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H Kwon

Publications and source records attributed to H Kwon.

71 records · Page 4Linked to original sources
PubMed1994

Facilitated binding of TATA-binding protein to nucleosomal DNA.

BINDING of the TATA-binding protein (TBP) to the TATA box is required for transcription from many eukaryotic promoters in gene expression. Regulation of this binding is therefore likely to be an important determinant of promoter activity. Incorporation of the TATA sequence into nucleosomes dramatically reduces transcription initiation, presumably because of stereochemical constraints on binding of general transcription factors. Biochemical and genetic studies imply that cellular factors such as yeast SWI/SNF are required for activator function and might alter chromatin structure. One step that could be regulated during the activation process is TBP binding in chromatin 12, 13. We show here that binding of TBP to the TATA sequence is severely inhibited by incorporation of this sequence into a nucleosome. Inhibition can be overcome by ATP-dependent alterations in nucleosomal DNA structure mediated by hSWI/SNF, a putative human homologue of the yeast SWI/SNF complex. Additionally, the orientation of the TATA sequence relative to the surface of the histone core affects the access of TBP. We propose that the dynamic remodelling of chromatin structure to allow TBP binding is a key step in the regulation of eukaryotic gene expression.

Adenosine Triphosphatases↗
PubMed1994

Myosin filament ATPase is enhanced by intramolecularly cross-linked actin.

Reaction of rabbit skeletal muscle F-actin with the lysine-directed photolabile cross-linker, N-5-azido-2-nitrobenzoyloxy succinimide was limited to Lysine-328 and Lysine-326, with Lysine-328 being labelled to a greater extent. Photolysis of the modified actin enhanced the actin-activated MgATPase activity of filamentous scallop myosin 3-4-fold more than unmodified actin, without affecting calcium sensitivity. Unphotolysed modified actin behaved as untreated actin, indicating that photolysis was essential for the effect. The actin-activated ATPase of filamentous rabbit myosin was similarly increased by photolysed N-5-azido-2-nitrobenzoyloxy succinimide-modified actin. After photolysis in either the monomeric (G-) or filamentous (F-) form, N-5-azido-2-nitrobenzoyloxy succinimide-modified actin moved as a monomeric (42 kDa) species on SDS gels, and depolymerized and polymerized readily, demonstrating that any cross-linking event produced by photolysis must be intramolecular. In contrast to the substantial increase in actin-activated ATPase activity observed when photolysed ANB-NOS-modified actin was added to filamentous myosin, the enhancement was not observed with the soluble HMM and S-1 fragments of myosin. Photolysed modified actin showed only poor movement on a rabbit HMM-coated surface in vitro motility assays. These results can be explained if the internally cross-linked G-actin subunits which comprise only a fraction of the actin population, either weaken the actin-actin contacts or have an increased affinity for myosin.

Actins↗
PubMed1994

A new family of murine retroviral vectors with extended multiple cloning sites for gene insertion.

Murine retroviral vectors with multiple unique cloning sites in the body and 3' long terminal repeat (LTR) are described. The various alterations to the vectors include changing the gag+ start codon (AUG) to a stop codon (UAA), a deletion of 468 bp from the envelope region, and an additional 387-bp deletion of the promoter and enhancer sequences from the 3' LTR. Multiple cloning sites in the body and 3' LTR facilitate double-copy vector construction. The hygromycin resistance and luciferase genes were subcloned into the body and 3' LTR to evaluate effects of vector modifications and effects of insert location (body vs. LTR and same orientation vs. reverse orientation with respect to the vector LTRs) on virus titer. The results indicate the modifications or insert position do not negatively influence potential vector titer and expression capacity. The described vectors have potentially useful characteristics for gene therapy studies.

3T3 Cells↗
PubMed1993

Sulfotransferase-mediated activation of 4-hydroxy- and 3,4-dihydroxy-3,4-dihydrocyclopenta[c,d]pyrene, major metabolites of cyclopenta[c,d]pyrene.

Cyclopenta[c,d]pyrene, a ubiquitous environmental and occupational pollutant, has been reported to be metabolically activated through epoxidation at the 3,4 double bond in the cyclopenta ring to produce an electrophilic and mutagenic cyclopenta[c,d]pyrene-3,4-epoxide. 4-Hydroxy-3,4-dihydrocyclopenta[c,d]-pyrene (4-HDCPP) and 3,4-dihydroxy-3,4-dihydrocyclopenta[c,d]pyrene (3,4-DHDCPP) are known to be major metabolites of cyclopenta[c,d]pyrene, which appear to be derived from cyclopenta[c,d]pyrene-3,4-epoxide. The present study was undertaken to determine whether 4-HDCPP or 3,4-DHDCPP can be further activated via the formation of reactive benzylic sulfuric acid ester metabolites. Thus, when 4-HDCPP or 3,4-DHDCPP was incubated with calf thymus DNA in the presence of rodent liver cytosol and the sulfo group donor, 3'-phosphoadenosine-5'-phosphosulfate, a significant covalent DNA binding was observed. This cytosol- and 3'-phosphadenosine-5'-phosphosulfate-dependent DNA binding was inhibited by 2,6-dichloro-4-nitrophenol and dehydroepiandrosterone, suggesting the involvement of both phenol and hydroxysteroid sulfotransferases in the activation of 4-HDCPP and 3,4-DHDCPP. A gender difference was observed for the hepatic cytosolic sulfotransferase activity for 4-HDCPP in rats (i.e., male > female). Of the two isomers of 3,4-DHDCPP, the trans-diol produced DNA adducts to a much greater extent than did the cis counterpart by sulfotransferase. 4-HDCPP and 3,4-DHDCPP were also mutagenic toward bacteria in the presence of hepatic cytosol and 3'-phosphadenosine-5'-phosphosulfate. The chemically synthesized sulfuric acid ester 4-sulfooxy-3,4-DCPP was directly mutagenic without any activation system. The data from this study suggest that sulfotransferase plays an important role in the activation of those secondary benzylic hydroxyl metabolites derived from cyclopenta[c,d]pyrene-3,4-epoxide and, possibly, from epoxides of other aromatic hydrocarbons.

Animals↗
PubMed1993

Determination of monosaccharides in glycoproteins by reverse-phase high-performance liquid chromatography.

This report examines monosaccharide composition and content using the reverse-phase high-performance liquid chromatographic separation of p-aminobenzoic ethyl ester derivatives of neutral and amino sugars released from glycoproteins. After acid hydrolysis of glycoproteins with 2 M trifluoroacetic acid, sugars are derivatized with p-aminobenzoic ethyl ester (ABEE), which strongly absorbs uv light at 254 nm in the presence of sodium cyanoborohydride. Reverse-phase (RP)-HPLC of the ABEE-sugar derivatives is performed on Pico.Tag column in an isocratic mode. RP-HPLC conditions are optimized by using ternary mixture as a mobile phase and 45 degrees C as a column temperature. This procedure is very useful for the simultaneous analysis of neutral and amino sugars in a single chromatographic step using RP-HPLC without reacetylation of deacetylated amino sugars, which were produced by acid hydrolysis, and additional chromatography to remove side products. Monosaccharide composition and contents determined on glycoproteins by this method were very comparable to those previously obtained by other techniques.

4-Aminobenzoic Acid↗
PubMed1992

Effects of human serum on rat cumulus-oocyte complex functions in vitro: oocyte activation and endocrine secretions.

Secretion of hormones and oocyte meiotic events were assessed following in vitro culture of ovulated rat cumulus-oocyte complexes (COCs) in media containing different types of human serum. Both toxic and nontoxic (determined by mouse embryo test) samples of fetal cord or adult female serum were utilized for these experiments. After short-term culture (4.5 hr), with or without COCs, medium containing 10% serum was collected and analyzed for its content of estradiol, progesterone, and prostaglandin E (PGE), and oocytes were cytologically evaluated for spontaneous activation (second polar-body extrusion). Activation of oocytes occurred in all media tested. Steroids (progesterone and estradiol) levels were markedly elevated in culture medium containing cord serum as compared to medium containing adult female serum. The progesterone content of culture medium decreased after incubation of COCs with cord serum and increased when incubated with adult female serum. Little or no prostaglandin was detected in any control media. However, COCs secreted prostaglandin during culture in all media. COCs secreted estradiol when cultured in medium containing cord but not adult female serum. Results demonstrate that two types of serum utilized for in vitro culture of COCs varied markedly in their hormone content and differentially affected the secretion of hormones by COCs during culture. The results are discussed in relation to the success of IVF procedures.

Adult↗
PubMed1992

Role of gizzard myosin light chains in calcium binding.

The contraction of molluscan and vertebrate smooth muscles is regulated by myosin. Although the myosin and its associated two subunits, the regulatory light chain and the essential light chain, constitute the Ca2+ regulatory system in both types of muscles, the mechanisms by which Ca2+ signal is transduced are quite different. In molluscan muscles, the direct binding of Ca2+ to the regulatory system triggers muscle contraction. In vertebrate smooth muscles, however, phosphorylation of the regulatory light chain is the major triggering mechanism. We measured Ca2+ binding in gizzard myosin and in hybrids of scallop myosin containing gizzard regulatory light chain or in hybrids of scallop regulatory domain containing gizzard essential light chain. Isolated chicken gizzard myosin did not bind Ca2+ in the range of pCa 8.0 to 5.0 in the presence of 2 mM MgCl2, supporting the lack of the specific Ca(2+)-binding site in gizzard myosin. Phosphorylation of the regulatory light chain did not generate a specific (Ca2+)-binding site. The hybrid scallop myosin containing gizzard regulatory light chain showed a similar Ca2+ binding as native scallop myosin with a one to one stoichiometry of Ca2+ to myosin head saturating at about pCa 6.0 at pH 7.6. In contrast, the hybrid scallop regulatory domain containing gizzard essential light chain did not bind Ca2+ either at pCa 6.0 or at pCa 8.0. Control preparations reconstituted with scallop essential light chains bound 0.69 mol per mol Ca2+ at pCa 6.0 with no binding at pCa 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Voronoi binding site model of a polycyclic aromatic hydrocarbon binding protein.

A three-dimensional Voronoi binding site model has been formulated from a series of competitors for the binding site on a recently isolated polycyclic aromatic hydrocarbon binding protein (PBP) from mouse liver. The PBP binds polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P), with high affinity and shows other characteristics associated with receptor-ligand complexes. Altogether, the in vitro binding constant of seven molecules were used to deduce the geometry and the energetics of a possible site model consisting of five regions: one tetrahedron-shaped finite central hydrophobic pocket, one infinite region representing access to the solvent, and three strongly repulsive regions representing the sterically forbidden walls of the pocket. The model then predicted the binding energies correctly for nine additional competitors and suggests that competition of monoaromatic (benzene) derivatives with B[a]P would be weak.

Animals↗
PubMed1990

Isolation of the regulatory domain of scallop myosin: role of the essential light chain in calcium binding.

The regulatory domain of scallop myosin, consisting of a regulatory light chain (R-LC), an essential light chain (E-LC), and a portion of heavy chain, occupies the neck region of myosin. This domain is directly involved in the regulation of molluscan muscle contraction, which is triggered by direct Ca2+ binding to myosin. We have isolated a soluble functional complex (regulatory complex) comprised of R-LC, E-LC, and a 10-kDa heavy chain fragment in a 1:1:1 stoichiometry by clostripain digestion of the myosin head (papain subfragment 1). N termini of the heavy chain fragments were either leucine-812 or valine-817. The isolated complex retained the specific Ca2(+)-binding site and bound Ca2+ with a similar affinity and selectivity as myosin. The individual components of the regulatory complex were isolated after complete denaturation with guanidine hydrochloride. The regulatory complex was reconstituted from isolated light chains and the heavy chain fragment. The renatured complex regained Ca2+ binding quantitatively. To elucidate the function of the E-LC in Ca2+ binding, we constructed hybrid regulatory complexes. The hybrid complexes reconstituted with molluscan E-LC and R-LC regained the specific Ca2(+)-binding site, whereas the hybrid complex formed with rabbit skeletal E-LC [alkali LC 2 (A2-LC)] and scallop R-LC did not. The results demonstrate that E-LCs from myosins regulated by direct Ca2+ binding are required for the specific Ca2+ binding in the molluscan muscle.

Animals↗
PubMed1989

Isolation of a cDNA encoding the human GM2 activator protein.

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.

Amino Acid Sequence↗
PubMedDate not supplied

NF-kappaB activation and HIV-1 induced apoptosis.

HIV infection leads to the progressive loss of CD4+ T cells and the near complete destruction of the immune system in the majority of infected individuals. High levels of viral gene expression and replication result in part from the activation of NF-kappaB transcription factors, which in addition to orchestrating the host inflammatory response also activate the HIV-1 long terminal repeat. NF-kappaB induces the expression of numerous cytokine, chemokine, growth factor and immunoregulatory genes, many of which promote HIV-1 replication. Thus, NF-kappaB activation represents a double edged sword in HIV-1 infected cells, since stimuli that induce an NF-kappaB mediated immune response will also lead to enhanced HIV-1 transcription. NF-kappaB has also been implicated in apoptotic signaling, protecting cells from programmed cell death under most circumstances and accelerating apoptosis in others. Therefore, activation of NF-kappaB can impact upon HIV-1 replication and pathogenesis at many levels, making the relationship between HIV-1 expression and NF-kappaB activation multi-faceted. This review will attempt to analyse the many faces and functions of NF-kappaB in the HIV-1 lifecycle.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Microsomal metabolism of cyclopenta[cd]pyrene. Characterization of new metabolites and their mechanism of formation.

Oxidation of cyclopenta[cd]pyrene (CPP) by mouse and human liver microsomes was used to produce several previously undescribed metabolites, which were separated and isolated by reversed-phase HPLC. Three of these, 3,4-dihydroCPP-c-3,4-diol, 4-hydroxy-3,4-dihydroCPP, and 4-oxo-3,4-dihydroCPP, were fully characterized by GC-MS and UV spectroscopic analysis as well as by total synthesis. Two additional pairs of metabolites were identified as isomeric tetrahydrotetrols and dihydrotriols by GC-MS analysis of their trimethylsilyl derivatives. Their UV spectra were recorded and found to agree with the structure assignments. The tetrahydrotetrols were further characterized by the fact that either 3,4- or 9,10-trans-dihydrodiol could serve as their precursor, indicating that they are the two diastomeric 3,4,9,10-tetrahydroCPP-t-3,4-t-9,10-tetrols. The dihydrotriols were shown to possess t-3,4-dihydrodiol functionality. As found previously using rat liver microsomes, the most abundant metabolite was 3,4-dihydroCPP-t-3,4-diol. It was produced with one enantiomer in severalfold excess over the other, and the major enantiomer was shown to have 3R,4R configuration by exciton chirality circular dichroism. Microsomal oxidation of [4-2H]CPP, which was synthesized for this study, was used to determine the mechanisms of formation of 4-oxo- and 4-hydroxy-3,4-dihydroCPP. The ketone was produced without detectable retention of deuterium label, eliminating the NIH shift as a possible mechanism. The alcohol was shown to arise by NADPH-dependent reduction of both the ketone and another intermediate presumed to be the 3,4-epoxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Oxidation of cyclopenta[cd]pyrene by human and mouse liver microsomes and selected cytochrome P450 enzymes.

The metabolism of the environmental pollutant and suspected human carcinogen, cyclopenta[cd]pyrene (CPP), was investigated. Human liver microsomes from three individuals were examined, as well as CD-1 mouse liver microsomes. Five new metabolites recently identified in our lab, 4-hydroxy-3,4-dihydroCPP, 3,4-dihydroCPP-cis-3,4-diol, 4-oxo-3,4-dihydroCPP, 3,4,9,10-tetrahydroCPP-trans-3,4-trans-9,10-tetrol, and trans-3,4-dihydroCPP-3, 4,x-triols, as well as the known major metabolite, 3,4-dihydroCPP-trans-3,4-diol, were all observed from the incubations of human liver microsomes and CPP. Even though all three human samples were capable of producing all the metabolites identified from the mouse liver microsomal incubations of CPP, the quantity of each metabolite varied among the microsomal samples. In an attempt to explain the variation among human liver samples, the microsomes derived from genetically engineered cells containing specific cytochrome P450 isozyme cDNAs were employed. It was found that the 3,4-cyclopenta double bond can be oxidized by the cytochrome P450 enzymes 1A1, 1A2, and 3A4. The 9,10 K-region double bond was not efficiently oxidized by cytochrome P450 1A1, but by P450 1A2 either from CPP or from the t-3,4-dihydrodiol. The lack of catalytic activity of 3A4 toward the t-3,4-dihydrodiol, despite its high activity toward CPP oxidation to tetrahydrotetrols, suggests the possibility of two dihydrodiol epoxides, 3,4-dihydrodiol 9,10-epoxide and 9,10-dihydrodiol 3,4-epoxide, of CPP.

Animals↗