Effects of environmental salinity on gill endothelin receptor expression in the killifish, Fundulus heteroclitus

We recently determined that rapid changes in environmental salinity alter endothelin-1 (EDN1) mRNA levels in the euryhaline killifish, Fundulus heteroclitus, so we hypothesized that EDN1 may be a local regulator of gill ion transport in teleost fishes. The purpose of the present study was to examine the effects of changes in environmental salinity on the gill endothelin receptors: EDNRA, EDNRB, and EDNRC. Using quantitative real-time PCR, we determined that after a fresh water (FW) to seawater (SW) transfer, there is a two to threefold increase in gill EDNRA and EDNRB mRNA levels. Likewise, we found a two to three fold increase in gill EDNRA and EDNRB protein concentration. In addition, killifish that have acclimated to FW for 30 days had significantly lower EDNRA mRNA and protein levels than SW killifish. ENDRA were immunolocalized to the mitochondrion-rich cells of the killifish gill, suggesting that EDN1 signaling cascades may affect MRC function. EDNRB were found throughout the gill vasculature and on lamellar pillar cells. We previously immunolocalized EDN1 to the pillar cell suggesting that EDN1 acts as an autocrine signaling molecule and potentially regulates pillar cell tone and lamellar perfusion. We conclude that EDN1 is physiologically active in the teleost gill, and regulated by environmental salinity. Future functional studies examining the physiological role of this system are necessary to completely understand EDN1 in the fish gill.     

Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2alpha-induced luteolysis of the sheep corpus luteum

Three experiments were designed to examine the mechanisms that govern prostaglandin (PGF2alpha)-induced regression of the sheep corpus luteum. Evidence is presented supporting the involvement of endothelin 1 (EDN1) in PGF2alpha-induced luteolysis. Experiment 1 measured effects of PGF2alpha when actions of EDN1 were blocked by sustained administration of a type-A endothelin (EDNRA) or type-B endothelin (EDNRB) antagonist in vivo. Experiment 2 examined antisteroidogenic actions of PGF2alpha and EDN1 in the presence of an EDNRA or EDNRB antagonist in Day-8 luteal minces. In experiment 3, luteal cellular expression of EDNRA and EDNRB was determined immunohistochemically. Relative gene expression of EDNRA and EDNRB receptors was examined by real-time RT-PCR in Day-8 sheep corpora lutea. EDNRA, but not EDNRB, participated in antisteroidogenic actions of EDN1. During the first 12 h after PGF2alpha-induced luteolysis, EDNRA antagonist did not prevent a decline in serum progesterone concentrations. Early actions of PGF2alpha are either direct or mediated by something other than EDN1. However, beyond 12 h after PGF2alpha, serum progesterone concentrations increased in EDNRA antagonist-treated animals until they were the same as saline-treated controls, whereas an EDNRB antagonist had no effect in vivo or in vitro. The EDNRA antagonist negated the antisteroidogenic actions of EDN1 but only partially abolished the actions of PGF2alpha in vitro. In contrast, the EDNRB antagonist was ineffective in abolishing antisteroidogenic actions of EDN1 and PGF2alpha. Whereas real-time RT-PCR demonstrated high expression of EDNRA and low expression of EDNRB, immunohistochemically, only EDNRA was located in small steroidogenic, endothelial, and smooth muscle cells. In summary, studies in ovine corpora lutea provided strong evidence that: 1) EDNRA, but not EDNRB, mediates antisteroidogenic actions of EDN1, 2) actions of PGF2alpha are both independent of and dependent upon mediation by EDN1, and 3) small steroidogenic cells are targets for antisteroidogenic effects of EDN1. Furthermore, the results from experiment 1 suggest that the intermediary role of EDN1 may be more important in later stages of luteal regression.     

Molecular evolution of the vertebrate hexokinase gene family: Identification of a conserved fifth vertebrate hexokinase gene

Hexokinases (HK) phosphorylate sugar immediately upon its entry into cells allowing these sugars to be metabolized. A total of four hexokinases have been characterized in a diversity of vertebrates—HKI, HKII, HKIII, and HKIV. HKIV is often called glucokinase (GCK) and has half the molecular weight of the other hexokinases, as it only has one hexokinase domain, while other vertebrate HKs have two. Differing hypothesis has been proposed to explain the diversification of the hexokinase gene family. We used a genomic approach to characterize hexokinase genes in a diverse array of vertebrate species and close relatives. Surprisingly we identified a fifth hexokinase-like gene, HKDC1 that exists and is expressed in diverse vertebrates. Analysis of the amino acid sequence of HKDC1 suggests that it may function as a hexokinase. To understand the evolution of the vertebrate hexokinase gene family we established a phylogeny of the hexokinase domain in all of the vertebrate hexokinase genes, as well as hexokinase genes from close relatives of the vertebrates. Our phylogeny demonstrates that duplication of the hexokinase domain, yielding a HK with two hexokinase domains, occurred prior to the diversification of the hexokinase gene family. We also establish that GCK evolved from a two hexokinase domain-containing gene, but has lost its N-terminal hexokinase domain. We also show that parallel changes in enzymatic function of HKI and HKIII have occurred.     

Gene Duplications and Evolution of Vertebrate Voltage-Gated Sodium Channels

Voltage-gated sodium channels underlie action potential generation in excitable tissue. To establish the evolutionary mechanisms that shaped the vertebrate sodium channel α-subunit (SCNA) gene family and their encoded Na_v1 proteins, we identified all SCNA genes in several teleost species. Molecular cloning revealed that teleosts have eight SCNA genes, compared to ten in another vertebrate lineage, mammals. Prior phylogenetic analyses have indicated that the genomes of both teleosts and tetrapods contain four monophyletic groups of SCNA genes, and that tandem duplications expanded the number of genes in two of the four mammalian groups. However, the number of genes in each group varies between teleosts and tetrapods, suggesting different evolutionary histories in the two vertebrate lineages. Our findings from phylogenetic analysis and chromosomal mapping of Danio rerio genes indicate that tandem duplications are an unlikely mechanism for generation of the extant teleost SCNA genes. Instead, analyses of other closely mapped genes in D. rerio as well as of SCNA genes from several teleost species all support the hypothesis that a whole-genome duplication was involved in expansion of the SCNA gene family in teleosts. Interestingly, despite their different evolutionary histories, mRNA analyses demonstrated a conservation of expression patterns for SCNA orthologues in teleosts and tetrapods, suggesting functional conservation. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Axel Meyer]     

Intermedin, a novel calcitonin family peptide that exists in teleosts as well as in mammals: a comparison with other calcitonin/intermedin family peptides in vertebrates

Endocrine regulation in vertebrates is critical for the adaptation and regulation of homeostasis. The G protein-coupled receptor (GPCR) signaling transduction system represents one of the most ancient forms of cell surface signaling. Recently, comparative sequence analysis has aided in the identification and pairing of a variety of ligand/GPCR signaling systems. Among the ligands of type II GPCRs, the calcitonin family peptides including calcitonin, alpha-calcitonin gene-related peptide (alphaCGRP), betaCGRP, adrenomedullin, and amylin are among the best studied hormones, and the founding member, calcitonin, was originally identified and isolated from teleosts. This unique group of peptides shares a conserved tertiary structure with an N-terminal disulfide-bridged ring. In mammals, these peptides signal through two closely related type II GPCRs and three unique receptor activity-modifying proteins. Recently, based on the analysis of multiple vertebrate genomes, we identified a novel calcitonin/CGRP family peptide named intermedin. Here we show that in humans the five paralogous family genes, calcitonin, CGRP, amylin, adrenomedullin, and intermedin, evolved before the emergence of modern vertebrates, and that teleost genomes carry multiple copies of these co-evolved hormone genes. Sequence comparison showed that each of these genes is highly conserved in different vertebrates and that multiple copies of these peptides in teleosts could be derived from ancient genome duplication and/or lineage-specific intragenic duplications. The present article provides an overview of the calcitonin/intermedin family peptides found in teleost and mammalian genomes, and describes their putative functions. In addition, we demonstrate that one of the intermedin orthologs deduced from the pufferfish (Fugu rubripes) genome shares a conserved signaling activity with mammalian intermedin. The combined results indicate that the physiology associated with each of these family peptides likely evolved during early vertebrate evolution and diverged to serve select physiological functions in different vertebrates.     

Methionine adenosyltransferase as a useful molecular systematics tool revealed by phylogenetic and structural analyses

Structural and phylogenetic relationships among Bacteria and Eukaryota were analyzed by examining 292 methionine adenosyltransferase (MAT) amino acid sequences with respect to the crystal structure of this enzyme established for Escherichia coli and rat liver. Approximately 30% of MAT residues were found to be identical in all species. Five highly conserved amino acid sequence blocks did not vary in the MAT family. We detected specific structural features that correlated with sequence signatures for several clades, allowing taxonomical identification by sequence analysis. In addition, the number of amino acid residues in the loop connecting beta-strands A2 and A3 served to clearly distinguish sequences between eukaryotes and eubacteria. The molecular phylogeny of MAT genes in eukaryotes can be explained in terms of functional diversification coupled to gene duplication or alternative splicing and adaptation through strong structural constraints. Sequence analyses and intron/exon junction positions among nematodes, arthropods and vertebrates support the traditional Coelomata hypothesis. In vertebrates, the liver MAT I isoenzyme has gradually adapted its sequence towards one providing a more specific liver function. MAT phylogeny also served to cluster the major bacterial groups, demonstrating the superior phylogenetic performance of this ubiquitous, housekeeping gene in reconstructing the evolutionary history of distant relatives.     

Effect of SNP Polymorphisms of EDN1, EDNRA,and EDNRB Gene on Ischemic Stroke

The objective of this study is to investigate the association between SNP polymorphisms of endothelin-1 (EDN1) and endothelin receptor (EDNRA and EDNRB) gene and ischemic stroke (IS) in the Chinese Han population in northern. A case–control study was introduced. We genotyped eight SNPs (rs1800541, rs2070699, and rs5370 in EDN1 gene; rs1801708, rs5333, and rs5335 in EDNRA gene; and rs3818416 and rs5351 in EDNRB gene) and calculated their polymorphic distribution in control group, IS group, and the IS subgroups. In male population, EDN1 gene rs2070699 G allele increased the incidence risk to 1.78 times (P = 0.009; OR 1.78; 95 % CI 1.15–2.75) and the risk of morbidity of rs5370 T allele carrying increased to 1.49 times (P = 0.048; OR 1.49; 95 % CI 1.00–2.21). EDNRA gene mutation rs5335 homozygous CC morbidity risk was significantly lower (P = 0.016; OR 0.52; 95 % CI 0.31–0.88). In the female population, the mutant homozygous AA cancer risk was significantly higher than G allele carriers (P = 0.019; OR 2.65; 95 % CI 1.18–6.00) on EDNRA gene rs1801708. In EDN1 gene, T allele of rs5370 and G allele of rs2070699 may be IS incidence risk factors in Northern Han male population. A allele of rs1801708 in EDNRA gene can increase the risk of IS in Northern Han women population.     

Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, andexpression analysis

Background  

In recent years, the relaxin family of signaling molecules has been shown to play diverse roles in mammalian physiology, but little is known about its diversity or physiology in teleosts, an infraclass of the bony fishes comprising ~ 50% of all extant vertebrates. In this paper, 32 relaxin family sequences were obtained by searching genomic and cDNA databases from eight teleost species; phylogenetic, molecular evolutionary, and syntenic data analyses were conducted to understand the relationship and differential patterns of evolution of relaxin family genes in teleosts compared with mammals. Additionally, real-time quantitative PCR was used to confirm and assess the tissues of expression of five relaxin family genes in Danio rerio and in situ hybridization used to assess the site-specific expression of the insulin 3-like gene in D. rerio testis.     

Mito-nuclear genetic comparison in a Wolbachia infected weevil: insights on reproductive mode, infection age and evolutionary forces shaping genetic variation

Background

The composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events (R1, R2 in all vertebrates, R3 in teleosts) and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse.

Results

In a genomic survey where we manually annotated and analyzing sequences from more than 300 SLC1 genes (from more than 40 vertebrate species), we found evidence for an interesting evolutionary history of this gene family. While human and mouse genomes contain 7 SLC1 genes, in prototheria, sauropsida, and amphibia genomes up to 9 and in actinopterygii up to 13 SLC1 genes are present. While some of the additional slc1 genes in ray-finned fishes originated from R3, the increased number of SLC1 genes in prototheria, sauropsida, and amphibia genomes originates from specific genes retained in these lineages. Phylogenetic comparison and microsynteny analyses of the SLC1 genes indicate, that theria genomes evidently lost several SLC1 genes still present in the other lineage. The genes lost in theria group into two new subfamilies of the slc1 gene family which we named slc1a8/eaat6 and slc1a9/eaat7.

Conclusions

The phylogeny of the SLC1/EAAT gene family demonstrates how multiple genome reorganization and duplication events can influence the number of active genes. Inactivation and preservation of specific SLC1 genes led to the complete loss of two subfamilies in extant theria, while other vertebrates have retained at least one member of two newly identified SLC1 subfamilies.     

Nme protein family evolutionary history, a vertebrate perspective

Background  

The Nme family, previously known as Nm23 or NDPK, is involved in various molecular processes including tumor metastasis and some members of the family, but not all, exhibit a Nucleoside Diphosphate Kinase (NDPK) activity. Ten genes are known in humans, in which some members have been extensively studied. In non-mammalian species, the Nme protein family has received, in contrast, far less attention. The picture of the vertebrate Nme family remains thus incomplete and orthology relationships with mammalian counterparts were only partially characterized. The present study therefore aimed at characterizing the Nme gene repertoire in vertebrates with special interest for teleosts, and providing a comprehensive overview of the Nme gene family evolutionary history in vertebrates.     

Variants of endothelin-1 and its receptors in atopic asthma.

Endothelin-1 (ET-1) is a 21 amino acid peptide released from several types of bronchial cells. It operates through two types of receptors, type A(ET-RA) and type B(ET-RB) and has various activities in the pathophysiology of atopic asthma. These genes are localised on different chromosomes where genome-wide searches have identified linkage for atopic asthma, thus supporting the candidacy of ET-1 and its receptors for atopic asthma. A genetic association study was performed with variants of these three genes in both British (n = 300) and Japanese populations (n = 200). No significant association was found between variants of EDN1 and EDNRB genes, and atopic asthma in either population. However, variants of EDNRA gene showed a marginal association with atopy [odds = 0.39(95% CI: 0.17-0.89), p = 0.022, Pc = 0.066], especially with antigen specific IgE levels [odds = 0.31 (95% CI: 0.20-0.77), p = 0.006, Pc = 0.018] in the British population. These findings suggest that EDNRA is a major candidate locus for atopy on chromosome 4.     

Phylogenetic and Evolutionary Analyses of the Frizzled Gene Family in Common Carp (Cyprinus carpio) Provide Insights into Gene Expansion from Whole-Genome Duplications

In humans, the frizzled (FZD) gene family encodes 10 homologous proteins that commonly localize to the plasma membrane. Besides being associated with three main signaling pathways for cell development, most FZDs have different physiological effects and are major determinants in the development process of vertebrates and. Here, we identified and annotated the FZD genes in the whole-genome of common carp (Cyprinus carpio), a teleost fish, and determined their phylogenetic relationships to FZDs in other vertebrates. Our analyses revealed extensive gene duplications in the common carp that have led to the 26 FZD genes that we detected in the common carp genome. All 26 FZD genes were assigned orthology to the 10 FZD genes of on-land vertebrates, with none of genes being specific to the fish lineage. We postulated that the expansion of the FZD gene family in common carp was the result of an additional whole genome duplication event and that the FZD gene family in other teleosts has been lost in their evolution history with the reason that the functions of genes are redundant and conservation. Through the expression profiling of FZD genes in common carp, we speculate that the ancestral gene was likely capable of performing all functions and was expressed broadly, while some descendant duplicate genes only performed partial functions and were specifically expressed at certain stages of development.     

Molecular evolution of the polyamine oxidase gene family in Metazoa

ABSTRACT: BACKGROUND: Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. RESULTS: We analysed 36 SMO, 26 APAO and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all the SMOs and APAOs from vertebrates. The two vertebrate monophyletic clades clustered strictly mirroring the organismal phylogeny of fishes, amphibians, reptiles, birds, and mammals. Evidences from comparative genomic analysis, structural evolution and functional divergence in a phylogenetic framework across Metazoa suggested an evolutionary scenario where the ancestor PAO coding sequence, present in invertebrates as an orthologous gene, has been duplicated in the vertebrate branch to originate the paralogous SMO and APAO genes. A further genome evolution event concerns the SMO gene of placental, but not marsupial and monotremate, mammals which increased its functional variation following an alternative splicing (AS) mechanism. CONCLUSIONS: In this study the explicit integration in a phylogenomic framework of phylogenetic tree construction, structure prediction, and biochemical function data/prediction, allowed inferring the molecular evolutionary history of the PAO gene family and to disambiguate paralogous genes related by duplication event (SMO and APAO) and orthologous genes related by speciation events (PAOs, SMOs/APAOs). Further, while in vertebrates experimental data corroborate SMO and APAO molecular function predictions, in invertebrates the finding of a supported phylogenetic clusters of insect PAOs and the co-occurrence of two PAO variants in the amphioxus urgently claim the need for future structure-function studies.     

Evolutionary history of the vertebrate mitogen activated protein kinases family

Background

The mitogen activated protein kinases (MAPK) family pathway is implicated in diverse cellular processes and pathways essential to most organisms. Its evolution is conserved throughout the eukaryotic kingdoms. However, the detailed evolutionary history of the vertebrate MAPK family is largely unclear.

Methodology/Principal Findings

The MAPK family members were collected from literatures or by searching the genomes of several vertebrates and invertebrates with the known MAPK sequences as queries. We found that vertebrates had significantly more MAPK family members than invertebrates, and the vertebrate MAPK family originated from 3 progenitors, suggesting that a burst of gene duplication events had occurred after the divergence of vertebrates from invertebrates. Conservation of evolutionary synteny was observed in the vertebrate MAPK subfamilies 4, 6, 7, and 11 to 14. Based on synteny and phylogenetic relationships, MAPK12 appeared to have arisen from a tandem duplication of MAPK11 and the MAPK13-MAPK14 gene unit was from a segmental duplication of the MAPK11-MAPK12 gene unit. Adaptive evolution analyses reveal that purifying selection drove the evolution of MAPK family, implying strong functional constraints of MAPK genes. Intriguingly, however, intron losses were specifically observed in the MAPK4 and MAPK7 genes, but not in their flanking genes, during the evolution from teleosts to amphibians and mammals. The specific occurrence of intron losses in the MAPK4 and MAPK7 subfamilies might be associated with adaptive evolution of the vertebrates by enhancing the gene expression level of both MAPK genes.

Conclusions/Significance

These results provide valuable insight into the evolutionary history of the vertebrate MAPK family.     

Hearing Aid for Vertebrates via Multiple Episodic Adaptive Events on Prestin Genes

Auditory detection is essential for survival and reproduction of vertebrates, yet the genetic changes underlying the evolution and diversity of hearing are poorly documented. Recent discoveries concerning prestin, which is responsible for cochlear amplification by electromotility, provide an opportunity to redress this situation. We identify prestin genes from the genomes of 14 vertebrates, including three fishes, one amphibian, one lizard, one bird, and eight mammals. An evolutionary analysis of these sequences and 34 previously known prestin genes reveals for the first time that this hearing gene was under positive selection in the most recent common ancestor (MRCA) of tetrapods. This discovery might document the genetic basis of enhanced high sound sensibility in tetrapods. An investigation of the adaptive gain and evolution of electromotility, an important evolutionary innovation for the highest hearing ability of mammals, detects evidence for positive selections on the MRCA of mammals, therians, and placentals, respectively. It is suggested that electromotility determined by prestin might initially appear in the MRCA of mammals, and its functional improvements might occur in the MRCA of therian and placental mammals. Our patch clamp experiments further support this hypothesis, revealing the functional divergence of voltage-dependent nonlinear capacitance of prestin from platypus, opossum, and gerbil. Moreover, structure-based cdocking analyses detect positively selected amino acids in the MRCA of placental mammals that are key residues in sulfate anion transport. This study provides new insights into the adaptation and functional diversity of hearing sensitivity in vertebrates by evolutionary and functional analysis of the hearing gene prestin.     

Proposal to transfer 'Aegyptianella ranarum', an intracellular bacterium of frog red blood cells, to the family Flavobacteriaceae as 'Candidatus Hemobacterium ranarum' comb. nov

'Aegyptianella ranarum' (order Rickettsiales), an ultrastructurally defined small, Gram-negative rod, is known to replicate in the red blood cells of frogs. Heretofore, this bacterium has not been characterized genetically. We cloned and sequenced the 16S rRNA (1310 bp) and gyrB (718 bp) genes of 'A. ranarum' from a Canadian frog blood specimen. In situ hybridization (with an 'A. ranarum' 16S rRNA gene polymerase chain reaction product as probe) and electron microscopy confirmed that 'A. ranarum' forms cytoplasmic inclusions in frog erythrocytes. blast comparisons with GenBank 16S rRNA and gyrB sequences showed that both 'A. ranarum' genes were most similar (91% and 67% identity) to those of Chryseobacterium meningosepticum, a bacterium in the family Flavobacteriaceae. In contrast, 'A. ranarum' 16S rRNA shared only 61% identity with Aegyptianella pullorum. Phylogenetic analyses of these genes using phylip supported 'A. ranarum' as a member of Flavobacteriaceae, but suggested that its cladistic sibling may be Bergeyella zoohelcum or Weeksella virosa, rather than C. meningosepticum. We propose to classify 'Aegyptianella ranarum' as 'Candidatus Hemobacterium ranarum' in the family Flavobacteriaceae. Our results provide a starting point for studies of related intraerythrocytic bacterial infections in frogs.