Heme synthase (ferrochelatase) catalyzes the removal of iron from heme and demetalation of metalloporphyrins

The red pigments in meat products, including cooked cured ham, arise from the reaction of myoglobin with nitric oxide generated from exogenous nitrite. Since carcinogenic nitrosoamines may be generated by the treatment of meats with nitrite, the production of nitrite-free meat products is an attractive alternative. Raw dry-cured (Parma) hams are produced by the treatment of meats with salts other than nitrite. Analysis of pigments in raw dry-cured hams reveals that the main pigment is zinc protoporphyrin, suggesting that the conversion of heme to zinc protoporphyrin occurs via an iron-removal reaction from myoglobin heme during the processing of raw hams. Purification of the iron-removal enzyme showed that it was identical to ferrochelatase. Recombinant ferrochelatase in combination with NADH-cytochrome b5 reductase catalyzed NADH-dependent iron-removal reaction from hemin and hemoproteins. Metal ions such as zinc and cobalt were also removed from the corresponding metalloporphyrins. The addition of zinc ions led to the formation of zinc protoporphyrin. In cultured cells, the conversion of zinc mesoporphyrin to mesoheme was observed to be dependent on ferrochelatase and could be markedly induced during erythroid differentiation. This is the first demonstration of a new enzyme reaction, the reverse reaction of ferrochelatase, which may contribute to a new route of the recycling of protoporphyrin and heme in cells.     

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.     

Organization of the Mitochondrial Genome of a Deep-Sea Fish, Gonostoma gracile (Teleostei: Stomiiformes): First Example of Transfer RNA Gene Rearrangements in Bony Fishes

We determined the complete nucleotide sequence of the mitochondrial genome (except for a portion of the putative control region) for a deep-sea fish, Gonostoma gracile. The entire mitochondrial genome was purified by gene amplification using long polymerase chain reaction (long PCR), and the products were subsequently used as templates for PCR with 30 sets of newly designed, fish-universal primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products showed that the genome contained the same 37 mitochondrial structural genes as found in other vertebrates (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes), with the order of all rRNA and protein-coding genes, and 19 tRNA genes being identical to that in typical vertebrates. The gene order of the three tRNAs (tRNA^Glu, tRNA^Thr, and tRNA^Pro) relative to cytochrome b, however, differed from that determined in other vertebrates. Two steps of tandem duplication of gene regions, each followed by deletions of genes, can be invoked as mechanisms generating such rearrangements of tRNAs. This is the first example of tRNA gene rearrangements in a bony fish mitochondrial genome. Received August 5, 1998; accepted February 19, 1999.     

The phylogenetic position of an undescribed paedomorphic clupeiform taxon: mitogenomic evidence

Molecular characters may offer a useful alternative to confidently estimate the phylogenetic position of paedomorphic taxa otherwise difficult to place based on morphology because of the reduction or absence of characters in their larvae-like adult stage. Here, we sequenced the complete mitogenome of a remarkable undescribed marine paedomorphic clupeiform fish to gain insight into its phylogenetic position. Of a length of 17,507 bp, this mitogenome exhibits a unique gene order within the Teleostei because of the inversion of the contiguous tRNA^Gln and tRNA^Ile within the IQM region and the presence of a putative second control region inserted between these tRNAs. Mitogenomic data from 27 clupeiform species and 22 non-clupeiform species were subjected to partitioned maximum likelihood and Bayesian analyses. All resultant phylogenetic trees strongly supported the placement of this undescribed taxon within the order Clupeiformes, suborder Clupeoidei, and the family Clupeidae, as the sister group of the tribe Spratelloidini (Jenkinsia + Spratelloides) of the subfamily Dussumieriinae. Together, they form a monophyletic group with Chirocentrus and, possibly, Etrumeus. Despite its overall resemblance to Sundasalanx, this undescribed taxa (Clupeidae gen. et sp. indet.) is not closely related to that genus and represents an independent paedomorphic lineage within the Clupeoidei. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

基于Rag1基因序列变异的鲤形目鱼类系统发育重建:采样策略对生命之树的影响

The phylogenetic relationships of species are fundamental to any biological investigation, including all evolutionary studies. Accurate inferences of sister group relationships provide the researcher with an historical framework within which the attributes or geographic origin of species (or supraspecific groups) evolved. Taken out of this phylogenetic context, interpretations of evolutionary processes or origins, geographic distributions, or speciation rates and mechanisms, are subject to nothing less than a biological experiment without controls. Cypriniformes is the most diverse clade of freshwater fishes with estimates of diversity of nearly 3,500 species. These fishes display an amazing array of morphological, ecological, behavioral, and geographic diversity and offer a tremendous opportunity to enhance our understanding of the biotic and abiotic factors associated with diversification and adaptation to environments. Given the nearly global distribution of these fishes, they serve as an important model group for a plethora of biological investigations, including indicator species for future climatic changes. The occurrence of the zebrafish, Danio rerio, in this order makes this clade a critical component in understanding and predicting the relationship between mutagenesis and phenotypic expressions in vertebrates, including humans. With the tremendous diversity in Cypriniformes, our understanding of their phylogenetic relationships has not proceeded at an acceptable rate, despite a plethora of morphological and more recent molecular studies. Most studies are pre-Hennigian in origin or include relatively small numbers of taxa. Given that analyses of small numbers of taxa for molecular characters can be compromised by peculiarities of long-branch attraction and nodal-density effect, it is critical that significant progress in our understanding of the relationships of these important fishes occurs with increasing sampling of species to mitigate these potential problems. The recent Cypriniformes Tree of Life initiative is an effort to achieve this goal with morphological and molecular (mitochondrial and nuclear) data. In this early synthesis of our understanding of the phylogenetic relationships of these fishes, all types of data have contributed historically to improving our understanding, but not all analyses are complementary in taxon sampling, thus precluding direct understanding of the impact of taxon sampling on achieving accurate phylogenetic inferences. However, recent molecular studies do provide some insight and in some instances taxon sampling can be implicated as a variable that can influence sister group relationships. Other instances may also exist but without inclusion of more taxa for both mitochondrial and nuclear genes, one cannot distinguish between inferences being dictated by taxon sampling or the origins of the molecular data.     

Heterotypy in the N-terminal region of growth/differentiation factor 5 (GDF5) mature protein during teleost evolution

Heterotypy is now recognized as a generative force in the formationof new proteins through modification of existing proteins. Wereport that heterotypy in the N-terminal region of the maturegrowth/differentiation factor 5 (GDF5) protein occurred duringevolution of teleosts. N-terminal length variation of GDF5 wasfound among teleost interfamilies and interorders but not withinteleost families or among tetrapods. We further show that increaseof proline and glutamine to the N-terminal region of matureGDF5 occurred in Eurypterygii, the higher lineage of teleosts.Because the basic amino acids, believed to control diffusion,are conserved in this region across all species examined, wesuggest that the N-terminal elongation of the mature GDF5 proteinduring evolution has altered the protein diffusion in Eurypterygii,leading to high concentrations of the protein in the joint ofthe pharyngeal skeleton, the location of cartilage formationduring development.     

Physiological roles of the beta-substituted alanine synthase gene family in Arabidopsis

The beta-substituted alanine (Ala) synthase (Bsas) family in the large superfamily of pyridoxal 5'-phosphate-dependent enzymes comprises cysteine (Cys) synthase (CSase) [O-acetyl-serine (thiol) lyase] and beta-cyano-Ala synthase (CASase) in plants. Nine genomic sequences encode putative Bsas proteins in Arabidopsis thaliana. The physiological roles of these Bsas isoforms in vivo were investigated by the characterization of T-DNA insertion mutants. Analyses of gene expression, activities of CSase and CASase, and levels of Cys and glutathione in the bsas mutants indicated that cytosolic Bsas1;1, plastidic Bsas2;1, and mitochondrial Bsas2;2 play major roles in Cys biosynthesis. Cytosolic Bsas1;1 has the most dominant contribution both in leaf and root, and mitochondrial Bsas2;2 plays a significant role in root. Mitochondrial Bsas3;1 is a genuine CASase. Nontargeted metabolome analyses of knockout mutants were carried out by a combination of gas chromatography time-of-flight mass spectrometry and capillary electrophoresis time-of-flight mass spectrometry. The level of gamma-glutamyl-beta-cyano-Ala decreased in the mutant bsas3;1, indicating the crucial role of Bsas3;1 in beta-cyano-Ala metabolism in vivo.