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W C Song

Publications and source records attributed to W C Song.

31 records · Page 2Linked to original sources
PubMed1998

Expression of estrogen sulfotransferase in MCF-7 cells by cDNA transfection suppresses the estrogen response: potential role of the enzyme in regulating estrogen-dependent growth of breast epithelial cells.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfonation of estrogens at the 3-hydroxyl position by use of 3'-phosphoadenosine-5'-phosphosulfate as an activated sulfate donor. Although largely known and studied as a phase II metabolic enzyme with prominent expression in the liver, the high substrate specificity of EST (with a high Vmax/Km value for estrogen) suggests that expression of the enzyme in extrahepatic, estrogen target tissues, such as the breast epithelium, may constitute an effective mechanism for local estrogen regulation as well. In this study, we have evaluated the physiological significance of EST expression by cDNA transfection studies with use of the estrogen-dependent MCF-7 breast cancer cell line as a model system. We show that expression of EST in MCF-7 cells effectively reduces the cells' response to physiological concentrations of estradiol (10 nM) by up to 70% as determined in an estrogen-responsive reporter gene assay. In addition, we demonstrate that expression of EST similarly inhibits estrogen-stimulated DNA synthesis and cell proliferation by 21% and 46%, respectively. (The thymidine incorporation rate was measured 3 days after and the cell numbers were counted 8 days after transfection.) These results provide direct evidence for the functional significance of in situ EST expression in the breast epithelium and suggest that abnormal regulation of the enzyme may have pathological implications in the development and maintenance of hormone-dependent breast carcinomas.

Breast↗
PubMed1998

Early isotonic saline resuscitation from uncontrolled hemorrhage in rats.

BACKGROUND: Attempts to modify traditional fluid resuscitation have been based on animal models that evaluate several variables including anesthesia. This study presents the effects of early saline resuscitation from severe uncontrolled hemorrhage unanesthetized rats. METHODS: Sixty-three female Sprague-Dawley rats were equally divided into three groups: group A, nonresuscitated; and groups B and C, resuscitated ;with isotonic saline (40 and 80 mL/kg, respectively). Hemodynamics, blood loss, survival time, and mortality were recorded for 360 minutes after the hemorrhage, which was initiated by 75% resection of the tail. RESULTS: In group C, 80 mL/kg of saline significantly lowered mortality (24% vs 76% and 71% for groups A and B, respectively) with concomitant increases in mean survival time (241 +/- 103 min vs 146 +/- 108 and 175 +/- 92 min for groups A and B, respectively). There were no statistically significant differences in blood loss, hematocrit, or hemodynamic parameters among the groups. CONCLUSIONS: Early and adequate isotonic saline resuscitation of unanesthetized rats improved outcome despite continuing hemorrhage. The significantly lower mortality rate and increased survival time were not a result of transiently improved arterial pressure and did not correlate with blood loss. No significant bleeding increases were noted in the resuscitated groups.

Animals↗
PubMed1997

Cellular localization and regulation of expression of testicular estrogen sulfotransferase.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the specific sulfonation of estrogens at the 3-hydroxyl position using 3'-phosphoadenosine-5'-phosphosulfate as an activated sulfate donor. Sulfated estrogens no longer bind to the estrogen receptor and are, therefore, hormonally inactive. Although liver has been considered a primary site for steroid sulfotransferase activities, we previously have cloned the mouse EST complementary DNA and found the enzyme to be expressed abundantly in the testis of normal mice. In this study we show by reverse transcription-PCR that EST is also expressed in the testes of rat and man, suggesting that testicular expression of EST may be a common phenomenon among different species. Using a purified polyclonal antibody raised against the bacterially expressed mouse EST protein, we demonstrate by immunohistochemistry that EST is localized selectively to the androgen-producing Leydig cells within the mouse testis. Additionally, we show that Leydig cell expression of EST is under the control of the pituitary hormone LH and is regulated differentially during development. In contrast to the high level of expression in mature intact animals, EST is not present in Leydig cells of hypophysectomized mice or in Leydig cells of fetal and prepubertal (day 5 or 17) mouse testes. Administration of hCG to hypophysectomized mice restored the testicular expression of EST. Together, these results suggest that testicular expression of EST may play an important role in male reproduction, conceivably by modulating the activity of locally synthesized estrogen in the testis of a sexually mature animal.

Adult↗
PubMed1996

Mouse decay-accelerating factor: selective and tissue-specific induction by estrogen of the gene encoding the glycosylphosphatidylinositol-anchored form.

Neonatal exposure of mice to estrogen (diethylstilbestrol) results in a high incidence (90%) of uterine tumor later in life. In an effort to screen for estrogen-regulated genes in the uterus of the neonatal mouse, we have isolated a murine homologue of the human decay-accelerating factor (DAF), a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein and a member of the regulators of complement activation family of proteins that function to prevent autologous complement-mediated tissue damage. The induced mouse DAF cDNA has a 64% sequence identity with the human counterpart at the nucleotide level and a 50% identity in the deduced amino acid sequence. It consists of 390 amino acids and contains four short consensus repeats of internal homology characteristic of human DAF. It also contains a hydrophobic C-terminal that most likely serves as a signal for GPI anchor attachment. Sequence comparison with the recently reported mouse DAF cDNAs confirmed that the estrogen-inducible gene corresponds to the mouse GPI DAF gene. The induction of mouse DAF by estrogen is tissue specific and can be mimicked by the antiestrogen tamoxifen. Furthermore, the regulation of uterine DAF expression by estrogen is limited to the GPI DAF gene. The transmembrane DAF gene is not expressed in the mouse uterus, either with or without estrogen stimulation. These results suggest that the two mouse DAF genes are differentially regulated, and that the GPI-anchored DAF may play important roles in estrogen responses and other physiologic or pathophysiologic processes of the female reproductive system.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of a testis-specific estrogen sulfotransferase and aberrant liver expression in obese and diabetogenic C57BL/KsJ-db/db mice.

Sulfation represents a major pathway for the inactivation of steroid hormones such as estrogens and is catalyzed by a group of enzymes called sulfotransferases. Aberrant regulation of an estrogen sulfotransferase has been demonstrated previously in the livers of obese and diabetogenic C57BL/KsJ-db/db strain mice. In this paper, we report the molecular cloning and functional characterization of a full-length complementary DNA for estrogen sulfotransferase from mouse testis. The mouse estrogen sulfotransferase complementary DNA encodes 295 amino acids. It shares 88%, 77%, 75%, and 68% identity in amino acid sequence with the rat liver, human liver, guinea pig adrenal, and bovine placental estrogen sulfotransferase, respectively. The mouse enzyme was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The fusion protein was affinity purified, and milligram quantities of pure enzyme were obtained after cleavage of the fusion protein with thrombin. The expressed enzyme exhibits a high substrate specificity toward estrogens, including estradiol and estrone. Neither dehydroepiandrosterone, pregnenolone, testosterone, nor a simple phenolic compound, 4-nitrophenol appears to be a substrate. Northern hybridization indicates that messenger RNA (1.3 kilobases) for the estrogen sulfotransferase is expressed exclusively in the testes in control C57BL/KsJ mice. However, both the messenger RNA and protein are dramatically induced in the livers of obese and diabetogenic C57BL/KsJ-db/db mice. In contrast to the liver, the constitutive expression of the enzyme in the testis is not affected by the db/db genotype. These results recapitulate the species-specific nature in the tissue distribution of estrogen sulfotransferase and suggest complex regulatory mechanisms in its expression under normal and pathophysiological conditions.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of an allene oxide synthase: a cytochrome P450 specialized for the metabolism of fatty acid hydroperoxides.

Allene oxide synthases convert lipoxygenase-derived fatty acid hydroperoxides to unstable allene epoxides. In plants, an allene oxide is a precursor of the growth regulator jasmonic acid. Previously, we showed that an allene oxide synthase from flaxseed has the spectral properties of a cytochrome P450. The relationship to the P450 gene family is now established from the primary structure deduced from the cDNA. The encoded protein of 536 amino acids has segments at the C terminus that match certain well conserved regions in cytochrome P450s. The heme-binding cysteine is recognizable at position 489. However, there are unprecedented modifications in this region, with substitution of two of the three most highly conserved amino acids. Also very unusual is the absence of a conserved threonine that normally helps form the O2-binding pocket in cytochrome P450s. Notably, O2 is not involved in the allene oxide synthase reaction and, furthermore, the enzyme is known to have a weak interaction with CO. While allene oxide synthases are usually described as microsomal, the flax cDNA encodes a 58-amino acid signal sequence characteristic of a mitochondrial or chloroplast transit peptide. Therefore, the enzyme is a type I P450 and most likely is located in chloroplasts. Overall, the flax allene oxide synthase has < or = 25% identity to other P450s; it belongs to a newly discovered gene family, to be designated CYP74. The flaxseed enzyme is prototypical of this family of enzymes that remain to be characterized in plants and animals.

Amino Acid Sequence↗
PubMed1993

Formation of epoxyalcohols by a purified allene oxide synthase. Implications for the mechanism of allene oxide synthesis.

The allene oxide synthase (hydroperoxide dehydrase) of flaxseed is a cytochrome P450 that exhibits an exceptionally high catalytic turnover (> or = 1000/s) for hydroperoxy substrates. In a previous study, using a crude extract of flaxseed, we detected a secondary activity that could offer an insight into the mechanism of the enzymatic transformation of hydroperoxides. We observed that the substrate 8R-hydroxy-15S-hydroperoxyeicosa-5,9,11,13,17-pentaenoic acid is converted not only to allene oxide, but also to epoxyalcohol derivatives (Brash, A. R., Baertschi, S. W., and Harris, T. M. (1990) J. Biol. Chem. 265, 6705-6712). The transformation of hydroperoxides to epoxyalcohols has been investigated extensively in other systems, and heterolytic or homolytic cleavage of the hydroperoxide is associated with characteristic rearrangements and stereochemistry of the epoxyalcohol products. Using the purified enzyme, we established that the epoxyalcohols are products of the allene oxide synthase. Their structures were determined by UV, gas chromatography-mass spectrometry, and NMR. The major epoxyalcohol is 8R,13R-dihydroxy-14R,15S-epoxyeicosa-5Z,9E ,11Z,17Z-tetraenoic acid, a trans-epoxide with an alpha-hydroxyl in the relative threo configuration. Two minor products are the corresponding 11E isomer and a cis-epoxide identified as 8R,13-dihydroxy-14S,15S-epoxyeicosa-5Z,9E,11E,++ +17Z-tetraenoic acid. Gas chromatography-mass spectrometry analysis of a reaction with [18O2]hydroperoxide substrate indicated complete retention of the hydroperoxy oxygens in the epoxyalcohol products. Mechanistic precedents support a homolytic hydroperoxide cleavage as the initial step in the synthesis of these epoxyalcohols. We suggest that the same process initiates allene oxide synthesis, a conclusion that is also most compatible with the known chemistry of cytochromes P450.

Alcohols↗
PubMed1991

Allene oxide and aldehyde biosynthesis in starfish oocytes.

Allene oxides are a very unusual type of epoxide that, in biological systems, are formed by the enzymic dehydration of fatty acid hydroperoxides (lipoxygenase products). This reaction occurs widely in plants, in which allene oxide synthesis is a key step in the conversion of linolenic acid to jasmonic acid, the plant growth regulator. We report biosynthesis of the allene oxide (8R)-8,9-epoxyeicosa-(5Z,9,11Z,14Z)-tetraenoic acid via the (8R)-lipoxygenase metabolism of arachidonic acid in starfish oocytes. Formation of the allene oxide was deduced from high pressure liquid chromatography, UV, gas chromatography-mass spectrometry and 1H-NMR analyses of the precise structure and mechanism of biosynthesis of its major hydrolysis product, the alpha-ketol 8-hydroxy-9-ketoeicosa-(5Z,11Z,14Z)-trienoic acid. A second enzymic activity detected in the oocytes (hydroperoxide lyase) cleaves specifically the (8R)-hydroperoxy substrate into C7 and C13 fragments, identified as the hydroxyacid, (5Z)-7-hydroxyheptenoic acid, and two aldehydes, (2E,4Z,7Z)-tridecenal and its 4E isomer. Discovery of the allene oxide synthase and hydroperoxide lyase marks the first definitive localization of these enzymic activities to an animal cell. It was established previously that the (8R)-lipoxygenase metabolite (8R)-HETE will activate the maturation (re-initiation of meiosis) of starfish oocytes. The individual 8-lipoxygenase products may be involved at distinct stages of cell development.

Aldehydes↗
PubMed1991

Investigation of the allene oxide pathway in the coral Plexaura homomalla: formation of novel ketols and isomers of prostaglandin A2 from 15-hydroxyeicosatetraenoic acid.

Prostaglandin A2 is a major constituent of the gorgonian Plexaura homomalla, and there is evidence that its biosynthesis involves a noncyclooxygenase pathway. The coral contains an 8(R)-lipoxygenase and an allene oxide synthase; from arachidonic acid, the sequential action of these enzymes gives an allene epoxide, the cyclization of which forms an analogue of prostaglandin A2 (PGA2) with no 15-hydroxyl group. In this study we examined the metabolic fate of 15-hydroxyeicosatetraenoic acid (15-HETE), which via analogous reactions could lead to PGA2. The 8(R)-lipoxygenase metabolized preferentially the 15(R) enantiomer of 15-HETE, and this reaction was stimulated fivefold by including 1 M NaCl in the incubation. Further enzymic steps were detected by comparing the metabolic profiles of the 8(R)-hydroperoxy-15(R)-hydroxy intermediate with that of its 8(S),15(S) enantiomer. Two main products were formed exclusively from the 8(R),15(R) enantiomer: an allene epoxide and the comparatively stable epoxide, 8,9-epoxy-10,15-dihydroxyeicosa-5,11,14-trienoic acid. Formation of the allene oxide was inferred from detection of its hydrolysis and cyclization products. It cyclized to give two isomers of PGA2 which have a "cis" arrangement of the side chains. The main hydrolysis product (8,15-dihydroxy-9-ketoeicosa-5,11,13-trienoic acid) was unstable and prone to oxygenation, giving 8,14,15-trihydroxy-9-ketoeicosa-5,10,12-trienoic acids after reduction of the 14-hydroperoxide. We conclude that metabolism of a 15-hydroxy eicosanoid is a potential route to the A series prostaglandins, although the low yield and lack of stereochemical control suggest that this is not the natural pathway of biosynthesis in P. homomalla. Unexpectedly, the major end products of the pathway are trihydroxy ketols and the single diastereomer of a stable epoxyalcohol.

Animals↗
PubMed1991

Purification of an allene oxide synthase and identification of the enzyme as a cytochrome P-450.

Fatty acid hydroperoxides (lipoxygenase products) are metabolized to allene oxides by a type of dehydrase that has been detected in plants, corals, and starfish oocytes. The allene oxides are unstable epoxide precursors of more complex products such as jasmonic acid, the plant growth hormone. Characterization of the dehydrase enzyme of flaxseed revealed that it is a 55-kilodalton hemoprotein. The spectral characteristics of this dehydrase revealed it to be a cytochrome P-450. It operates with the remarkable activity of greater than or equal to 1000 turnovers per second. The results establish a new catalytic activity for a cytochrome P-450 and illustrate the cooperation of different oxygenases in pathways of fatty acid metabolism.

Chromatography, Gel↗
PubMed1990

Identification of novel hydroxy fatty acids in the barnacle Balanus balanoides.

The ability of the barnacle Balanus balanoides tissues to produce eicosanoid hatching factors from endogenous polyunsaturated fatty acids has been investigated. GC-MS analysis of an active HPLC fraction from the trihydroxy fatty acid band on TLC revealed the presence of a number of trihydroxy fatty acids and two compounds which were tentatively identified as chlorinated dihydroxy fatty acids. The identified trihydroxy fatty acids are 10,11,12-trihydroxy-5,8,14-eicosatrienoic acid, 10,11,12-trihydroxy-5,8,14,17-eicosatetraenoic acid, 13,14,15-trihydroxy-5,8,11,17-eicosatetraenoic acid and 12,13,14-trihydroxy-4,7,10,16,19-docosapentaenoic acid. The tentatively identified chlorinated dihydroxy fatty acids are 9-chloro- and/or 11-chloro-8,12-dihydroxyeicosatetraenoic acid. The formation of these compounds is evidence of lipoxygenase activities in Balanus balanoides and their identification will facilitate the understanding of the roles eicosanoids play in barnacle physiology, especially with regard to the larval hatching process.

Animals↗