Maternal methylosome protein 50 is essential for embryonic development in medaka Oryzias latipes.

Methylosome protein 50 (Mep50) is a protein that is rich in WD40 domains, which mediate and regulate a variety of physiological processes in organisms. Previous studies indicated the necessity of Mep50 in embryogenesis in mice Mus musculus and fish. This study aimed to further understand the roles of maternal Mep50 in early embryogenesis using medaka Oryzias latipes as a model. Without maternal Mep50, medaka zygotes developed to the pre-early gastrula stage but died later. The transcriptome of the embryos at the pre-early gastrula stage was analyzed by RNA sequencing. The results indicated that 1572 genes were significantly upregulated and 741 genes were significantly downregulated in the embryos without maternal Mep50. In the differentially expressed genes (DEGs), the DNA-binding proteins, such as histones and members of the small chromosome maintenance complex, were enriched. The major interfered regulatory networks in the embryos losing maternal Mep50 included DNA replication and cell cycle regulation, AP-1 transcription factors such as Jun and Fos, the Wnt pathway, RNA processing, and the extracellular matrix. Quantitative RT-PCR verified 16 DEGs, including prmt5, H2A, cpsf, jun, mcm4, myc, p21, ccne2, cdk6, and col1, among others. It was speculated that the absence of maternal Mep50 could potentially lead to errors in DNA replication and cell cycle arrest, ultimately resulting in cell apoptosis. This eventually resulted in the failure of gastrulation and embryonic death. The results indicate the importance of maternal Mep50 in early embryonic development, particularly in medaka fish.

Methylosome protein 50 is necessary for oogenesis in medaka.

Methylosome protein 50 (Mep50) functions as a partner to protein arginine methyltransferase 5. MEP50 serves as a coactivator for both the androgen receptor and estrogen receptor in humans. Mep50 plays a crucial role in the development of germ cells in Drosophila. The precise role of Mep50 in oogenesis remains unclear in vertebrates. The objective of this study was to investigate the role of Mep50 in oogenesis in medaka fish. Disruption of Mep50 resulted in impaired oogenesis and the formation of multiple oocyte follicles in medaka. RNA-seq analysis revealed significant differential gene expression in the mutant ovary, with 4542 genes up-regulated and 1264 genes down-regulated. The regulated genes were found to be enriched in cellular matrices and ECM-receptor interaction, the Notch signaling pathway, the PI3K-Akt signaling pathway, the MAPK signaling pathway, the Hippo signaling pathway, and the Jak-Stat pathway, among others. In addition, the genes related to the hypothalamus-pituitary-gonad axis, steroid metabolism, and IGF system were impacted. Furthermore, the mutation of mep50 caused significant alterations in alternative splicing of pre-mRNA in ovarian cells. Quantitative RT-PCR results validated the findings from RNA-seq analysis in the specific genes, including akt2, map3k5, yap1, fshr, cyp17a, igf1, ythdc2, cdk6, and col1, among others. The findings of this study demonstrate that Mep50 plays a crucial role in oogenesis, participating in a diverse range of biological processes such as steroid metabolism, cell matrix regulation, and signal pathways. This may be achieved through the regulation of gene expression via mRNA splicing in medaka ovarian cells.

Evaluation of microstructure, dissolution rate, and oral bioavailability of paclitaxel poloxamer 188 solid dispersion.

Poor solubility is a major challenge for enhancing the oral bioavailability and clinical application of many drugs, including the broad-spectrum chemotherapy drug paclitaxel (PTX). A practical approach to improving the solubility of insoluble drugs is through the use of solid dispersion (SD). This study aimed to investigate the potential of the triblock copolymer, poloxamer 188 (P188), as a carrier for preparing solid dispersion of paclitaxel using spray drying technology. We systematically studied its microstructure, dissolution behavior in vitro, and pharmacokinetics. Our findings demonstrate that PTX exists in an amorphous state in copolymer composed of polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) P188, with stronger miscibility with hydrophobic PPO segments. All three in vitro dissolution models revealed that the release rate of drugs in SD was significantly higher compared to that of physical mixtures (PM) as well as raw drugs. Furthermore, our pharmacokinetic results showed that the area under the curve(AUC) of PTX in SD was 6 times higher than that of active pharmaceutical ingredient(API), 4.5 times higher than PM, and the highest blood drug concentration (Cmax) reached 357.51 ± 125.54 (ng/mL), approximately 20 times higher than API. Overall, our findings demonstrate that the dissolution rate of amorphous PTX in SD significantly improves, effectively enhancing the oral bioavailability of PTX.

Mep50 is essential for embryonic development in medaka fish.

Mep50 as a partner promotes the activity and substrate affinity of Prmt5. Prmt5 and Mep50 function together in multiple bioprocesses of the cells. Both Prmt5 and Mep50 are necessary for maintenance of the stem cells and are indispensable in the embryogenesis in the mammals. However, the role of Mep50 is rarely studied in fish. This study was to investigate the role of Mep50 in embryonic development of medaka. Medaka mep50 was mutated by genomic editing with CRISPR-Cas9 technology. Two mutants with a deletion of 22 and 46 bp separately in mep50 caused premature stopping of translation. The homozygotes of these mutant fish were obtained by self-crossing of the heterozygotes. These homozygotic mutants could reproduce embryos but the offspring were not viable. The apoptotic cells were significantly more in the mutant embryos than that in the wild type indicated by TUNEL assay. Quantitative RT-PCR showed that the expression of oct4 and sox2 were significantly decreased, but p53 was increased in the mutant embryos. These results suggest that disruption of mep50 severely interferes with embryogenesis and mep50 is necessary for embryonic development by maintaining stem cells and repression of apoptosis in medaka.

Gene duplication, conservation, and divergence of activating transcription factor 5 gene in zebrafish.

Activating transcription factor 5 (Atf5) is a member of the ATF/CREB family of transcription factors and involved in diverse cellular functions and diseases in mammals. However, the function of atf5 remains largely unknown in fish. Here, we report the expression pattern and function of duplicated atf5 genes in zebrafish. The results showed that the gene structures of zebrafish atf5a and atf5b were similar to their mammalian orthologs. Zebrafish Atf5a and Atf5b shared an amino acid sequence identity of 40.7%. Zebrafish atf5a and atf5b had maternal origin with dynamic expression during embryonic development. Zebrafish atf5a mRNA is mainly enriched in olfactory epithelium, midbrain, and hindbrain, while zebrafish atf5b mRNA is mainly detected in midbrain, hindbrain, and liver during embryogenesis. The results of acute hypoxia experiment showed that atf5a mRNA was significantly upregulated in the brain, liver, and muscle, while atf5b mRNA was just increased significantly in the brain. Functional analysis showed that knockdown of atf5a affects the development of the ciliated neurons in zebrafish embryos. The effect was enhanced when atf5a MO was co-injected with atf5b MO. The development of ciliated neurons in zebrafish embryos was not affected by injection of atf5b MO alone. atf5a knockdown also affects the development of early-born olfactory neurons. The effects caused by atf5a knockdown could be rescued by atf5b mRNA. These results suggest that the duplicated atf5 genes may have evolved divergently and play redundant biological roles in the development of olfactory sensory neurons in zebrafish.

Regulation of bone metabolism mediated by ß-adrenergic receptor and its clinical application.

Recent studies have confirmed that ß-adrenergic receptors (ß-ARs) are expressed on the surface of osteoblasts and osteoclasts, and that the sympathetic nervous system can regulate bone metabolism by activating them. ß-AR blockers (BBs) are commonly used in the treatment of cardiovascular diseases in the elderly. It is important to investigate whether BBs have a beneficial effect on bone metabolism in the treatment of cardiovascular diseases, so as to expand their clinical application. This article reviews the effects of BB on bone metabolism and the progress of clinical research.

Alternative splicing of medaka bcl6aa and its repression by Prdm1a and Prdm1b.

Bcl6 and Prdm1 (Blimp1) are a pair of transcriptional factors that repressing each other in mammals. Prdm1 represses the expression of bcl6 by binding a cis-element of the bcl6 gene in mammals. The homologs of Bcl6 and Prdm1 have been identified in teleost fish. However, whether these two factors regulate each other in the same way in fish like that in mammals is not clear. In this study, the regulation of bcl6aa by Prdm1 was investigated in medaka. The mRNA of bcl6aa has three variants (bcl6aaX1-X3) at the 5'-end by alternative splicing detected by RT-PCR. The three variants can be detected in adult tissues and developing embryos of medaka. Prdm1a and prdm1b are expressed in the tissues and embryos where and when bcl6aa is expressed. The expression of prdm1a was high while the expression of bcl6aa was low, and vice versa, detected in the spleen after stimulation with LPS or polyI:C. In vitro reporter assay indicated that bcl6aa could be directly repressed by both Prdm1a and Prdm1b in a dosage-dependent manner. After mutation of the key base, G, of all predicted binding sites in the core promoter region of bcl6aa, the repression by Prdm1a and/or Prdm1b disappeared. The binding site of Prdm1 in the bcl6aa gene is GAAAA(T/G). These results indicate that both Prdm1a and Prdm1b directly repress the expression of bcl6aa by binding their binding sites where the 5'-G is critical in medaka fish.

The Proteins Interacting with Prmt5 in Medaka (Oryzias latipes) Identified by Yeast Two-Hybridization.

BACKGROUND: Prmt5 plays major role in regulation of gene expression, RNA processing, cell growth and differentiation, signal transduction, germ cell development, etc., in mammals. Prmt5 is also related to cancer. Knowing the proteins interacting with Prmt5 is important to understand Prmt5's function in cells. Although there have been reports on proteins binding with Prmt5 in mammals, the partner proteins of Prmt5 in fish are still unclear. OBJECTIVES: The objective was to obtain proteins that bind with Prmt5 in medaka, a model fish. METHODS: Yeast two hybridization was adopted to achieve the objective. Medaka Prmt5 was used as a bait to fish the prey, binding proteins in a cDNA library of medaka. Co-immunoprecipitation and in silicon analysis were performed to study the interaction of medaka Mep50 and Prmt5. RESULTS: Eight proteins were identified to bind with Prmt5 from 69 preliminary positive colonies. The binding proteins are methylosome protein 50 (Mep50), apolipoprotein A-I-like (Apo-AI), PR domain containing protein 1a with zinc fingers (Prdm1a), Prdm1b, T-cell immunoglobulin mucin family member 3 (Tim-3), phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (Paics), NADH dehydrogenase subunit 4 (ND4) and sciellin (Scl). Co-immunoprecipitation confirmed the interaction of medaka Prmt5 and Mep50. Predicted structures of medaka Prtm5 and Mep50 are similar to that of human PRMT5 and MEP50. CONCLUSION: Medaka Mep50, Prdm1a, Prdm1b, Apo-AI, Tim-3, Paics, ND4, and Scl bind with Prmt5.

Identification, expression pattern, and immune response of Tim-1 and Tim-4 in embryos and adult medaka (Oryzias latipes).

T-cell immunoglobulin (Ig) and mucin domain-containing 1 (Tim-1) and Tim-4 are two members of the Tim family. In mammals, Tim-1 and Tim-4 are proteins mainly expressed in immune cells and are associated with immune response. In the present study, medaka Oryzias latipes' Tim-1 (OlTim-1) and OlTim-4 were identified and characterized using bioinformatics analyses. With the use of reverse-transcription polymerase chain reaction, the expression profiles of OlTim-1 and OlTim-4 were examined in embryos and adult fish and in immune tissues following the intraperitoneal injection of stimulants. The results revealed that OlTim-1 possesses a cytoplasmic region, a transmembrane region, a mucin domain, and an Ig-like domain, while OlTim-4 is composed of two Ig-like domains and a mucin domain, but without the transmembrane region and cytoplasmic region. OlTim-1 and OlTim-4 expressions are detectable from the gastrula stage on, indicating that they are zygotic genes. Furthermore, OlTim-1 and OlTim-4 are expressed ubiquitously in the adult. Administration of immune stimulants, namely lipopolysaccharides and polyinosinic:polycytidylic acid, significantly increased the expression levels of OlTim-1 and OlTim-4 in the liver and intestine within 1 day and in the head, kidney, and spleen within 3 to 4 days postinjection. These results suggest that OlTim-1 and OlTim-4 are possibly involved in both innate and adaptive immunities.

Identification, expression and functional analysis of prmt7 in medaka Oryzias latipes.

Arginine methylation is an important posttranslational modification and catalyzed by a family of protein arginine methyltransferases (PRMTs). PRMT7 is the type III PRMT and produces solely monomethylarginine products. PRMT7 has been found to play important roles in multiple biological processes in mammals. However, the expression pattern and function of Prmt7 remain largely unknown in fish. In this study, we characterized the medaka prmt7 gene and determined its expression pattern and function during embryogenesis and germ cell development. The results showed that the chromosomal location and gene structure of medaka prmt7 were similar to its mammalian orthologs. Comparisons of deduced amino acid sequences indicated that medaka Prmt7 was a homolog of human PRMT7 with two methyltransferase domains. Reverse transcription-polymerase chain reaction (RT-PCR) and real time RT-PCR revealed that medaka prmt7 had maternal origin with continuous and dynamical expression during embryonic development. Whole-mount in situ hybridization analysis observed that the transcripts of prmt7 were ubiquitous at morula and gastrula stage, and were later riched in the brain and otic vesicles during embryogenesis. In the adult stage, prmt7 messenger RNA was detected in all examined tissues with the high levels in the ovary and testis. The expression of prmt7 in the gonads was restricted to oocytes of the ovary and spermatids/sperm of the testis. Functional analysis showed that knockdown of medaka prmt7 did not reduce the total number of primordial germ cells (PGCs) in vivo but significantly affected PGCs distribution during embryonic development. These results indicate that prmt7 may be involved in germ cell development in medaka.

Fundc1 is necessary for proper body axis formation during embryogenesis in zebrafish.

FUN14 domain-containing protein 1 (FUNDC1) is a mitochondrial outer membrane protein which is responsible for hypoxia-induced mitophagy in mammalian cells. Knockdown of fundc1 is known to cause severe defects in the body axis of a rare minnow. To understand the role of Fundc1 in embryogenesis, we used zebrafish in this study. We used bioimaging to locate zebrafish Fundc1 (DrFundc1) with MitoTracker, a marker of mitochondria, and/or CellLight Lysosomes-GFP, a label of lysosomes, in the transfected ovary cells of grass carp. The use of Western blotting detected DrFundc1 as a component of mitochondrial proteins with endogenous COX IV, LC3B, and FUNDC1 in transgenic human embryonic kidney 293 T cells. DrFundc1 induced LC3B activation. The ectopic expression of Drfundc1 caused cell death and apoptosis as well as impairing cell proliferation in the 293 T cell line, as detected by Trypan blue, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and incorporation of BrdU. DrFundc1 up-regulated expression of both autophagy- and apoptosis-related genes, including ATG5, ATG7, LC3B, BECLIN1, and BAX in transgenic 293 T cells. A knockdown of Drfundc1 using short hairpin RNA (shRNA) led to midline bifurcation with two notochords and two spinal cords in zebrafish embryos. Co-injection of Drfundc1 mRNA repaired defects resulting from shRNA. Knockdown of Drfundc1 resulted in up- or down-regulation of genes related to autophagy and apoptosis, as well as decreased expression of neural genes such as cyclinD1, pax2a, opl, and neuroD1. In summary, DrFundc1 is a mitochondrial protein which is involved in mitophagy and is critical for typical body axis development in zebrafish.

Bcl6aa and bcl6ab are ubiquitously expressed and are inducible by lipopolysaccharide and polyI:C in adult tissues of medaka Oryzias latipes.

B-cell lymphoma-6 (Bcl6) is a transcriptional repressor that plays important roles in various physiological activities such as innate and adaptive immune response, lymphocyte differentiation, and cell cycle regulation in mammals. Two homologs of Bcl6a, namely Bcl6aa and Bcl6ab, are identified in teleost fish including medaka Oryzias latipes. The expression profiles of bcl6aa and bcl6ab in medaka were studied using reverse-transcription polymerase chain reaction and in situ hybridization. The transcripts of bcl6aa and bcl6ab were detected from very early embryos such as the four-cell stage until hatching. Bcl6aa and bcl6ab were clearly detected in the embryonic body from 5 days postfertilization onward by in situ hybridization. Bcl6aa was specifically expressed in the retina, whereas bcl6ab was expressed in entire embryonic body. The results referred to that both bcl6aa and bcl6ab originate maternally in the zygotes and may play major roles in embryogenesis of medaka. The transcripts of bcl6aa and bcl6ab were detected in all examined adult tissues, including immune organs such as the gill, spleen, kidney, liver, and intestine. The expression of bcl6aa and bcl6ab in the liver, spleen, head-kidney, and intestine could be upregulated or downregulated by lipopolysaccharide and polyriboinosinic-polyribocytidylic acid. These results indicate that both bcl6aa and bcl6ab may be involved in immune response in medaka.

Expression of the alternative splicing variants of bcl6b in medaka Oryzias latipes.

Bcl6B, also known as BAZF, plays important roles in the immune response, repression of cancers, and maintenance of spermatogonial stem cells in mammals. In this study, the homologous gene bcl6b and its 5 alternative splicing variants, namely bcl6bX1 to bcl6bX5, were isolated from medaka fish, Oryzias latipes. Medaka bcl6b possesses conserved domains such as BTB domain, RD2 domain and four zinc fingers. Medaka bcl6bX1 to bcl6bX3 possess all three previously mentioned domains with minor differences in sequences. Medaka bcl6bX4 possesses only the BTB domain due to premature stopping, and bcl6bX5 possesses both the BTB domain and zinc fingers without the RD2 domain. Medaka bcl6b was expressed in the tissues including the brain, heart, gill, muscle, spleen, kidney, intestine, ovary and testes of adult fish. Medaka bcl6b was expressed in the embryos from very early stage, and could be detected clearly in the developing eyes by RT-PCR and in situ hybridization. Medaka bcl6b could respond to the stimuli of polyI:C and LPS in the kidney and spleen. Medaka bcl6bX1 to bcl6bX3 were the majority of the variants expressed in the adult tissues and the embryos, and were the major response to the stimulation of polyI:C and LPS in the spleen. These results suggested that bcl6b, including its isoforms, could function in various tissues and embryogenesis. Moreover, bcl6b might be a factor for immune response in medaka.

Expression pattern and functional analysis of fundc1 in rare minnow (Gobiocypris rarus).

Fundc1 is a mitochondrial outer membrane protein and plays important roles in mitochondria fission and hypoxia-induced mitophagy in mammalian cells. However, there is no relevant report of fundc1 in fish. In the present study, we cloned a 942bp fundc1 cDNA from rare minnow. The cDNA, designated as Grfundc1 cDNA, contains an open reading frame (ORF) of 459bp which encodes a polypeptide of 152 amino acid residues. Comparisons of deduced amino acid sequences demonstrated that Grfundc1 was highly homologous with those of other vertebrates. RT-PCR and real time PCR detection revealed that the transcripts of Grfundc1 were not detectable in the unfertilized eggs and had high levels at blastula and gastrula stages. Whole mount in situ hybridization analysis observed that Grfundc1 was ubiquitously expressed at early stage and later riched in specific regions, such as brain, branchial arch, eye and somite during embryogenesis. Grfundc1 was expressed in all the tissues of rare minnow adult, including brain, liver, gill, eyes, heart, kidney, intestine, muscle, testis and ovary. The expression of Grfundc1 in the brain, gill, heart and eye of rare minnow adult was significantly down-regulated by hypoxia. Similar hypoxic response was observed in the rare minnow embryos at 48hpf following hypoxia exposure. Functional analysis showed that knockdown of Grfundc1 significantly caused defects in the body axis and dorsal neural tissues of rare minnow embryos. These results indicate that Grfundc1 may play important roles in embryogenesis in fish.

Expression of mep50 in adult and embryos of medaka fish (Oryzias latipes).

Protein arginine methylation is important for gene regulation and biological processes. Methylosome protein 50 (Mep50) is identified as a partner of protein arginine methyltransferase 5 (Prmt5), a major enzyme capable of symmetric dimethylation, in mammals and Xenopus. The isolation and characterization of medaka mep50 were reported in this paper. Medaka Mep50 is a homolog of human MEP50 with six WD40 domains. Medaka mep50 was ubiquitously expressed in the adult tissues and had maternal origin with continuous and dynamical expression during embryonic development detected by RT-PCR and in situ hybridization. A strong interaction of medaka Mep50 and Prmt5 was shown by yeast two hybridization. The expression pattern of mep50 is similar to that of prmt5 in medaka. The results suggested that medaka Mep50 could be a partner of Prmt5 and might play major roles in a variety of tissues in medaka.

Germ cell-specific expression of dead end (dnd) in rare minnow (Gobiocypris rarus).

Rare minnow (Gobiocypris rarus) is an emerging model fish in China, and the development of its gonads is still elusive. Germ cell-specific genes are conserved in animals. Dead end (Dnd) was first documented as a germ granule component in zebrafish. Here, we report the cloning and expression profile of dnd in rare minnow. RT-PCR results showed that dnd is expressed specifically in the gonads of both sexes, is maternal in origin and is expressed continuously during embryogenesis. Dnd mRNA could be detected exclusively in the germ cells of the testis and ovary. Temporal expression of dnd mRNA is similar to that of vasa and dnd in zebrafish during embryogenesis. Taken together, dnd mRNA is restricted to the germ cells of rare minnow.

Identification and expression profiles of prdm1 in medaka Oryzias latipes.

Mouse Prdm1, also known as Blimp1, plays important roles in maturation and survival of lymphoid cells, as well as in organogenesis of muscle, limb, sensor organs and primordial germ cells. The homologues of mouse prdm1 have been identified in a diverse of animals including zebrafish and fugu. Here, we report the identification and expression profiles of two homologues of prdm1, namely prdm1a and prdm1b in medaka, Oryzias latipes. The transcripts of prdm1a and prdm1b were detectable in all the tissues including immune organs such as gill, spleen, kidney, liver and intestine that we have checked on. The transcripts of prdm1a could be detected in the embryonic shield at mid-gastrula stage and later in the somite, eye, otic vesicle, branchial arches, fin, intestine and cloaca during embryogenesis using in situ hybridization. Moreover, the expression of prdm1a in the liver of both medaka and zebrafish could be up-regulated by the immune stimuli including lipopolysaccharide, polyI:C and the grass carp reovirus, similarly to the up-regulation of IL1B. These results indicate that Prdm1a may play important roles in embryogenesis and also in immune response in fish.

Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus.

Fish have diverse sex determination and differentiation. DMRT1 and aromatase are conserved in the phyla and play pivotal roles in sex development. Gobiocypris rarus is a small fish used as a model in aquatic toxicology in China and has been used to study the effects of environmental endocrine disruptors on gene expression, but its sexual development remains elusive. Here, we report the full-length cDNA of G. rarus dmrt1 and its expression along with the expression of cyp19a1a and cyp19a1b, two genes encoding gonad and brain type aromatases, in adults and during ontogenesis. Both cyp19a1a and dmrt1 are expressed in the ovary and testis but show sexual dimorphism. Expression of cyp19a1a in the ovary is higher than in testes and dmrt1 follows the opposite pattern. Juvenile gonad histology changes at 15 days after hatching. The dimorphic expression of dmrt1 and cyp19a1a appears from 5 days after hatching, which is earlier than histological change. cyp19a1b is expressed coordinately with cyp19a1a until 15 days after hatching. These results show that dmrt1 and cyp19a1a play important roles in sex determination and sex differentiation in G. rarus.

Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization.

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. In this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGFbeta1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XlBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia.

Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress.

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O(2)) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.