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M Y Chiang

Publications and source records attributed to M Y Chiang.

30 records · Page 2Linked to original sources
PubMed1993

Regulation of 5-lipoxygenase and 5-lipoxygenase-activating protein expression in HL-60 cells.

5-Lipoxygenase performs the first two enzymic reactions in the biosynthetic pathway for the leukotrienes. We have utilized HL-60 cells to study the mechanisms regulating expression of 5-lipoxygenase and the recently described 18 kDa membrane protein, 5-lipoxygenase-activating protein (FLAP). Differentiation of HL-60 cells into granulocyte-like cells with dimethyl sulphoxide (Me2SO), retinoic acid or dibutyryl cyclic AMP (Bt2-cAMP) resulted in a 2-3-fold increase in 5-lipoxygenase enzyme activity and a 4-fold increase in leukotriene B4 synthesis. Differentiation of HL-60 cells into monocyte-like cells with 1,25-dihydroxyvitamin D3 induced 5-lipoxygenase activity 5-fold, but leukotriene B4 production was only increased 2-fold. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells failed to induce 5-lipoxygenase or leukotriene B4 production. Examination of the levels of the transcript encoding 5-lipoxygenase and FLAP demonstrated that differentiation of HL-60 cells into granulocytes resulted in a co-ordinate induction of 5-lipoxygenase and FLAP mRNA. In contrast, differentiation of HL-60 cells into monocytes resulted in discordant regulation of 5-lipoxygenase and FLAP mRNA. Treatment with 1,25-dihydroxyvitamin D3 resulted in a 6-fold increase in 5-lipoxygenase mRNA and a 1.3-fold increase in FLAP mRNA, while treatment with phorbol ester did not induce 5-lipoxygenase mRNA but did increase FLAP mRNA 2-fold. The transcriptional rate of the 5-lipoxygenase and FLAP genes did not change upon Me2SO or 1,25-dihydroxyvitamin D3 treatment, suggesting that the increase of the mRNA coding for these proteins was not due to transcriptional activation of their respective genes. The mRNA half-life for 5-lipoxygenase did not change significantly upon treatment with Me2SO or 1,25-dihydroxyvitamin D3. The FLAP mRNA half-life increased from approx. 3.5 h to 4.5 h in cells treated with either Me2SO or 1,25-dihydroxyvitamin D3. These data suggest that expression of 5-lipoxygenase and FLAP is controlled by a post-transcriptional event other than stabilization of the mRNA.

5-Lipoxygenase-Activating Proteins↗
PubMed1992

Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides.

We have investigated the use of a cationic lipid preparation to enhance antisense oligonucleotide activity in human umbilical vein endothelial cells. A liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was found to increase by at least 1000-fold the potency of an antisense oligonucleotide (ISIS 1570) that hybridizes to the AUG translation initiation codon of human intercellular adhesion molecule-1. In the presence of 8 microM DOTMA, 6-15-fold more 35S-ISIS 1570 associated with cells, at oligonucleotide concentrations from 0.01 to 5 microM, than did in the absence of DOTMA. Both 35S-ISIS 1570 association with cells and antisense activity were increased as a function of DOTMA concentration and with increasing time of incubation with the cationic lipid. Fluorescein-labeled ISIS 1570 was used to assess the intracellular distribution of the oligonucleotide in the presence and absence of DOTMA. In the absence of DOTMA, the oligonucleotide localized to discrete structures in the cytoplasm of the cell, resulting in a punctate fluorescence pattern. In the presence of DOTMA, cellular fluorescence markedly increased and the oligonucleotide localized within the nucleus, as well as to discrete structures in the cytoplasm. Accumulation of the oligonucleotide in the nucleus in the presence of DOTMA was time and temperature dependent. Nuclear accumulation was inhibited by preincubation of the cells with monensin but not chloroquine, NH4Cl, nocodazole, colcemid, or brefeldin A. These data demonstrate that cationic lipids increase antisense activity by increasing the amount of oligonucleotide associated with cells and altering intracellular distribution of the oligonucleotide.

Base Sequence↗
PubMed1991

Antisense oligonucleotides inhibit intercellular adhesion molecule 1 expression by two distinct mechanisms.

Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 1939, which targeted specific sequences in the 3'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of 35S-labeled proteins. Treatment of cells with ISIS 1939 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 1939 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 1939 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 1939 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 1939, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.

Amino Acid Sequence↗
PubMed1991

Independent cellular and ontogenetic expression of mRNAs encoding three alpha polypeptides of the rat GABAA receptor.

Previous studies have shown that several distinct but related polypeptides can serve as alpha subunits of functional GABAA receptors. Furthermore, the diversity of these polypeptides at least partially accounts for the functional heterogeneity of GABAA receptors. In this paper, we report the results of in situ hybridization studies using probes derived from our recently reported cDNAs for alpha 1, alpha 2, and alpha 4 GABAA receptor polypeptides. We show that the mRNAs that encode these isoforms have distinct regional and cellular distributions and are present at widely varying levels within the rat brain. In addition, our Northern blot analyses indicate that each of these three alpha mRNAs has a distinct pattern of ontogenetic regulation. Differential regulation of alpha polypeptide isoforms may lead to changes in GABAA receptor function during ontogeny as well as to distinct cellular responses to GABA and GABA-related drugs.

Animals↗
PubMed1991

Sequence and regional distribution of the mRNA encoding the alpha 2 polypeptide of rat gamma-aminobutyric acidA receptors.

A cDNA from a rat hippocampal cDNA library encodes an isoform of the alpha polypeptide of the gamma-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the alpha 2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor alpha polypeptides and shares 71-76% identity with previously described rat alpha polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the alpha 2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined "BZ type II" receptors. Other workers have previously shown that the alpha polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of alpha 2 mRNAs in rat brain suggests that the alpha 2 subunit may indeed be involved in the BZ type II receptors.

Animals↗
PubMed1991

Leukotriene receptors and mechanisms of signal transduction.

To better understand the mechanisms by which leukotriene tone is regulated, we have characterized the mechanisms of genetic regulation of 5-lipoxygenase in HL60 cells induced to differentiate with dimethyl sulfoxide (DMSO) and compared a number of rat and human 5-lipoxygenase introns. We demonstrate that differentiation of HL60 cells with DMSO results in coordinate induction of 5-LO and 5-LO activating protein (FLAP). The production of LTB4, 5-LO protein, 5-LO mRNa and FLAP RNA increased coordinately. However, two approaches demonstrated no increase in the initiation of transcription of 5-LO and FLAP pre-mRNA and no changes in the mRNA half lives. Moreover, cycloheximide inhibits the induction of the mRNAs and proteins. Thus, we suggest that 5-LO and FLAP are coordinately regulated in HL60 cells via mechanisms involving changes in RNA processing. To better understand potential mechanisms involved, we have cloned and sequenced several human 5-LO introns and compared them to analogous rat 5-LO introns. A number of regions of potential regulatory significances are conserved and may be important in controlling the rate of pre-mRNA processing.

5-Lipoxygenase-Activating Proteins↗
PubMed1989

A novel alpha subunit in rat brain GABAA receptors.

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.

Amino Acid Sequence↗
PubMed1983

Absolute configuration of a chiral CHD group via neutron diffraction: confirmation of the absolute stereochemistry of the enzymatic formation of malic acid.

Neutron diffraction has been used to monitor the absolute stereochemistry of an enzymatic reaction. (-)(2S)malic-3-d acid was prepared by the action of fumarase on fumaric acid in D2O. After a large number of cations were screened, it was found that (+)(R) alpha-phenylethylamine forms the large crystals necessary for a neutron diffraction analysis. The subsequent structure determination showed that (+)(R) alpha-phenylethylammonium (-)(2S)malate-3-d has an absolute configuration of R at the CHD site (i.e., the C3 carbon of malate). This result confirms the absolute stereochemistry of fumarate-to-malate transformation as catalyzed by the enzyme fumarase.

Malates↗