in all samples tested, representing a soluble form of MT1-MMP, which cannot be ascribed to alternative splicing, as northern blotting showed only one transcript. With various biochemical methods, it was shown that this species occurs in a latent form bearing the N-terminal prodomain, and, additionally, it is bound to TIMP-2, which appeared to be bound via its C-terminal domain to a site different from the active site. Cell ELISA and immunohistochemical analysis revealed that, besides fibroblasts, all other cells, such as inflammatory, epithelial, endothelial, giant and cancer cells, express MT1-MMP on their plasma membrane as a proenzyme. Taking into account the proteolytic abilities of MT1-MMP, the latent sMT1-MMP–TIMP-2 complex could be considered as a new interstitial collagenase. However, the exact role, the production mechanism and the cell origin of this complex remain to be elucidated.

The complement system in vertebrates plays a crucial role in the elimination of pathogens. To regulate complement on self-tissue and to prevent spontaneous activation and systemic depletion, complement is controlled by both fluid-phase and membrane-bound inhibitors. One such inhibitor, complement factor I (CFI) regulates complement by proteolytic cleavage of components C3b and C4b in the presence of specific cofactors. Complement factor H (CFH), the main cofactor for CFI, regulates the alternative pathway of complement activation by acting in the breakdown of C3b to iC3b. To gain further insight into the origin of C3 regulation in bony fish we have cloned and characterized the CFI and CFH1 cDNAs in the rainbow trout (Oncorhynchus mykiss). In this study we report the primary sequence, the tissue expression profile, the polypeptide domain architecture and the phylogenetic analysis of trout CFI and CFH1 genes. The deduced amino acid sequences of trout CFI and CFH1 polypeptides exhibit 42% and 32% identity with human orthologs, respectively. RNA expression analysis showed that CFI is expressed differentially in trout tissues, while liver is the main source of CFH1 expression. Our data indicate that factor H and I genes have emerged during evolution as early as the divergence of teleost fish.

Purpose Hepatic ischemia/reperfusion injury can lead to remote lung injury by inducing oxidative stress and inflammation. this study aims to investigate whether support of liver function with a bioartificial liver can attenuate remote lung injury after extended hepatectomy. Methods Fourteen domestic pigs were subjected to liver ischemia for 150 minutes and 70–75% hepatectomy. Six hours after initiation of hepatic reperfusion the animals were randomly allocated to a 6-hour treatment with a bioartificial liver (group B, n=7) or observation (group C, n=7). Hemodynamic and metabolic parameters were monitored for 24 hours following reperfusion. Lung biopsies were used for histological, nitrotyrosine and mrNA analysis. Results Oxygenation gradually deteriorated in group C, but was not significantly impaired in group B. Histological evaluation revealed improvements in alveolar collapse, necrotized pneumonocytes and lymphocyte infiltration in group B. Nitrotyrosine content of the lung was lower in group B compared to group C (55±12 vs. 132±22 nM/mg protein, pConclusions Application of a bioartificial liver was associated with improvement in several parameters of post-hepatectomy lung injury. the mechanisms appear to involve reduced nitrosative stress and attenuation of the native inflammatory process in the lung.

The hyper-IgE syndrome (HIES) is a rare primary immunodeficiency characterized by a highly elevated serum IgE, recurrent staphylococcal skin abscesses and cyst-forming pneumonia. Non-immunological abnormalities, including a distinctive facial appearance, hyperextensive joints, scoliosis, fracture following minor trauma, and the retention of primary teeth are also observed in many patients. Recently, it was shown that heterozygous mutations in signal transducer and activator of transcription 3 (STAT3), can cause autosomal-dominant HIES. Here we identify and characterize a novel mutation in the DNA-binding domain of STAT3 in a patient with hyper-IgE syndrome. Sequence analysis revealed a de novo heterozygous transition of a G-to-A, causing a substitution of a glycine residue for an aspartic acid in the translated sequence (G342D). The patient has normal levels of STAT3, which is able to translocate to the nucleus upon IL-6 stimulation. However, enzyme-linked DNA-protein interaction analysis showed that the G342D mutation affects the binding ability of STAT3 to target DNA sequences. In addition, as shown by qRT-PCR, the mutation abrogates the STAT3-dependent transcription of the retinoid-related orphan receptor gammat (ROR gammat) gene, an indispensable transcription factor for the commitment of naive CD4+ T cells to the Th17 lineage. These data suggest that the novel G342D mutation affects the binding of STAT3 on DNA and the STAT3-dependent expression of ROR gammat mRNA, leading to the HIES phenotype.

Age-related macular degeneration (AMD) is the leading cause of severe visual loss among people over 60 years old. The lack of a broadly effective treatment for AMD underscores the need to identify causative biomarkers that could serve as preventive targets. Thus far, two major susceptibility loci for AMD have been identified, CFH T1277C and LOC387715 G270T. The primary goal of the present study was to elucidate whether these polymorphisms are major genetic determinants of AMD in a Greek population. A clinic-based, case-control association study was conducted, comprising 100 Greek patients with early and late-stage AMD and 115 independent controls of Caucasian origin. All participants underwent clinical examination including best-corrected visual acuity, intraocular pressure, and dilated fundus examination. Moreover, they were genotyped for CFH T1277C and LOC387715 G270T polymorphisms, by direct sequencing and ARMS PCR, respectively. The frequency of the CFH 1277C allele was significantly higher in AMD patients in comparison with controls while the odds ratios (ORs) for AMD were 4.4–5.5. Statistical comparison of early and advanced AMD patients, on the basis of CFH genotype, revealed that the CFH 1277C allele was associated with both subgroups when compared with the controls (P

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.

Vitronectin is a major cell adhesion glycoprotein that is found in plasma and the extracellular matrix. Vitronectin consists of an N-terminal somatomedin B domain and two hemopexin-like domains and controls functions including cell adhesion, migration, haemostasis and immune defence. In order to study the molecular evolution of the complement lytic pathway regulation, we have cloned and characterized the vitronectin gene from rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout vitronectin exhibits 45%, 46%, 47% and 63% identity with human, chicken, Xenopus and zebrafish orthologs, respectively. The domain architecture of the trout vitronectin, consisting of a somatomedin B domain and two hemopexin-like domains, resembles that of mammalian vitronectins. Analysis of partial genomic clones shows that trout vitronectin gene exhibits the same exon-intron organization profile as the human ortholog gene. The trout vitronectin gene is probably present as a single copy in the trout genome, showing a differential expression pattern among tissues investigated.

CD59, the major inhibitor of the complement membrane attack complex, is an 18-20 kDa glycoprotein, linked to the membrane via a glycosylphosphatidylinositol (GPI)-anchor. It restricts binding of C9 to the C5b-8 complex, preventing the formation of the complement membrane attack complex C5b-9. In this study we report the cloning of a second CD59-like gene in the rainbow trout, Oncorhynchus mykiss (referred to as CD59-2 and the previously deposited trout CD59-like gene as CD59-1). Trout CD59-2 is 56% identical to CD59-1 at the amino acid level. Both of trout CD59s show the highest identity score (54%) with putative CD59-like molecules from other teleost, and the overall identity with their mammalian orthologs is less than 30%. Trout CD59s are expressed in brain, heart, intestine, kidney, liver and spleen. Particularly, CD59-2 is abundant in trout brain, while CD59-1 seems to be absent in the trout spleen. Moreover, both of trout CD59 genes seems to be present as a single copy in trout genome.

Mannose-binding lectin (MBL) is a C-type lectin which participates in the innate immune system as an activator of the complement system and as opsonin after binding to certain carbohydrate structures on microorganisms and pathogens. C-type lectins are all Ca(2+)-dependent molecules and they share a tightly folded carbohydrate recognition domain (CRD). In this report the isolation and characterisation of cDNA transcripts encoding two mannose-binding lectin isoforms MBL-1 and MBL-2 from rainbow trout (Oncorhynchus mykiss) is presented. The deduced amino acid sequences of trout MBL-1 and MBL-2 (185 and 186 aa, respectively) present 83% identity to each other, exhibiting the highest identity score 46, 46 and 42% with the Atlantic salmon, shishamo smelt and zebrafish counterparts, respectively. The identity to birds and mammalian MBLs ranges from 25 to 33%. The trout MBL-1 and MBL-2 contain the EPN motif of mannose-binding C-type lectins, important for mannose specificity and they are expressed exclusively in liver and spleen, respectively.

Of the 35 proteins, enzymes, receptors and regulatory components of the complement system, C8gamma is unique in that it is the only lipocalin. C8gamma is a subunit of the C8 molecule, which is one of the five components (C5b, C6, C7, C8 and C9) that interact as a consequence of complement activation to form the membrane attack complex. Until now, C8gamma has been characterized only in mammalian species. In order to elucidate the phylogeny of this molecule, we have cloned the C8gamma subunit in rainbow trout (Oncorhynchus mykiss), a teleost fish representing a critical point in the evolutionary divergence of the complement system. The deduced amino acid sequence of trout C8gamma shows significant identity (37%) to the human C8gamma homolog and much lower to the other known lipocalins. The lipocalin domain is present and all the cysteine residues are conserved. The trout C8gamma gene is probably present as a single copy in the trout genome showing a differential expression pattern among tissues investigated.

During evolution two general systems of immunity have emerged: innate or, natural immunity and adaptive (acquired), or specific immunity. The innate system is phylogenetically older and is found in some form in all multicellular organisms, whereas the adaptive system appeared about 450 million years ago and is found in all vertebrates except jawless fish. The complement system in higher vertebrates plays an important role as an effector of both the innate and the acquired immune response, and also participates in various immunoregulatory processes. In lower vertebrates complement is activated by the alternative and lectin pathways and is primarily involved in the opsonization of foreign material. The Agnatha (the most primitive vertebrate species) possess the alternative and lectin pathways while cartilaginous fish are the first species in which the classical pathway appears following the emergence of immunoglobulins. The rest of the poikilothermic species, ranging from teleosts to reptilians, appear to contain a well-developed complement system resembling that of the homeothermic vertebrates. It seems that most of the complement components have appeared after the duplication of primordial genes encoding C3/C4/C5, fB/C2, C1s/C1r/MASP-1/MASP-2, and C6/C7/C8/C9 molecules, in a process that led to the formation of distinct activation pathways. However, unlike homeotherms, several species of poikilotherms (e.g. trout) have recently been shown to possess multiple forms of complement components (C3, factor B) that are structurally and functionally more diverse than those of higher vertebrates. We hypothesize that this remarkable diversity has allowed these animals to expand their innate capacity for immune recognition and response. Recent studies have also indicated the possible presence of complement receptors in protochordates and lower vertebrates. In conclusion, there is considerable evidence suggesting that the complement system is present in the entire lineage of deuterostomes, and regulatory complement components have been identified in all species beyond the protochordates, indicating that the mechanisms of complement activation and regulation have developed in parallel.

To gain further insight into the evolutionary history of the complement proteins C3, C4, and C5 we have now cloned the fifth component of complement from a rainbow trout (Oncorhynchus mykiss) liver cDNA library; this is the first report of C5 cloning in a species other than human and mouse. The deduced amino acid sequence of a partial cDNA clone (2.25 kb), representing approximately 44% of the coding sequence, showed 60 and 58% similarity to human and mouse C5, respectively. To validate the molecular information derived from the cloning we developed an improved purification protocol. Mass spectrometric analysis of C5 tryptic digests yielded peptide signals that matched theoretical protein sequence derived from the partial cDNA. Northern blot analysis of RNA from various tissues showed the presence of a single mRNA transcript in trout liver and Southern blot analysis indicated that the gene coding for C5 is present as a single copy in the trout genome. The presence of C5 in trout suggests that C3, C4, and C5 must have diverged before the appearance of teleost fish.

We have previously identified and characterized three distinct trout C3 proteins (C3-1, C3-3 and C3-4) that differ in their electrophoretic mobility, glycosylation patterns, reactivity with monospecific C3 antibodies, partial amino acid sequence and binding to various complement activators. To study the structural elements that determine the observed functional differences, we have cloned and sequenced the three C3 isoforms. Comparison of the deduced amino acid sequences showed that the sequence identity/similarity of C3-3 to C3-4 is 76/81%, whereas those of C3-3 and C3-4 to C3-1 are 55/67% and 54/67%, respectively. It is interesting that the beta-chain of C3-4 contains two insertions of 65 (residues 504-569) and 23 amino acids (residues 123-146), while the beta-chain of C3-1 contains a 14-amino acid insertion (residues 143-157). The C3 convertase cleavage site (Arg-Ser) is conserved in the three trout isoforms; however, the factor I cleavage sites are Arg-Ala (for C3-1 and C3-4) and Arg-Thr (C3-3) instead of Arg-Ser at position 1281 of human C3, and Arg-Thr (C3-1, C3-3) instead of Arg-Ser for C3-4 at position 1298 of human C3. Of special interest is the absence of the His(1126) and Glu(1128) (human C3 numbering) from C3-4 and of Glu(1128) from C3-3. These residues are thought to play an important role in determining the binding specificity of the thioester-containing proteins. Accordingly, we postulate that the distinct binding reactions of the trout C3 isoforms with various complement activators could be due at least in part to the observed changes in the His and Glu residues.

In all other species analyzed to date, the functionally active form of complement component C3 exists as the product of a single gene. We have now identified and characterized three functional C3 proteins (C3-1, C3-3, and C3-4) in trout that are the products of at least two distinct C3 genes. All three proteins are composed of an alpha-and a beta-chain and contain a thioester bond in the alpha-chain. However, they differ in their electrophoretic mobility, glycosylation, reactivity with monospecific C3 antibodies, and relative ability to bind to various surfaces (zymosan, Escherichia coli, erythrocytes). A comparison of the partial amino acid sequences of the three proteins showed that the amino acid sequence identity/similarity of C3-3 to C3-4 is 87/91%, while that of C3-3 and C3-4 to C3-1 is 51.5/65.5% and 60/73% respectively. Thus, trout possess multiple forms of functional C3 that represent the products of several distinct genes and differ in their ability to bind covalently to various complement activators.

The development and maturation of the nervous system are the results of highly specific interactions among developing neurons with their cellular and substrate environment. These interactions regulate cell proliferation and differentiation, neuroblast migration, axonal growth and guidance, target recognition and synapse formation. The development, over the past decade, of the methods of molecular biology, gives the opportunity for the study of these interactions in the molecular level. This kind of studies will give insight not only in the regulatory mechanisms of the establishment of form and pattern within the central nervous system (CNS) but also in very important properties of it, as it is plasticity and regeneration. Within this frame of studies it has been recently reported the isolation of cDNA clones from a λgt11 cDNA expression library made from NGF-treated PC12 celisi. The screening of the library was done with polyclonal rabbit and guinea pig anti-NILE (NGF Inducible Large External-Glycoprotein) antibodies the characteristics of which are previously described2,3. Two of these clones are modestly but reproducibly up-regulated by NGF in PC12 cells.