Biomedical subjects

Guor Mour Her

Publications and source records attributed to Guor Mour Her.

8 recordsLinked to original sources

PubMed2006

Betanodavirus induces phosphatidylserine exposure and loss of mitochondrial membrane potential in secondary necrotic cells, both of which are blocked by bongkrekic acid.

In this study, we show how the red spotted grouper nervous necrosis virus (RGNNV) causes loss of mitochondrial membrane potential and promotes host secondary apoptotic necrosis. RGNNV viral proteins such as protein alpha (42 kDa) and protein A (110 kDa) were quickly expressed between 12 h and 24 h postinfection (p.i.) in GL-av cells. Annexin V staining revealed that the NNV infection of GL-av cells induced phosphatidylserine (PS) externalization and development of bulb-like vesicles (bleb formation) at 24 h p.i. NNV infection also induced DNA fragmentation detectable by TUNEL assay between 12 h (8%) and 72 h (32%) p.i. Bongkrekic acid (1.6 microM; BKA) blocked permeability of the mitochondrial permeability transition pore, but cyclosporine A (CsA) did not block secondary necrosis. Finally, secondary necrotic cells were not engulfed by neighboring cells. Our data suggest that RGNNV induces apoptotic death via opening the mitochondrial permeability transition pore thereby triggering secondary necrosis in the mid-apoptotic phase.

Apoptosis↗

PubMed2006

Imbalance in liver homeostasis leading to hyperplasia by overexpressing either one of the Bcl-2-related genes, zfBLP1 and zfMcl-1a.

Apoptosis is an essential part of normal embryonic development in vertebrates, and it is involved in sculpturing organs and controlling cell populations. In previous studies, we identified two novel proteins, zfBLP1 and zfMcl-1a, which are similar to those of the Bcl-2 family as a group of evolutionarily conserved proteins that regulate cellular anti-apoptosis. To evaluate the effect of dysregulated hepatocyte apoptosis during zebrafish hepatogenesis, we demonstrate the transgenic overexpression of either zfBLP1 or zfMcl-1a in zebrafish larval liver. Results showed that 18%-43% of larvae overexpressed zfBLP1 and that 16%-37% of larvae overexpressed zfMc1-1a in the liver leading to liver hyperplasia in 5-day postfertilization (dpf) zebrafish larvae. Histologically, zebrafish larvae exhibiting liver hyperplasia displayed a normal type of hepatocyte and the same cell numbers in their two liver buds compared with only one liver bud of wild-type larvae. Of interest, the expression of cyclin genes (A2, B, D1, and E), hepatocyte nuclear factor genes (HNF-1alpha, beta, -3beta, and 4alpha), and oncogenic markers (P53, c-myc, beta-catenin, N-ras, and gankyrin) were up-regulated, while the expression of C/EBP-alpha was down-regulated in a zfMcl-1a-mediated anti-apoptotic process of the liver. Increased cell death and proliferation was found in both hepatic cells of zebrafish larvae overexpressing either zfBLP1 or zfMcl-1a. However, those zebrafish larvae with liver hyperplasia only lived approximately 10 days. (This finding may have been due to liver abnormalities that led to failure of liver function.) In conclusion, transgenic overexpression of zfBLP1 or zfMcl-1a in zebrafish larvae interrupts regulation of the homeostatic balance between cell proliferation and programmed cell death during hepatogenesis and leads to liver hyperplasia.

Animals↗

PubMed2004

Up-regulation of muscle-specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knock-down of myostatin-1.

Myostatin, a secreted growth and differentiation factor (GDF-8) belongs to transforming growth factor (TGF-beta) superfamily that plays as a negative regulator of skeletal muscle development and growth. Recently, myostatin has been isolated from fish; however, its role in muscle development and growth remains unknown. Here, we present the expression of myostatin during development and the effects of its knock-down on various genes such as muscle regulatory transcription factors (MRFs), muscle-specific proteins (MSP), and insulin-like growth factors (IGFs). The myostatin expression was found to be maternal as it starts in one-cell stage onward. The reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and Southern and Northern blots demonstrated that the myostatin expression is not only restricted to skeletal muscle, but it expressed all the tested tissues. Expression of myostatin was effected by using antisense morpholinos resulted in significant phenotypic difference in stages 18 and 20 hours postfertilization (hpf). To confirm the specificity of myostatin morpholino, furthermore, a rescue experiment was conducted. The length as well as width of somites was increased with almost no gap in between the somites. In addition, it deserves to mention that this is a first animal model that shows changes in the size of the somites. Moreover, analyses of MRFs, MSP, and IGFs in the knock-down embryos by RT-PCR revealed the up-regulation of MyoD, Myogenin, and Mck transcription, whereas IGF-2 transcription showed mild response with no effect on IGF-1, Desmin, and Myf5. In situ hybridization showed that there was an increase in the number of somites from 3 to 4 at 13 and 22 hpf. Taken together, these data suggest that myostatin plays a major role during myogenesis, apart from inhibition of proliferation as well as differentiation.

Animals↗

PubMed2004

Functional conserved elements mediate intestinal-type fatty acid binding protein (I-FABP) expression in the gut epithelia of zebrafish larvae.

Intestinal-type fatty acid binding protein (I-FABP) plays an important role in the intracellular binding and trafficking of long chain fatty acids in the intestine. The aim of this study, therefore, was to elucidate the regulation and spatiotemporal expression of the I-FABP gene during zebrafish larval development. We performed in vivo reporter-gene analysis in zebrafish by using a transient and transgenic approach. Green fluorescent protein-reporter analyses revealed that the proximal 192-bp region of the I-FABP promoter is sufficient to direct intestine-specific expression during zebrafish larval development. Functional dissection of a 41-bp region within this 192-bp promoter revealed that one C/EBP and two GATA-like binding sites, along with a novel 15-bp element within it are required for I-FABP gene expression in vivo. In addition, the six consensus sites (CCACATCAGCATGAA) in the 15-bp element are critical for I-FABP gene regulation in the zebrafish gut epithelia. Comparison analyses of the orthologous 15-bp element from mammalian I-FABP genes suggests that these mammalian elements are functionally equivalent to the zebrafish 15 element. These results provide the first in vivo evidence that these binding sites (C/EBP and GATA) and the novel 15-bp element contribute to intestine-specific gene expression and that they are functionally conserved across vertebrate evolution.

Animals↗

PubMed2004

Zebrafish intestinal fatty acid binding protein (I-FABP) gene promoter drives gut-specific expression in stable transgenic fish.

Mammalian intestinal fatty acid-binding protein (I-FABP) is a small cytosolic protein and is thought to play a crucial role of intracellular fatty acid trafficking and metabolism in gut. To establish an in vivo system for investigating its tissue-specific regulation during zebrafish intestinal development, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a transgenic strategy to generate gut-specific transgenic zebrafish with green/red fluorescent intestine. The 4.5-kb 5'-flanking sequence of zebrafish I-FABP gene was sufficient to direct fluorescent expression in intestinal tube, first observed in 3 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. In all five transgenic lines 45-52% of the F2 inheritance rates were consistent with the ratio of Mendelian segregation. These fish can also provide a valuable resource of labeled adult intestinal cells for in vivo or in vitro studies. Finally, it is possible to establish an in vivo system using these fish for screening genes required for gut development. genesis 38:26-31, 2004.

Animals↗

PubMed2003

In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio).

Mammalian liver fatty acid binding protein (L-FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid trafficking and metabolism. To better understand its tissue-specific regulation during zebrafish hepatogenesis, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a green fluorescent protein (GFP) transgenic strategy to generate liver-specific transgenic zebrafish. The 2.8-kb 5'-flanking sequence of zebrafish L-FABP gene was sufficient to direct GFP expression in liver primordia, first observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42-51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp-1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development.

Animals↗

PubMed2003

435-bp liver regulatory sequence in the liver fatty acid binding protein (L-FABP) gene is sufficient to modulate liver regional expression in transgenic zebrafish.

Liver fatty acid binding protein (L-FABP) is a small protein that is thought to play an important role in the intracellular binding and trafficking of long chain fatty acids in the liver. Expression of the gene encoding the zebrafish liver fatty acid binding protein is regulated by a 435-bp distal region (-1944 to -1510) of the L-FABP promoter. The 435-bp sequence is sufficient for gene activation in the liver primordia (or bud) and continues to be active in the adult liver when positioned adjacent to the SV40 basal promoter and linked directly to green fluorescent protein. The 435-bp sequence region has two distinct liver regulatory elements, A (-1944 to -1623) and B (-1622 to -1510), and contains multiple putative consensus binding sites. The element A sequence includes two consensus HFH and one HNF-1alpha site and the element B sequence includes one consensus HNF-3beta site. Deletion of an internal 435-bp fragment (-1944 to -1510) including the A and B elements totally ablated the liver-specific activity of the zebrafish L-FABP gene promoter. Deletion of either of the two elements reduces the liver activity. Mutation of the HNF-1alpha site or either of the two HFH sites in the A element or the HNF-3beta site in the B element significantly altered specificity in the liver primordia of transient expression embryos. The importance of the HNF-1alpha consensus binding site in the A element and the HNF-3beta consensus binding site in the B element within the 435-bp distal region of the L-FABP promoter region suggests that combinatorial interactions between multiple regulatory factors are responsible for the gene expression of L-FABP in the liver.

Animals↗

PubMed2003

Proximal upstream region of zebrafish bone morphogenetic protein 4 promoter directs heart expression of green fluorescent protein.

We examined the activity of the bone morphogenetic protein 4 (BMP4) promoter in zebrafish embryos via transient and stable transgenic expression analyses in order to obtain a better understanding of the regulation of BMP4 tissue-specific expression. Transient expression studies showed that the 9.0-kb BMP4 promoter/upstream region drove green fluorescent protein (GFP) expression mainly in the heart. Deletion analyses indicated the existence of multiple regulatory elements in the 7.5-kb BMP4 promoter/proximal upstream region. In addition, a coinjection experiment further demonstrated the 2.4-kb Bgl II-Hind III DNA region contains major positive regulatory elements. In addition, stable transgenic lines were established to further confirm the heart-specificity of this segment in BMP4 promoter. The results showed that GFP was mainly localized in the myocardium of developing ventricles of 48-hpf (hours postfertilization), 72-hpf, and 100-hpf transgenic F(1) embryos. Together, these results indicate that the 7.5-kb BMP4 promoter/proximal upstream region specifically contains regulatory elements for BMP4 expression in the heart, while regulatory elements for other endogenous BMP4-expressing tissues may reside in more distal regions and/or in introns.

Animals↗