Biomedical subjects

Chang-Jen Huang

Publications and source records attributed to Chang-Jen Huang.

At least 19 recordsLinked to original sources

PubMed2007

Massive expansion of EBV+ monoclonal T cells with CD5 down regulation in EBV-associated haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) comprises primary and secondary forms; the secondary form is most commonly triggered by the Epstein-Barr virus (EBV; EBV-HLH). Patients with EBV-HLH usually exhibit oligoclonal or monoclonal T cell proliferation, which may mimic T cell lymphoproliferative disorder (T-LPD). This article reports on EBV-HLH in a 17-month-old girl with an extreme surge of reactive T lymphocytosis (absolute count 167x10(9)/l) with CD5 down regulation. Bone marrow aspirate and trephine contained florid haemophagocytosis and massive infiltration of CD3+ Epstein-Barr virus-encoded RNA+ lymphocytes, as seen by double labelling. These lymphocytes were monoclonal for EBV and T cell receptor gamma chain gene rearrangement. The patient responded dramatically to intravenous immunoglobulin, interferon alpha2b, ganciclovir and prednisolone, suggesting restoration of her immune system and eradication of the clonal T cells through these immunoregulatory agents. Thus, careful clinicopathological correlation is warranted in the interpretation of immunophenotyping and clonality data in T cell proliferation in association with EBV-HLH to avoid erroneous diagnosis of T-LPD.

CD5 Antigens↗

PubMed2006

Heart-targeted overexpression of Nip3a in zebrafish embryos causes abnormal heart development and cardiac dysfunction.

We transiently expressed a proapoptotic protein, Nip3a, by a heart-specific BMP4 promoter in zebrafish embryos and generated two variants of embryos with abnormal heart phenotypes (A and B). Embryos with phenotype A heart defects showed hypoplastic or elongated ventricles, elongated or enlarged atriums with no normal cardiac looping resulting a significant longer SV-BA distance, and bradycardia at 48 h post-fertilization (hpf). Embryos with phenotype B heart defects showed an enlarged fluid-filled pericardium, severe hypoplasia, non-contracting ventricles, and elongated or enlarged slowly beating atriums with no normal looping. Histological sections further revealed the absence of a proper atrioventricular boundary and no endocardial cells lining this region in both 48- and 72-hpf Nip3a-overexpressing embryos, implicating defective endocardial cushion formation. These phenotypes are reminiscent of atrioventricular canal defects in humans. In addition, induced apoptotic myocardium cells were clustered in the presumptive atrioventricular boundary as well as in the adjacent ventricle and atrium of 48- and 72-hpf Nip3a-overexpressing embryos. Nip3a expression was readily detected in 80% epiboly BMP4-Nip3a-injected embryos, and defects in heart development were observed in both the linear heart tube and subsequent chamber formation stages. These results showed that myocyte apoptosis is a universal pathogenic factor for congenital heart failure using zebrafish as a model organism.

Animals↗

PubMed2006

Effects of cadmium on structure and enzymatic activity of Cu,Zn-SOD and oxidative status in neural cells.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder disease. Ten percent of the ALS patients are congenital (familial ALS), and the other 90% are sporadic ALS (SALS). It has been shown that mutations found in the Cu,Zn-SOD cause 20% of the familial ALS due to its low enzyme activity. We hypothesized that heavy metals may interfere the structure of Cu,Zn-SOD protein to suppress its activity in some of the SALS. In this study, we expressed and characterized the recombinant human Cu,Zn-SOD under various concentrations of Cu(2+), Zn(2+), and Cd(2+). By atomic absorption spectrophotometry, we demonstrated that adding of cadmium significantly increased the content of cadmium ion, but reduced its Zn(2+) content and enzyme activity of the Cu,Zn-SOD protein. The data of circular dichroism spectra demonstrated that the secondary structure of Cu,Zn-SOD/Cd is different from Cu,Zn-SOD, but close to apo-SOD. In addition to the effect of cadmium on Cu,Zn-SOD, cadmium was also shown to induce neural cell apoptosis. To further investigate the mechanism of neural cell apoptosis induced by cadmium, we used proteomics to analyze the altered protein expressions in neural cells treated with cadmium. The altered proteins include cellular structural proteins, stress-related and chaperone proteins, proteins involved in reactive oxygen species (ROS), enzyme proteins, and proteins that mediated cell death and survival signaling. Taken together, in this paper, we demonstrate that cadmium decreases the content of Zn(2+), changes the conformation of Cu,Zn-SOD protein to decrease its enzyme activity, and causes oxidative stress-induced neural cell apoptosis.

Animals↗

PubMed2006

Biochemical characterization and cloning of transglutaminases responsible for hemolymph clotting in Penaeus monodon and Marsupenaeus japonicus.

To investigate the shrimp blood clotting enzyme, a transglutaminase in the hemocytes of Penaeus monodon (abbreviated as TGH) was purified. TGH is an abundant homodimeric cytosolic protein with 84.2 kDa subunits. It clotted shrimp plasma and incorporated fluorescent dansylcadaverine into succinyl casein upon activation by CaCl(2) in vitro. IC(50) for the activation was 3 mM, which is below the shrimp plasma Ca(2+) level. Showing similar properties as other type II transglutaminase, TGH was particularly unstable after activation. MALDI-TOF/TOF mass-analyses of tryptic peptides of P. monodon TGH confirmed its identity to STG I (AY074924) previously cloned. A possible allele of the other isozyme STG II (AY771615) has also been cloned from the P. monodon cDNA and designated as PmTG. The predicted PmTG protein sequence is 58% similar to that of STG I and 99.2% to that of STG II. Likewise, a novel enzyme Mj-TGH was purified and cloned from Marsupenaeus japonicus hemocytes. Results of sequence alignment and phylogenetic analyses of these transglutaminases suggest that STG I and Mj-TGH are 83% identical and orthologous to each other, while PmTG/STG II and a previously cloned M. japonicus transglutaminase (AB162767) are their paralogs. Protein of the latter two could not be isolated, their regulated expression was discussed.

Amino Acid Sequence↗

PubMed2006

Requirement of nuclear localization and transcriptional activity of p53 for its targeting to the yolk syncytial layer (YSL) nuclei in zebrafish embryo and its use for apoptosis assay.

We expressed zebrafish p53 protein fused to GFP by a neuron-specific HuC promoter in zebrafish embryos. Instead of displaying neuronal expression patterns, p53-GFP was targeted to zebrafish YSL nuclei. This YSL targeting is p53 sequence-specific because GFP fusion proteins of p63 and p73 displayed neuronal-specific patterns. To dissect the underlying mechanisms, various constructs encoding a series of p53 mutant proteins under the control of different promoters were generated. Our results showed that expression of p53, in early zebrafish embryo, is preferentially targeted to the nuclei of YSL, which is mediated by importin. Similarly, this targeting is abrogated when p53 nuclear localization signal is disrupted. In addition, the transcriptional activity of p53 is required for this targeting. We further showed that fusion of pro-apoptotic BAD protein to p53-GFP led to apoptosis of YSL cells, and subsequent imperfect microtubule formation and abnormal blastomere movements.

Animals↗

PubMed2005

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish.

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.

Amino Acid Sequence↗

PubMed2005

Glycomic survey mapping of zebrafish identifies unique sialylation pattern.

Functional genomics and proteomics studies of the developmental glycobiology of zebrafish are greatly hampered by the current lack of knowledge on its glycosylation profile. To furnish the requisite structural basis for a more insightful functional delineation and genetic manipulation, we have initiated a survey mapping of the possible expression of stage-specific glycoconjugates in zebrafish. High-sensitivity mass spectrometry (MS) analysis in conjunction with the usual array of enzymatic and chemical derivatization was employed as the principal method for rapid differential mapping of the glycolipids and sequentially liberated N- and O-glycans from the total extracts. We demonstrated that all developmental stages of the zebrafish under investigation, from fertilized eggs to hatched embryos, synthesize oligomannosyl types of N-glycans, as well as complex types with additionally beta4-galactosylated, Neu5Ac/Neu5Gc monosialylated Lewis x termini. A combination of collision-induced dissociation (CID)-MS/MS and nuclear magnetic resonance (NMR) analyses led to the identification of an abundant and unusual mucin-type O-glycosylation, based on a novel sequence Fucalpha1-3GalNAcbeta1-4(Neu5Ac/Neu5Gcalpha2-3)Galbeta1-3GalNAc. This core structure may be further oligosialylated, but exclusively in the earlier development stages. Similarly, MS and MS/MS analyses of the extracted glycolipid fraction revealed the presence of a heterogeneous family of oligosialylated lactosylceramide compounds. In contrast to the O-glycans, these glycolipids only appear in the later development stages, suggesting a complex pattern of regulation for sialyltransferase activities during zebrafish embryogenesis.

Animals↗

PubMed2005

Epithelial Ca(2+) channel expression and Ca(2+) uptake in developing zebrafish.

The purpose of the present work was to study the possible role of the epithelial Ca(2+) channel (ECaC) in the Ca(2+) uptake mechanism in developing zebrafish (Danio rerio). With rapid amplification of cDNA ends, full-length cDNA encoding the ECaC of zebrafish (zECaC) was cloned and sequenced. The cloned zECaC was 2,578 bp in length and encoded a protein of 709 amino acids that showed up to 73% identity with previously described vertebrate ECaCs. The zECaC was found to be expressed in all tissues examined and began to be expressed in the skin covering the yolk sac of embryos at 24 h postfertilization (hpf). zECaC-expressing cells expanded to cover the skin of the entire yolk sac after embryonic development and began to occur in the gill filaments at 96 hpf, and thereafter zECaC-expressing cells rapidly increased in both gills and yolk sac skin. Corresponding to ECaC expression profile, the Ca(2+) influx and content began to increase at 36-72 hpf. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater caused upregulation of the whole body Ca(2+) influx and zECaC expression in both gills and skin. Colocalization of zECaC mRNA and the Na(+)-K(+)-ATPase alpha-subunit (a marker for mitochondria-rich cells) indicated that only a portion of the mitochondria-rich cells expressed zECaC mRNA. These results suggest that the zECaC plays a key role in Ca(2+) absorption in developing zebrafish.

Amino Acid Sequence↗

PubMed2005

Identification of three mutations in the Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene with familial amyotrophic lateral sclerosis: transduction of human Cu,Zn-SOD into PC12 cells by HIV-1 TAT protein basic domain.

The most frequent genetic causes of amyotrophic lateral sclerosis (ALS) determined so far are mutations occurring in the gene coding for copper/zinc superoxide dismutase (Cu,Zn-SOD). The mechanism may involve the formation of hydroxyl radicals or malfunctioning of the SOD protein. Wild-type SOD1 was constructed into a transcription-translation expression vector to examine the SOD1 production in vitro. Wild-type SOD1 was highly expressed in Escherichia coli. Active SOD1 was expressed in a metal-dependent manner. To investigate the possible roles of genetic causes of ALS, a human Cu,Zn-SOD gene was fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain (RKKRRQRRR) of human immunodeficiency virus type 1 in a bacterial expression vector to produce a genetic in-frame Tat-SOD1 fusion protein. The expressed and purified Tat-SOD1 fusion proteins in E. coli can enter PC12 neural cells to observe the cellular consequences. Denatured Tat-SOD1 was successfully transduced into PC12 cells and retained its activity via protein refolding. Three point mutations, E21K, D90V, and D101G, were cloned by site-directed mutagenesis and showed lower SOD1 activity. In undifferentiated PC12 cells, wild-type Tat-SOD1 could prevent DNA fragmentation due to superoxide anion attacks generated by 35 mM paraquat, whereas mutant Tat-D101G enhanced cell death. Our results demonstrate that exogenous human Cu,Zn-SOD fused with Tat protein can be directly transduced into cells, and the delivered enzymatically active Tat-SOD exhibits a cellular protective function against oxidative stress.

Amyotrophic Lateral Sclerosis↗

PubMed2004

Zebrafish heparin-binding neurotrophic factor enhances neurite outgrowth during its development.

Heparin-binding neurotrophic factor (HBNF) is a secreted heparin-binding protein containing highly basic and cysteine-rich amino acid residues. In this study, we cloned the full-length HBNF cDNA from zebrafish and determined its genomic structure by bioinformatics analysis. Zebrafish HBNF gene is composed of five exons and four introns spanning approximately 82kb. RT-PCR analysis revealed that zebrafish HBNF transcript was highly expressed in adult brain and intestine tissues while less in other tissues. During embryogenesis, zebrafish HBNF transcript was observed to be moderately expressed at earlier stages with a gradual decline. Higher expression level was observed after hatching and maintaining this level into adulthood. The overall amino acid sequence of zebrafish HBNF shows 60% identity to human HBNF, but with approximately 40% identity to other midkine proteins. Like mammalian homolog, zebrafish HBNF could induce significant neurite outgrowth in PC12 cells without NGF stimulation. In addition, zebrafish HBNF was able to enhance extensive neurite outgrowth in zebrafish embryos during embryogenesis. In summary, a feasible in vivo assay for neurite outgrowth was established in zebrafish.

Amino Acid Sequence↗

PubMed2004

Functional characterization of the placental fusogenic membrane protein syncytin.

Syncytin is an envelope protein of the human endogenous retrovirus family W (HERV-W). Syncytin is specifically expressed in the human placenta and mediates trophoblast cell fusion into the multinucleated syncytiotrophoblast layer. It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. Functional characterization of syncytin protein can aid understanding of the molecular mechanism underlying syncytin-mediated cell fusion. In this report, we studied the structure-function relationship of syncytin in 293T and HeLa cells transiently expressing wild-type syncytin or syncytin mutants generated by linker scanning and deletion mutagenesis. Of the 22 linker-inserted mutants, mutants InS51, InV139, InE156, InS493, InA506, and InL529 were fusogenic, suggesting that regions around amino acids S51, V139, and E156 in the SU subunit and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation. Of the 17 deletion mutants, nine mutants with deletions in the region from amino acids 479 to 538 were fusogenic. The deletion mutant DelI480, containing only the first four amino acid residues in the cytoplasmic domain, had enhanced fusogenic activity in comparison with the wild-type. In addition, two heptad repeat regions (HRA and B) were defined in the TM subunit of syncytin. A peptide inhibitor derived from the C-terminal heptad repeat region (HRB) was shown to potently inhibit syncytin-mediated cell fusion. Our results suggest that the cytoplasmic domain of syncytin is not essential for syncytin-mediated fusion but may play a regulatory role, and an intramolecular interaction between HRA and B is involved in the fusion process.

Base Sequence↗

PubMed2004

Transinactivation of the epidermal growth factor receptor tyrosine kinase and focal adhesion kinase phosphorylation by dietary flavonoids: effect on invasive potential of human carcinoma cells.

Focal adhesion kinase (FAK), a member of a growing family of structurally distinct protein tyrosine kinases (PTK), has been linked to specific phosphorylation events, and the elevation of FAK activity in human carcinoma cells correlated with increased invasive potential. Transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase activity is proposed to stimulate cell migration and the subsequent activation of downstream signaling pathways. Quercetin (Qu) and luteolin (Lu), are potent PTK inhibitors as well as putative chemopreventive agents. The present work, we demonstrate that Qu and Lu at concentration of 20 microM transinactivated EGFR tyrosine kinase activity with marked reduction in phosphotyrosyl level of 170, 125, 65, 60 and 42 kDa cellular proteins, and induced apoptosis in MiaPaCa-2 cells. The 125 kDa protein was further identified as a FAK by immunoprecipitation and immunoblotting analyses. Tumor cells treated with Lu or Qu dampened the phosphorylation of FAK. In addition, our data clearly demonstrated that tumor cells responded to Qu and Lu by parallel reductions in the levels of phosphorylated FAK and the secreted matrix metalloproteinase (MMP) that may lead to the suppression of invasive potential and cell migration in vitro. While the molecular mechanism of FAK regulation of MMP secretion in tumor cells remains unclear, our results suggested that blockade of the EGFR-signaling pathway may contributed to the net effect. As suggested in the current study, targeting EGFR and FAK with the objective of modulating their regulatory pathways could offer prospects for the treatment of EGFR-responsive cancers in the future.

Carcinoma↗

PubMed2004

Identification of 5'-upstream region of pufferfish ribosomal protein L29 gene as a strong constitutive promoter to drive GFP expression in zebrafish.

The genomic structure of Tetraodon fluviatilis L29 gene was determined and its promoter activity was analyzed in COS-1 cells and zebrafish embryos. The TfL29 gene comprises four exons and three introns, spanning approximately 1.7kb. The 5(')-upstream 2.2-kb of the first exon contains 10 E-boxes and many putative binding motifs for transcription factors GATA-1, AML-1a, c-Myb, Oct-1, CdxA, and NRF-2. Promoter activity assay showed that the distal 2.2-kb fragment not only had high luciferase activity in COS-1 cells, but also strong and ubiquitous GFP expression in a variety of tissues in zebrafish embryos. On the other hand, there are no TATA or CAAT boxes within a 300-bp region upstream from the transcription initiation site. Although this region has high luciferase activity in COS-1 cells, it is not sufficient to drive GFP expression in zebrafish embryos. In this proximal 300-bp region, there are two E-boxes, two CdxA sites, and one NRF-2 site that is immediately downstream of the transcription start site.

Amino Acid Sequence↗

PubMed2004

Purification and cloning of an endogenous protein inhibitor of carp nephrosin, an astacin metalloproteinase.

Nephrosin is a newly discovered member of the astacin family. It is a secreted proteinase and is present in carp head kidney, kidney, and spleen, all of which are responsible for immune and hematopoietic functions in fish. A complex formed by nephrosin and its inhibitor was purified from carp kidney extract by heparin affinity column chromatography. The presence of the nephrosin-inhibitor complex in different tissues was examined by immunoblotting with polyclonal antisera against the purified nephrosin inhibitor and nephrosin. Both nephrosin and the nephrosin inhibitor were present mainly in gill, head kidney, kidney, and spleen. In addition, we have cloned the cDNA encoding the nephrosin inhibitor. There are two different cDNA clones possibly resulting from two different genes, and the long form contains unique tandem repeat sequences in the 3'-end. The deduced primary structure of nephrosin inhibitor is similar to that of fetuin-A, a mammalian protein present in blood, liver, cerebrospinal fluid, and cerebral cortex during fetal development. Treatment with both N-glycosidase F and O-glycosidase removed the carbohydrate moiety of the nephrosin inhibitor and decreased the apparent molecular mass from 40 to 30 kDa. The nephrosin inhibitor seems to be synthesized in liver and then secreted to the blood as a precursor. When it was distributed into hematopoietic tissues, it was processed from 67 to 40 kDa and acquired inhibitory activity. This processing phenomenon of fetuin has not been reported elsewhere. Importantly, the presence of an endogenous inhibitor of nephrosin is the first report of this kind for astacin enzymes. It is very likely that endogenous tissue inhibitors may also be present for the regulation of other astacin enzymes.

Amino Acid Sequence↗

PubMed2003

Characterization of two mosquito STATs, AaSTAT and CtSTAT. Differential regulation of tyrosine phosphorylation and DNA binding activity by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

Two mosquito STATs, AaSTAT and CtSTAT, have been cloned from Aedes albopictus and Culex tritaeniorhynchus mosquitoes, respectively. These two STATs are more similar to those of Drosophila, Anopheles, and mammalian STAT5 in the DNA binding and Src homology 2 domains. The mRNA transcripts are expressed at all developmental stages, and the proteins are present predominantly at the pupal and adult stages in both mosquitoes. Stimulation with lipopolysaccharide resulted in an increase of tyrosine phosphorylation and DNA binding activity of AaSTAT and CtSTAT as well as an increase of luciferase activity of a reporter gene containing Drosophila STAT binding motif in mosquito C6/36 cells. After being infected with Japanese encephalitis virus, nuclear extracts of C6/36 cells revealed a decrease of tyrosine phosphorylation and DNA binding activity of AaSTAT which could be restored by sodium orthovanadate treatment. Taking all of the data together, this is the first report to clone and characterize two mosquito STATs with 81% identity and to demonstrate a different response of tyrosine phosphorylation and DNA binding of these two STATs by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

Amino Acid Motifs↗

PubMed2003

Expression and promoter analysis of mouse mastrin gene.

Human astrin is a newly identified microtubule-associated protein, which is highly expressed in the testis. Silencing of astrin has resulted in growth arrest and apoptotic cell death. In this study, we describe the cloning and genomic structure of mastrin, the mouse counterpart to astrin. The overall mouse mastrin amino-acid sequence is 66% identical to human astrin. Mastrin protein was demonstrated to localize to mitotic spindles during mitosis. Genomic clones containing mastrin gene were isolated; the gene was found to have 24 exons spanning 24kb of genomic DNA. Deletion analysis of 5(')-flanking sequences demonstrated that the first 120bp proximal to the TATA-less promoter region is necessary for minimal transcription of the mouse mastrin gene.

3T3 Cells↗

PubMed2003

Allele-specific differential expression of a common adiponectin gene polymorphism related to obesity.

Adiponectin gene polymorphisms have recently been reported to be associated with obesity, insulin sensitivity, and the risk of type 2 diabetes. We examined a T94G polymorphism of the adiponectin gene in 245 ostensibly normal nondiabetic subjects. The G allele frequency was lower among subjects with higher BMI (> or =27) than in those with lower BMI. BMI was inversely correlated with the dose of G allele. Multivariate linear regression analyses showed that the adiponectin genotypes were significantly related to BMI after adjusting for age and gender. The dose of the G allele was associated with a reduction of approximately 1.12 kg/m(2) in BMI. We further found that the relative mRNA levels of G allele were consistently higher than those of T allele in the omental adipose tissue from 21 heterozygous subjects. Finally, we observed that the expression levels of adiponectin affected insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes. In conclusion, the allele-specific differential expression of this common polymorphism could be responsible for its biological effects observed in this and the other studies.

3T3-L1 Cells↗

PubMed2003

Cloning of zebrafish BAD, a BH3-only proapoptotic protein, whose overexpression leads to apoptosis in COS-1 cells and zebrafish embryos.

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS(84)AP, corresponding to S(136) in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish alpha-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.

Actins↗