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Biomedical subjects

S Choe

Publications and source records attributed to S Choe.

At least 19 recordsLinked to original sources
PubMed2003

Molecular basis for pseudo vitamin D-deficiency rickets in the Hannover pig.

The molecular basis for pseudo vitamin D deficiency rickets (PDDR) in the Hannover pig model was determined in the current study. Consistent with the inability of Hannover PDDR pigs to maintain ambient levels of 1,25-dihydroxyvitamin D (i.e., 1,25D), the bioactivation enzyme cytochrome P450C1 (or CYP27B1) was determined to contain coding-region deletions that rendered the enzyme ineffective due to frame-shift mutations and expression of a premature termination codon. Expression levels of P450C1mRNA were up-regulated in response to the low-1,25D high-parathyroid hormone state of the PDDR animals. In a complementary manner, cytochrome P450C24 mRNA was not detectable in PDDR pigs. Two different deletions were detected within the Hannover pig strain in which the P450C1 coding region contained either 173 bp or 329 bp deletions that resulted in the expression of non-sense products beginning within the I-helix region and extending through the truncated C-terminal domains. The boundaries for the deletion segments aligned with derived mRNA processing sites. This observation was consistent with an mRNA processing error as the causative factor for the coding-region deletions. Based upon the expression of a non-functional P450C1 enzyme, the Hannover pig model for PDDR was determined to be identical to the human disease in which enzyme-inhibitory mutations are the molecular basis for the calcium disorder.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2001

Crystal structure of Arp2/3 complex.

We determined a crystal structure of bovine Arp2/3 complex, an assembly of seven proteins that initiates actin polymerization in eukaryotic cells, at 2.0 angstrom resolution. Actin-related protein 2 (Arp2) and Arp3 are folded like actin, with distinctive surface features. Subunits ARPC2 p34 and ARPC4 p20 in the core of the complex associate through long carboxyl-terminal alpha helices and have similarly folded amino-terminal alpha/beta domains. ARPC1 p40 is a seven-blade beta propeller with an insertion that may associate with the side of an actin filament. ARPC3 p21 and ARPC5 p16 are globular alpha-helical subunits. We predict that WASp/Scar proteins activate Arp2/3 complex by bringing Arp2 into proximity with Arp3 for nucleation of a branch on the side of a preexisting actin filament.

Actin Cytoskeleton↗
PubMed2001

Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K+ channels.

Local anesthetics, commonly used for treating cardiac arrhythmias, pain, and seizures, are best known for their inhibitory effects on voltage-gated Na(+) channels. Cardiovascular and central nervous system toxicity are unwanted side-effects from local anesthetics that cannot be attributed to the inhibition of only Na(+) channels. Here, we report that extracellular application of the membrane-permeant local anesthetic bupivacaine selectively inhibited G protein-gated inwardly rectifying K(+) channels (GIRK:Kir3) but not other families of inwardly rectifying K(+) channels (ROMK:Kir1 and IRK:Kir2). Bupivacaine inhibited GIRK channels within seconds of application, regardless of whether channels were activated through the muscarinic receptor or directly via coexpressed G protein G(beta)gamma subunits. Bupivacaine also inhibited alcohol-induced GIRK currents in the absence of functional pertussis toxin-sensitive G proteins. The mutated GIRK1 and GIRK2 (GIRK1/2) channels containing the high-affinity phosphatidylinositol 4,5-bisphosphate (PIP(2)) domain from IRK1, on the other hand, showed dramatically less inhibition with bupivacaine. Surprisingly, GIRK1/2 channels with high affinity for PIP(2) were inhibited by ethanol, like IRK1 channels. We propose that membrane-permeant local anesthetics inhibit GIRK channels by antagonizing the interaction of PIP(2) with the channel, which is essential for G(beta)gamma and ethanol activation of GIRK channels.

Anesthetics, Local↗
PubMed2001

Potassium and sodium ions in a potassium channel studied by molecular dynamics simulations.

We have performed simulations of both a single potassium ion and a single sodium ion within the pore of the bacterial potassium channel KcsA. For both ions there is a dehydration energy barrier at the cytoplasmic mouth suggesting that the crystal structure is a closed conformation of the channel. There is a potential energy barrier for a sodium ion in the selectivity filter that is not seen for potassium. Radial distribution functions for both ions with the carbonyl oxygens of the selectivity filter indicate that sodium may interact more tightly with the filter than does potassium. This suggests that the key to the ion selectivity of KcsA is the greater dehydration energy of Na(+) ions, and helps to explain the block of KcsA by internal Na(+) ions.

Bacterial Proteins↗
PubMed2001

Rapid reductive destruction of hazardous organic compounds by nanoscale Fe0.

Fe0-mediated reductive destruction of hazardous organic compounds such as chlorinated organic compounds (COCs) and nitroaromatic compounds (NACs) in the aqueous phase is one of the latest innovative technologies. In this paper, rapid reductive degradation of COCs and NACs by synthesized nanoscale Fe0 in anaerobic batch systems was presented. The nanoscale Fe0, characterized by high specific surface area and high reactivity, rapidly transformed trichloroethylene (TCE), chloroform (CF), nitrobenzene (NB), nitrotoluene (NT), dinitrobenzene (DNB) and dinitrotoluene (DNT) under ambient conditions, which results in complete disappearance of the parent compounds from the aqueous phase within a few minutes. GC analysis reported that the main products of the dechlorination of TCE and CF were ethane and methane as well as that most of the nitro groups in NACs were reductively transformed to amine groups. These results suggest that the rapid reductive destruction by nanoscale Fe0 is potentially a viable in situ or aboveground treatment of groundwater contaminated with hazardous organic compounds including COCs and NACs.

Feasibility Studies↗
PubMed2001

Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis.

Plants unable to synthesize or perceive brassinosteroids (BRs) are dwarfs. Arabidopsis dwf4 was shown to be defective in a steroid 22alpha hydroxylase (CYP90B1) step that is the putative rate-limiting step in the BR biosynthetic pathway. To better understand the role of DWF4 in BR biosynthesis, transgenic Arabidopsis plants ectopically overexpressing DWF4 (AOD4) were generated, using the cauliflower mosaic virus 35S promoter, and their phenotypes were characterized. The hypocotyl length of both light- and dark-grown AOD4 seedlings was increased dramatically as compared to wild type. At maturity, inflorescence height increased >35% in AOD4 lines and >14% in tobacco DWF4 overexpressing lines (TOD4), relative to controls. The total number of branches and siliques increased more than twofold in AOD4 plants, leading to a 59% increase in the number of seeds produced. Analysis of endogenous BR levels in dwf4, Ws-2 and AOD4 revealed that dwf4 accumulated the precursors of the 22alpha-hydroxylation steps, whereas overexpression of DWF4 resulted in increased levels of downstream compounds relative to Ws-2, indicative of facilitated metabolic flow through the step. Both the levels of DWF4 transcripts and BR phenotypic effects were progressively increased in dwf4, wild-type and AOD4 plants, respectively. This suggests that it will be possible to control plant growth by engineering DWF4 transcription in plants.

Arabidopsis↗
PubMed2001

Identification of a small-molecule binding site at the dimer interface of the HIV integrase catalytic domain.

Integration of the reverse-transcribed HIV cDNA into the host DNA is a required step in viral replication. The virus-encoded integrase protein catalyzes the initial DNA breaking and joining reactions that mediate cDNA integration. Here, the identification by X-ray crystallography of a small-molecule binding site on the integrase catalytic domain is reported. The small-molecule family studied consists of a core of arsenic or phosphorus surrounded by four aromatic groups. Two arsenic derivatives were visualized bound to integrase. In each case, two molecules bound at symmetry-related sites on the catalytic domain dimer interface. The first compound studied, tetraphenyl arsonium, did not inhibit integrase. However, a synthetic compound substituting a catechol for one of the phenyl rings, dihydroxyphenyltriphenylarsonium, bound to the same site and did inhibit the enzyme. Changes in the vicinity of the catalytic site were seen with the inhibitory compound only, potentially explaining its mechanism of action. Further substituting phosphonium for arsonium yielded a compound with an IC(50) in the low micromolar range. These findings may be useful in designing new inhibitors of integrase, which is at present the only one of the three HIV enzymes for which clinically useful inhibitors are not available.

Amino Acid Substitution↗
PubMed2000

Externally added aFGF mutants do not require extensive unfolding for transport to the cytosol and the nucleus in NIH/3T3 cells.

Acidic fibroblast growth factor (aFGF) is transported to the cytosol and the nucleus when added to cells expressing FGF receptors, implying that aFGF must cross cellular membranes. Since protein translocation across membranes commonly requires extensive unfolding of the protein, we were interested in testing whether this is also necessary for membrane translocation of aFGF. We therefore constructed mutant growth factors with intramolecular disulfide bonds to prevent complete unfolding. Control experiments demonstrated that translocation of aFGF by the diphtheria toxin pathway, which requires extensive unfolding of the protein, was prevented by disulfide bond formation, indicating that the introduced disulfide bonds interfered with the unfolding of the growth factor. On the other hand, when the growth factor as such was added to cells expressing FGF receptors, the disulfide-bonded mutants were translocated to the cytosol and the nucleus equally well as wild-type aFGF. The possibility that the translocation of the mutants was due to reduction of the disulfide bonds prior to translocation was tested in experiments using an irreversibly cross-linked mutant. Also this mutant was transported to the cytosol and to the nucleus. The results suggest that extensive unfolding is not required for membrane translocation of aFGF.

3T3 Cells↗
PubMed2000

Identification of a binding site on the type II activin receptor for activin and inhibin.

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.

Activin Receptors↗
PubMed2000

Phosphorylation of Acanthamoeba actophorin (ADF/cofilin) blocks interaction with actin without a change in atomic structure.

LIM-kinase activated by GST-Pak1 phosphorylates Acanthamoeba actophorin stoichiometrically and specifically on serine 1. The atomic structure of phosphorylated actophorin determined by X-ray crystallography is essentially identical with the structure of unphosphorylated actophorin. We compared biochemical properties of phosphorylated actophorin, unphosphorylated actophorin and mutants of actophorin with serine 1 replaced by aspartic acid or alanine. Phosphorylation strongly inhibits interaction of actophorin with Mg-ADP- or Mg-ATP-actin monomers and Mg-ADP-actin filaments, so Ser1 phosphorylation directly blocks interaction of actin-depolymerizing factor (ADF)/cofilin proteins with actin. About 30 % of actophorin is phosphorylated in live amoebas grown in suspension culture. Phosphorylation of ADF/cofilin proteins by LIM-kinase or other enzymes will tend to stabilize actin filaments by inhibiting the ability of these proteins to sever and depolymerize older actin filaments that have hydrolyzed their bound ATP and dissociated the phosphate.

Acanthamoeba↗
PubMed2000

Kinetics of reductive denitrification by nanoscale zero-valent iron.

Zero-valent iron powder (Fe0) has been determined to be potentially useful for the removal of nitrate in the water environment. This research is aimed at subjecting the kinetics of denitrification by nanoscale Fe0 to an analysis of factors affecting the chemical denitrification of nitrate. Nanoscale iron particles with a diameter in the range of 1-100 nm, which are characterized by the large BET specific surface area to mass ratio (31.4 m2/g), removed mostly 50, 100, 200, and 400 mg/l of nitrate within a period of 30 min with little intermediates. Compared with microscale (75-150 microm) Fe0, end product is not ammonia but N2 gas. Kinetics analysis from batch studies revealed that the denitrification reaction with nanoscale Fe0 appeared to be a pseudo first-order with respect to substrate and the observed reaction rate constant (k(obs)) varied with iron content at a relatively low degree of application. The effects of mixing intensity (rpm) on the denitrification rate suggest that the denitrification appears to be coupled with oxidative dissolution of iron through a largely mass transport-limited surface reaction (<40 rpm).

Iron Compounds↗
PubMed2000

Potassium channel structure: domain by domain.

Since the determination of the structure of a bacterial potassium channel, the ion channel community has managed to gain momentum in the quest for a complete picture. The information is coming at a steady flow, on a domain by domain basis. Recent discoveries are starting to reveal clues to the complex manner in which potassium channels show enormous diversity of function and also to their methods of regulation. Currently, the structures of four domains are known, with the most recent addition being the Kvbeta structure. As efforts continue in the study of the transmembrane domains, especially the voltage-sensing apparatus, there has been a new realization with respect to the identification and role of the cytoplasmic domains in protein-protein interactions in particular. An additional discovery, considerably aided by recent genomic analysis, is that potassium channels comprising subunits with two pore regions and four transmembrane helices combined in a dimeric fashion are abundant and are probable targets for local anesthetics.

Animals↗
PubMed2000

Voltage dependent activation of potassium channels is coupled to T1 domain structure.

The T1 domain, a highly conserved cytoplasmic portion at the N-terminus of the voltage-dependent K+ channel (Kv) alpha-subunit, is responsible for driving and regulating the tetramerization of the alpha-subunits. Here we report the identification of a set of mutations in the T1 domain that alter the gating properties of the Kv channel. Two mutants produce a leftward shift in the activation curve and slow the channel closing rate while a third mutation produces a rightward shift in the activation curve and speeds the channel closing rate. We have determined the crystal structures of T1 domains containing these mutations. Both of the leftward shifting mutants produce similar conformational changes in the putative membrane facing surface of the T1 domain. These results suggest that the structure of the T1 domain in this region is tightly coupled to the channel's gating states.

Amino Acid Substitution↗
PubMed2000

Lesions in the sterol delta reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis.

The brassinosteroid (BR) biosynthetic pathway, and the sterol pathway which is prerequisite to the BR pathway, are rapidly being characterized because of the availability of a large number of characteristic dwarf mutants in Arabidopsis. Here we show that the Arabidopsis dwarf5 mutants are disrupted in a sterol Delta7 reduction step. dwf5 plants display the characteristic dwarf phenotype typical of other BR mutants. This phenotype includes small, round, dark-green leaves, and short stems, pedicels, and petioles. Metabolite tracing with 13C-labeled precursors in dwf5 verified a deficiency in a sterol Delta7 reductase activity. All six independent alleles contain loss-of-function mutations in the sterol Delta7 reductase gene. These include a putative mRNA instability mutation in dwf5-1, 3' and 5' splice-site mutations in dwf5-2 and dwf5-6, respectively, premature stop codons in dwf5-3 (R400Z) and dwf5-5 (R409Z), and a mis-sense mutation in dwf5-4 (D257N). The dwf5 plant could be restored to wild type by ectopic overexpression of the wild-type copy of the gene. Both the Arabidopsis dwf5 phenotype and the human Smith-Lemli-Opitz syndrome are caused by loss-of-function mutations in a sterol Delta7 reductase gene, indicating that it is required for the proper growth and development of these two organisms.

Alleles↗
PubMed2000

Biosynthetic pathways of brassinolide in Arabidopsis.

Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone --> 3-dehydro-6-deoxoteasterone --> 6-deoxotyphasterol --> 6-deoxocastasterone --> 6alpha-hydroxycastasterone --> castasterone --> BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone --> 3-dehydroteasterone --> typhasterol --> castasterone --> BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants.

Arabidopsis↗
PubMed1999

Domain movement in gelsolin: a calcium-activated switch.

The actin-binding protein gelsolin is involved in remodeling the actin cytoskeleton during growth-factor signaling, apoptosis, cytokinesis, and cell movement. Calcium-activated gelsolin severs and caps actin filaments. The 3.4 angstrom x-ray structure of the carboxyl-terminal half of gelsolin (G4-G6) in complex with actin reveals the basis for gelsolin activation. Calcium binding induces a conformational rearrangement in which domain G6 is flipped over and translated by about 40 angstroms relative to G4 and G5. The structural reorganization tears apart the continuous beta sheet core of G4 and G6. This exposes the actin-binding site on G4, enabling severing and capping of actin filaments to proceed.

Actins↗