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ACE2 orthologues in non-mammalian vertebrates (Danio, Gallus, Fugu, Tetraodon and Xenopus).

Angiotensin-converting enzyme 2 (ACE2), a newly identified member in the renin-angiotensin system (RAS), acts as a negative regulator of ACE. It is mainly expressed in cardiac blood vessels and the tubular epithelia of kidneys and abnormal expression has been implicated in diabetes, hypertension and heart failure. The mechanism and physiological function of this zinc metallopeptidase in mammals are not yet fully understood. Non-mammalian vertebrate models offer attractive and simple alternatives that could facilitate the exploration of ACE2 function. In this paper we report the in silico analysis of Ace2 genes from the Gallus (chicken), Xenopus (frog), Fugu and Tetraodon (pufferfish) genome assembly databases, and from the Danio (zebrafish) cDNA library. Exon ambiguities of Danio and Xenopus Ace2s were resolved by RT-PCR and 3'RACE. Analyses of the exon-intron structures, alignment, phylogeny and hydrophilicity plots, together with the conserved synteny among these vertebrates, support the orthologous relationship between mammalian and non-mammalian ACE2s. The putative promoters of Ace2 from human, Tetraodon and Xenopus tropicalis drove the expression of enhanced green fluorescent protein (EGFP) specifically in the heart tissue of transgenic Xenopus thus making it a suitable model for future functional genomic studies. Additionally, the search for conserved cis-elements resulted in the discovery of WGATAR motifs in all the putative Ace2 promoters from 7 different animals, suggesting a possible role of GATA family transcriptional factors in regulating the expression of Ace2.

Angiotensin-Converting Enzyme 2↗

Identification and analysis of cis-regulatory elements in development using comparative genomics with the pufferfish, Fugu rubripes.

The control of vertebrate development is facilitated by cis-regulatory sequences hardwired into the genome. Given that many developmental processes are strikingly similar across all backboned animals, it is reasonable to expect these sequences to be conserved at the nucleotide level, their potential for mutation being constrained by their function. Comparison between the genomes of highly divergent organisms allows such sequences to be identified and some of the most successful approaches have compared regions from the pufferfish, Fugu rubripes, with its distant mammalian relatives, rodents and humans. This review describes progress made in this kind of comparison, from small regions of individual genes, to whole genome alignments.

Animals↗

A new tetrodotoxin-producing actinomycete, Nocardiopsis dassonvillei, isolated from the ovaries of puffer fish Fugu rubripes.

Three puffer fishes, Fugu rubripes, collected from the Bohai Sea of China were examined for tetrodotoxin-producing microorganisms. An actinomycete isolated from the ovaries of the puffer fishes was found to produce tetrodotoxin. After being cultured at 28 degrees C for 7 days, cells were harvested by centrifuge and disrupted by ultrasonication. The toxin was purified from the cell lyzate by ultrafiltration, active charcoal column, Bio-gel-p2 and ion exchange column chromatography. Mouse neuroblastoma cell culture, thin-layer chromatography, fluorimetric spectrophotometry, UV-spectrophotometry and electrospray ionization mass spectrometry, together with mouse bioassay demonstrated that the isolated strain produced tetrodotoxin and related toxin during cultivation. Based on morphological, physiological, biochemical characteristics and 16S rDNA alignment, this strain was identified as Nocardiopsis dassonvillei. Our findings suggested that N. dassonvillei in the ovaries was closely related to the toxification of the puffer fish.

Actinobacteria↗

Toxicity and distribution of tetrodotoxin-producing bacteria in puffer fish Fugu rubripes collected from the Bohai Sea of China.

To investigate the relationship between the toxicity of puffer fish and the distribution of tetrodotoxin-producing bacteria in puffer fish Fugu rubripes collected from the Bohai Sea of China, bacteria were isolated from each organ (ovaries, livers, intestines and gallbladders) and screened for tetrodotoxin (TTX) production. 20 out of 36 isolated strains were found to produce TTX in vitro. In the organs of ovaries and livers whose toxicity is more potent than other organs, the number and toxicity of TTX-producing strains was greater than that of others. Most TTX-producing bacterial strains were identified as Bacillus spp. (19 strains) and Actinomycete spp. (1 strain) based on the morphological observation, physiological and biochemical characteristics and G+C content of DNA. The purified toxin was identified to be TTX by high performance liquid chromatography assay, thin-layer chromatography assay and electrospray ionization mass spectrometry analysis. Our results suggested that TTX-producing bacteria are closely related to the toxification of the puffer fish. More research is needed to elucidate the mechanism of TTX synthesis and the role of TTX in bacteria.

Actinomyces↗

Distribution of tetrodotoxin, saxitoxin, and their analogs among tissues of the puffer fish Fugu pardalis.

The anatomical distribution of tetrodotoxin (TTX), saxitoxin (STX) and their analogs (TTXs, STXs) in three female and three male specimens of the marine puffer fish Fugu pardalis from Miyagi Prefecture, 2005, Japan, were studied. 5-DeoxyTTX, 11-deoxyTTX, and 5,6,11-trideoxyTTX were quantified by liquid chromatography/mass spectrometry (LC/MS) for the first time, and other TTXs and STXs were determined by liquid chromatography-fluorescent detection (LC-FLD). As a result, 5,6,11-trideoxyTTX was found to be the major TTX analog in all tissues tested, whereas 5-deoxyTTX and 11-deoxyTTX were minor components. Especially, in female (n=3), the ratios of 5,6,11-trideoxyTTX to total of all TTX analogs (mole/mole) in ovaries (mean+/-SD, 0.42+/-0.055) were significantly larger than those in livers (0.17+/-0.025) (P<0.05). In contrary, the ratios of 4,9-anhydroTTX to total of all TTX analogs in livers (0.27+/-0.047) were significantly larger than those in ovaries (0.073+/-0.040) (P<0.01). The ratios of TTX to total of all TTX analogs were not significantly different between ovaries (0.47+/-0.078) and livers (0.55+/-0.067). In male (n=3), all these ratios were not significantly different between livers and testis. 4-S-CysteinylTTX was detected in liver, spleen, gall, and intestine in 1-6mole% of total of all TTX analogs, supporting our previous hypothesis that 4-S-cysteinylTTX is a metabolite of TTX.

Animals↗

Identification of cDNAs from Japanese pufferfish (Fugu rubripes) and Atlantic salmon (Salmo salar) coding for homologues to tetrapod prion proteins.

We identified cDNAs coding for homologues to tetrapod prion proteins (PrPs) in Atlantic salmon (Salmo salar) and Japanese pufferfish (Fugu rubripes), which were termed 'similar to PrPs' (stPrPs). Besides significant sequence homologies the fish stPrPs display characteristic structural features in common with tetrapod PrPs. In addition, two stPrPs were shown to be highly expressed in brain tissue. None of the so far identified PrP-homologues of fish resembles doppel. Hence, the duplication of the PrP gene, which generated doppel, may have occurred not in fish but later in the tetrapod lineage. The identification of fish PrPs provides a basis to address concerns about a possible susceptibility of fish to prion infections.

Amino Acid Sequence↗

New BEL-like LTR-retrotransposons in Fugu rubripes, Caenorhabditis elegans, and Drosophila melanogaster.

The BEL group of retroelements is present in greater numbers, variety and taxonomic range than may have been thought previously. In addition to the insects, nematodes and schistosomes, BEL-like elements are present in echinoderms, urochordates, and at least two highly diverged species of fish. We describe one new full-length BEL-like element in Fugu that we call Suzu, another in Drosophila that we call Tinker, and seven new families in C. elegans. Many of the C. elegans elements have an unusual insertion at the 5' end. The previously known Roo, TRAM and Telemac are also BEL-like retrotransposons. Some BEL-like elements have captured an envelope gene, probably from other retroelements in some cases but from a phlebovirus in one case.

Amino Acid Motifs↗

Serine/threonine phosphatases of the pufferfish, Fugu rubripes.

The compact genome of the Japanese pufferfish, Fugu rubripes, about 7.5 times smaller than the human genome, facilitates the isolation of vertebrate genes. We have used this genome to isolate and characterize members of the vertebrate serine/threonine protein phosphatase gene family. Our data reveal the presence of two isoforms of PP2A, alpha and beta, and PPX, as previously found in mammals.

Amino Acid Sequence↗

Identification of an achaete-scute homolog, Fash1, from Fugu rubripes.

Proteins of the achaete-scute family of transcription factors play important roles in neurogenesis in both invertebrates and vertebrates. Here, we report the cloning and characterization of a Japanese pufferfish, Fugu rubripes achaete-scute homolog 1, Fash1. Sequence alignment of the predicted amino acid sequence of Fash1 with other vertebrate homologs of the achaete scute homolog 1 subclass shows that the carboxyl 2/3 of the protein, including the basic helix-loop-helix, a putative nuclear localization signal and several consensus phosphorylation sites, is highly conserved. Strikingly, the similarity in this region between eight vertebrate species is close to 90%.

Amino Acid Sequence↗

Isolation and genomic characterization of the TUPLE1/HIRA gene of the pufferfish Fugu rubripes.

In an effort to obtain a small genomic construct for the generation of a HIRA transgenic mouse, we have isolated and sequenced the Fugu TUPLE1/HIRA gene. We have compared the gene organization and the proteins encoded in pufferfish and human and also searched for conserved DNA sequences that might be important in gene regulation. The pufferfish gene spans approx. 9 kb, which is approx. 11 times smaller than the human gene, owing to the reduced size of the introns. Like its human counterpart, it is organized into 25 exons. The majority of the splice sites are in identical positions to those found in the human gene, however, for three internal exons the positions of the splice sites are not directly comparable. The coding regions are almost identical in size and show a high degree of similarity, especially at the amino and carboxy termini. Comparisons of 5' and 3' sequences failed to detect similarities or sequences involved in regulation.

Amino Acid Sequence↗

Molecular cloning and expression of pyruvate kinase from globefish (Fugu rubripes) skeletal muscle.

A cDNA clone encoding pyruvate kinase (PK) was isolated from a skeletal muscle cDNA library of globefish (Fugu rubripes), which is a kind of lower vertebrate. The full-length cDNA of globefish skeletal muscle pyruvate kinase (FM-PK) is approximately 2 kb and encodes a protein comprising 530 amino acids. The FM-PK gene is spanning approximately 4.8 kb and consists of 11 exons. FM-PK mRNA was detected in muscle and heart using Northern blots. The recombinant FM-PK (rFM-PK) was expressed in a baculovirus-insect cell system and purified using ion-exchange chromatography. The purified rFM-PK was shown to exist a 230 kDa homotetramer composed of 57 kDa subunits. Gel filtration showed 230000 as the tetramer of the subunit. The apparent K(m) (or S(0.5)) and the Hill coefficient for phosphoenolpyruvate (PEP) and ADP are 0.14 mM, 1.3 and 0.30 mM 0.98 at pH 7.4, respectively, when the enzyme is saturated with the second substrate. The rFM-PK is strongly activated by fructose-1,6-bisphosphate, the apparent K(m) for PEP changes to 0.059 mM and the Hill coefficient to 1.1. ATP, which is the product of the enzyme reaction, inhibits activity. This is the first report to show the full-length cDNA and amino acid sequence of PK for a species of fish.

Amino Acid Sequence↗

Identification of 4-S-Cysteinyltetrodotoxin from the liver of the puffer fish, Fugu pardalis, and formation of thiol adducts of tetrodotoxin from 4,9-anhydrotetrodotoxin.

The metabolic pathway of tetrodotoxin (TTX), a powerful and specific voltage-gated sodium channel blocker, has not been well-clarified either in TTX-poisoned patients or in puffer fish. 4-S-CysteinylTTX (4-CysTTX) was isolated from the liver of the puffer fish, Fugu pardalis, as the first adduct of TTX with thiol. The structure was fully characterized by electrospray ionization-MS and two-dimensional NMR spectroscopy. The configuration of Cys in this compound was confirmed to be S (L-Cys) by application of the Marfey's method to cystine obtained from 4-CysTTX by iodine oxidation. We also found that 4-CysTTX was derived from 4,9-anhydroTTX by incubation with a large excess of Cys in aqueous buffer (pH 8.0) for 90 min at 40 degrees C in 33% yield by HPLC. GSH also reacted with 4,9-anhydroTTX to form 4-S-glutathionylTTX (4-GSTTX) in 39% yield under the same conditions, whereas TTX scarcely reacted with Cys and GSH. These reactions were strictly pH-dependent, giving the highest yield at pH 8.0. 4-GSTTX was converted to 4,9-anhydroTTX in 0.8 M sodium phosphate buffer (pH 8.0) at 25 degrees C. Its half-life was approximately 4 h. The minimum lethal doses of 4-CysTTX and 4-GSTTX to mice by ip injection were more than 140 and 860 microg/kg (n = 2), which were 14- and 86-fold larger than the LD(50) of TTX, respectively. 4-GSTTX was hydrolyzed to 4-CysTTX by gamma-glutamyltranspeptidase (Sigma, catalog no. G9270), which was supposed to contain cysteinylglycine dipeptidase. We also examined the effect of Cys or GSH coinjection (ip) with TTX to mice for detoxification of TTX and concluded that these coinjections did not reduce the toxicity of TTX.

Animals↗

Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome.

Cloning and sequencing techniques now allow us to characterize genes directly instead of having to deduce their properties from their effects. This new genetics reaches its apotheosis in the plan to obtain the complete DNA sequence of the human genome, but this is far beyond the capacity of present sequencing methods. Small 'model' genomes, 'such as those of Escherichia coli (4.7 megabases (Mb) and yeast (14 Mb), or even those of Caenorhabditis elegans (100 Mb) and Drosophila (165 Mb), are better scaled to existing technology. The yeast genome will contain genes with functions common to all eukaryotic cells, and those of simple multicellular organisms may throw light on the genetic specification of more complex functions. However, vertebrates differ in their morphology and development, so the ideal model would be a vertebrate genome of minimum size and complexity but with maximum homology to the human genome. Here we report the characterization of the small genome (400 Mb) of the tetraodontoid fish, Fugu rubripes. A random sequencing approach supported by gene probing shows that the haploid genome contains 400 Mb of DNA, of which more that 90% is unique. This genome is 7.5 times smaller than the human genome and because it has a similar gene repertoire it is the best model genome for the discovery of human genes.

Animals↗

Bioinformatic identification of polymerizing and transmembrane mucins in the puffer fish Fugu rubripes.

Mucins are large glycoproteins characterized by mucin domains that show little sequence conservation and are rich in the amino acids Ser, Thr, and Pro. To effectively predict mucins from genomic and protein sequences obtained from genome projects, we developed a strategy based on the amino acid compositional bias characteristic of the mucin domains. This strategy is combined with an analysis of other features commonly found in mucins. Our method has now been used to predict mucins in the puffer fish Fugu rubripes that were previously not identified or annotated. At least three gel-forming mucins were found with the same general domain structure as the human MUC2 mucin. In addition one transmembrane mucin was identified with SEA and EGF domains as found in the mammalian transmembrane mucins. These results suggest that the number of gel-forming mucins has been conserved during evolution of the vertebrates, whereas the family of transmembrane mucins has been markedly expanded in the higher vertebrates.

Animals↗

A tetrodotoxin-producing Vibrio strain, LM-1, from the puffer fish Fugu vermicularis radiatus.

Identification of tetrodotoxin (TTX) and its derivatives produced from a Vibrio strain in the intestine of the puffer fish Fugu vermicularis radiatus was performed by thin-layer chromatography, electrophoresis, high-performance liquid chromatography, and gas chromatography-mass spectrometry, together with a mouse bioassay for toxicity. It was demonstrated that the isolated bacterium produced TTX, 4-epi-TTX, and anhTTX during cultivation, suggesting that Vibrio strains are responsible for the toxification of the puffer fish.

Animals↗

Amnesia in relation to fugue states--distinguishing a neurological from a psychogenic basis.

A case of transient amnesia is described in which a patient reported memory loss for five days, during which he had wandered extensively. Analysis showed that he did not have selective amnesia for the public events which had occurred during the five days for which he professed memory loss. This finding was incompatible with our case having a neurologically based global memory disorder during the fugue state. These findings offer support for a distinction between personal and public episodic memory.

Adult↗

Comparative study of family 2 GPCRs in Fugu rubripes.

In this study, members of family 2 GPCRs, one of the largest families of receptors in vertebrates, were isolated and characterized in the genome of the Japanese pufferfish, Fugu rubripes, and compared with the orthologous genes in other vertebrates. Phylogenetic analysis carried out with all vertebrate family 2 GPCR members indicated that CALR/CGRPR and CRF are the most divergent receptor group within this family and that the remaining members appear to originate from a common ancestral gene precursor.

Animals↗