Biomedical subjects

S K Shyue

Publications and source records attributed to S K Shyue.

12 recordsLinked to original sources

PubMed2001

Adenovirus-mediated heme oxygenase-1 gene transfer inhibits the development of atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Increasing evidence supports the role of heme oxygenase-1 (HO-1) in cytoprotective response and iron homeostasis. The object of this study was to investigate whether adenovirus-mediated gene transfer of HO-1 in arteries reduces iron overload and inhibits lesion formation in apolipoprotein E (apoE)-deficient mice. METHODS AND RESULTS: Infection of rat aortic smooth muscle cells with adenovirus carrying the human HO-1 gene (Adv-HO-1) resulted in a high-level expression of HO-1 protein, which effectively reduced the hemin-induced iron overload in these cells. Adenovirus-mediated gene transfer in arteries in vivo was achieved by direct injection of Adv-HO-1 into the left ventricles of anesthetized animals. Transgene was expressed in the endothelium and aortic lesion of apoE-deficient mice after they had received recombinant adenovirus for 1 week and gradually decayed during the next 5 weeks. When young apoE-deficient mice (14 weeks old) received Adv-HO-1 (2.5 x 10(9) pfu) for 6 weeks, lesions that developed in the aortic root or aortic arch were significantly smaller than those in control littermates receiving empty viral vector. Furthermore, the iron deposition as well as tissue iron content was much less in aortic tissue of Adv-HO-1-treated mice. The inhibitory effect of HO-1 gene transfer on the progression of advanced lesions was also observed in older apoE-deficient mice (20 weeks old) receiving Adv-HO-1 intraventricularly. CONCLUSIONS: Overexpression of HO-1 in vascular cells facilitates iron metabolism and attenuates development of atherosclerosis in apoE-deficient mice.

Adenoviridae↗

PubMed2001

Colocalization and interaction of cyclooxygenase-2 with caveolin-1 in human fibroblasts.

Results from our previous study suggest that cyclooxygenase-2 (COX-2) induced by phorbol 12-myristate 13-acetate (PMA) may be localized to caveolae-like structures (Liou, J.-Y., Shyue, S.-K., Tsai, M.-J., Chung, C.-L., Chu, K.-Y., and Wu, K. K. (2000) J. Biol. Chem. 275, 15314-15320). In this study, we determined subcellular localization of COX-2 and caveolin-1 by confocal microscopy. COX-2 in human foreskin fibroblasts stimulated by PMA (100 nm) or interleukin-1beta (1 ng/ml) for 6 h was localized to plasma membrane in addition to endoplasmic reticulum and nuclear envelope. Caveolin-1 was localized to plasma membrane, and image overlay showed colocalization of COX-2 with caveolin-1. This was confirmed by the presence of COX-2 and caveolin-1 in the detergent-insoluble membrane fraction of cells stimulated by PMA. Immunoprecipitation showed complex formation of COX-2 with caveolin-1 in a time-dependent manner. A larger quantity of COX-2 was complexed with caveolin-1 in PMA-treated than in interleukin-1beta-treated cells. Purified COX-2 complexed with glutathione S-transferase-fused caveolin-1, which was not inhibited by the scaffolding domain peptide. Caveolin-1-bound COX-2 was catalytically active, and its activity was not inhibited by the scaffolding domain peptide. These results suggest that COX-2 induced by PMA and interleukin-1beta is colocalized with caveolin-1 in the segregated caveolae compartment. Because caveolae are rich in signaling molecules, this COX-2 compartment may play an important role in diverse pathophysiological processes.

Blotting, Western↗

PubMed2001

Selective augmentation of prostacyclin production by combined prostacyclin synthase and cyclooxygenase-1 gene transfer.

BACKGROUND: We tested the hypothesis that combined cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) gene transfer selectively augments prostacyclin production without a concurrent overproduction of other prostanoids. METHODS AND RESULTS: ECV304 cells were transfected with bicistronic pCOX-1/PGIS versus pCOX-1 or pPGIS, and prostanoids were analyzed. Contrary to the high prostaglandin E2 synthesis in pCOX-1 transfected cells, selective prostacyclin formation was noted with bicistronic plasmid transfection. Next, we determined the optimal ratio of Ad-COX-1 to Ad-PGIS by transfecting human umbilical vein endothelial cells with various titers of these 2 adenoviral constructs and determined the level of protein expression and prostanoid synthesis. Our results show that optimal ratios of adenoviral titers to achieve a large prostacyclin augmentation without overproduction of prostaglandin E2 or F2alpha were 50 to 100 plaque forming units (pfu) of Ad-COX-1 to 50 pfu of Ad-PGIS per cell. A higher Ad-PGIS to Ad-COX-1 ratio caused a paradoxical decline in prostacyclin synthesis. CONCLUSIONS: Prostacyclin synthesis can be selectively augmented by cotransfecting endothelial cells with an optimal ratio of COX-1 to PGIS. Combined COX-1 and PGIS gene transfer has the potential for therapeutic augmentation of prostacyclin.

6-Ketoprostaglandin F1 alpha↗

PubMed2001

NO modulates monocyte chemotactic protein-1 expression in endothelial cells under cyclic strain.

Endothelial cells (ECs) under hemodynamic forces increase intracellular reactive oxygen species (ROS) that modulate gene expression. We previously showed that NO attenuated the shear flow-induced gene level. The present study explored the role of endothelial NO in cyclic strain-treated ECs. Treatment of ECs with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, reduced cyclic strain-induced monocyte chemotactic protein (MCP)-1 expression. Conversely, exposure of ECs to an NO synthase inhibitor augmented MCP-1 mRNA levels. NO attenuated the binding of activator protein-1 to the 12-O-tetradecanoylphobol-13-acetate-responsive element (TRE) in the MCP-1 promoter region. ECs overexpressed with endothelial NO synthase (eNOS) inhibited cyclic strain-induced MCP-1 expression and MCP-1 promoter (-540 bp) activity. Consistently, ECs treated with SNAP or infected with adenovirus carrying eNOS reduced strain-induced superoxide levels. These strain-induced superoxide and MCP-1 expressions were greatly blunted by treating ECs with an NADPH oxidase inhibitor, diphenyleneiodonium chloride or apocynine, but not with a xanthine oxidase inhibitor. ECs infected with adenovirus carrying the dominant-negative mutant of Rac (RacN17), a component of NADPH oxidase, reduced the strain-induced superoxide and MCP-1 expression. In contrast, ECs transfected with a constitutively active Rac (RacV12) increased MCP-1 and 4x TRE promoter activities. However, ECs cotransfected with eNOS and RacV12 reduced those promoter activities. Consistently, the increases of superoxide levels and MCP-1 expression by overexpression of RacV12 were abolished after infecting ECs with eNOS. Our results show that NO from eNOS-inhibiting redox-sensitive MCP-1 expression is mediated via Rac-dependent NADPH oxidase by reducing ROS. This study provides a molecular basis to support the notion that endothelial NO acts as an antioxidant by negatively regulating redox-sensitive gene expression in ECs constantly under hemodynamic influence.

Animals↗

PubMed2000

Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells.

The subcellular colocalization of prostacyclin synthase (PGIS) with prostaglandin H synthase (PGHS) has not been delineated. To test the hypothesis that its colocalization with PGHS is crucial for prostacyclin synthesis, we determined subcellular locations of PGIS, PGHS-1, and PGHS-2 in bovine aortic endothelial cells by immunofluorescent confocal microscopy. PGIS and PGHS-1 were colocalized to nuclear envelope (NE) and endoplasmic reticulum (ER) in resting and adenovirus-infected bovine aortic endothelial cells. PGIS and PGHS-2 were also colocalized to ER in serum-treated or adenovirus-cyclooxygenase-2-infected cells. By contrast, PGIS was not colocalized with PGHS-2 in cells induced with phorbol 12-myristate 13-acetate where PGHS-2 was visualized primarily in vesicle-like structures. The lack of colocalization was accompanied by failed prostacyclin production. Resting ECV304 cells did not produce prostacyclin and had no detectable PGHS-1 and PGIS proteins. Confocal analysis showed abnormal colocalization of PGIS and PGHS-1 to a filamentous structure. Interestingly, the abundant PGIS and PGHS-1 expressed in adenovirus-infected ECV304 cells were colocalized to NE and ER, which synthesized a large quantity of prostacyclin. These findings underscore the importance of colocalization of PGHS and PGIS to ER and NE in prostacyclin synthesis.

Animals↗

PubMed1999

Modulation of inducible nitric oxide synthase induction by prostaglandin E2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages.

Prostaglandin E2 (PGE2) is the major cyclooxygenase metabolite in macrophages with complex proinflammatory and immunoregulatory properties. In the present study, we have compared the modulatory role of PGE2/cAMP-dependent signaling on induced nitric oxide (NO) production in two murine macrophages, J774 and RAW 264.7. With no effect on NO release by itself, PGE2 co-addition with lipopolysaccharide (LPS) resulted in a concentration-dependent enhancement in NO release and inducible NO synthase induction in J774, but not in RAW 264.7, macrophages. The potentiation effect of PGE2 in J774 cells was still seen when applied within 9 h after LPS treatment. Whereas RAW 264.7 macrophages release PGE2 with greater extent than J774 macrophages in response to LPS, indomethacin and NS-398, upon abolishing LPS-induced PGE2 release, caused a more obvious inhibition of NO release from J774 than RAW 264.7 cells. Thus, we suggest a higher positive modulatory role of PGE2--either endogenous or exogenous--on NO formation in J774 cells. Supporting these findings, exogenous PGE2 triggers cAMP formation in J774 cells with higher potency and efficacy. Of interest, dBcAMP also elicits higher sensitivity in potentiating NO release in J774 cells. We conclude that the opposite effect of PGE2/cAMP signaling on macrophage NO induction depends on its signaling efficacy and might be associated with the difference in endogenous PGE2 levels.

Animals↗

PubMed1998

Origins and antiquity of X-linked triallelic color vision systems in New World monkeys.

It is known that the squirrel monkey, marmoset, and other related New World (NW) monkeys possess three high-frequency alleles at the single X-linked photopigment locus, and that the spectral sensitivity peaks of these alleles are within those delimited by the human red and green pigment genes. The three alleles in the squirrel monkey and marmoset have been sequenced previously. In this study, the three alleles were found and sequenced in the saki monkey, capuchin, and tamarin. Although the capuchin and tamarin belong to the same family as the squirrel monkey and marmoset, the saki monkey belongs to a different family and is one of the species that is most divergent from the squirrel monkey and marmoset, suggesting the presence of the triallelic system in many NW monkeys. The nucleotide sequences of these alleles from the five species studied indicate that gene conversion occurs frequently and has partially or completely homogenized intronic and exonic regions of the alleles in each species, making it appear that a triallelic system arose independently in each of the five species studied. Nevertheless, a detailed analysis suggests that the triallelic system arose only once in the NW monkey lineage, from a middle wavelength (green) opsin gene, and that the amino acid differences at functionally critical sites among alleles have been maintained by natural selection in NW monkeys for >20 million years. Moreover, the two X-linked opsin genes of howler monkeys (a NW monkey genus) were evidently derived from the incorporation of a middle (green) and a long wavelength (red) allele into one chromosome; these two genes together with the (autosomal) blue opsin gene would immediately enable even a male monkey to have trichromatic vision.

Alleles↗

PubMed1998

Molecular genetics of spectral tuning in New World monkey color vision.

Although most New World monkeys have only one X-linked photopigment locus, many species have three polymorphic alleles at the locus. The three alleles in the squirrel monkey and capuchin have spectral peaks near 562, 550, and 535 nm, respectively, and the three alleles in the marmoset and tamarin have spectral peaks near 562, 556, and 543 nm, respectively. To determine the amino acids responsible for the spectral sensitivity differences among these pigment variants, we sequenced all exons of the three alleles in each of these four species. From the deduced amino acid sequences and the spectral peak information and from previous studies of the spectral tuning of X-linked pigments in humans and New World monkeys, we estimated that the Ala --> Ser, Ile --> Phe, Gly --> Ser, Phe --> Tyr, and Ala --> Tyr substitutions at residue positions 180, 229, 233, 277, and 285, respectively, cause spectral shifts of about 5, -2, -1, 8, and 15 nm. On the other hand, the substitutions His --> Tyr, Met --> Val or Leu, and Ala --> Tyr at positions 116, 275, and 276, respectively, have no discernible spectral tuning effect, though residues 275 and 276 are inside the transmembrane domains. Many substitutions between Val and Ile or between Val and Ala have occurred in the transmembrane domains among the New World monkey pigment variants but apparently have no effect on spectral tuning. Our study suggests that, in addition to amino acid changes involving a hydroxyl group, large changes in residue size can also cause a spectral shift in a visual pigment.

Amino Acid Substitution↗

PubMed1997

Prostacyclin synthase active sites. Identification by molecular modeling-guided site-directed mutagenesis.

Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin. In this study we have used a molecular modeling-guided site-directed mutagenesis to predict the active sites in substrate binding pocket and heme environment of PGIS. A three-dimensional model of PGIS was constructed using P450BM-3 crystal structure as the template. Our results indicate that residues Ile67, Val76, Leu384, Pro355, Glu360, and Asp364, which were suggested to be located at one side of lining of the substrate binding pocket, are essential for catalytic activity. This region containing beta1-1, beta1-2, beta1-3, and beta1-4 strands is predicted well by the model. At the heme region, Cys441 was confirmed to be the proximal axial ligand of heme iron. The conserved Phe and Arg in P450BM-3 were substituted by Leu112 and Asp439, respectively in PGIS. Alteration of Leu112 to Phe retained the activity, indicating that Leu112 is a functional substitution for Phe. In contrast, mutant Asp439 --> Ala exhibited a slight increase in activity. This result implies a difference in the heme region between P450BM-3 and PGIS. Our results also indicate that stability of PGIS expression is not affected by heme site or active site mutations.

Amino Acid Sequence↗

PubMed1995

Adaptive evolution of color vision genes in higher primates.

The intron 4 sequences of the three polymorphic alleles at the X-linked color photo-pigment locus in the squirrel monkey and the marmoset reveal that the alleles in each species are exceptionally divergent. The data further suggest either that each triallelic system has arisen independently in these two New World monkey lineages, or that in each species at least seven deletions and insertions (14 in the two species) in intron 4 have been transferred and homogenized among the alleles by gene conversion or recombination. In either case, the alleles in each species apparently have persisted more than 5 million years and probably have been maintained by overdominant selection.

Alleles↗

PubMed1994

Weak male-driven molecular evolution in rodents.

In humans and rodents the male-to-female ratio of mutation rate (alpha m) has been suggested to be extremely large, so that the process of nucleotide substitution is almost completely male-driven. However, our sequence data from the last intron of the X chromosome-linked (Zfx) and Y chromosome-linked (Zfy) zinc finger protein genes suggest that alpha m is only approximately 2 in rodents with a 95% confidence interval from 1 to 3. Moreover, from published data on oogenesis and spermatogenesis we estimate the male-to-female ratio of the number of germ cell divisions per generation to be approximately 2 in rodents, confirming our estimate of alpha m and suggesting that errors in DNA replication are the primary source of mutation. As the estimated alpha m for rodents is only one-third of our previous estimate of approximately 6 for higher primates, there appear to be generation-time effects--i.e., alpha m decreases with decreasing generation time.

Animals↗

PubMed1994

Intronic gene conversion in the evolution of human X-linked color vision genes.

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.

Animals↗