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J Yi

Publications and source records attributed to J Yi.

At least 55 records · Page 3Linked to original sources
PubMed1998

Molecular cloning and functional expression of a skeletal muscle dihydropyridine receptor from Rana catesbeiana.

In skeletal muscle the dihydropyridine receptor is the voltage sensor for excitation-contraction coupling and an L-type Ca2+ channel. We cloned a dihydropyridine receptor (named Fgalpha1S) from frog skeletal muscle, where excitation-contraction coupling has been studied most extensively. Fgalpha1S contains 5600 base pairs coding for 1688 amino acids. It is highly homologous with, and of the same length as, the C-truncated form predominant in rabbit muscle. The primary sequence has every feature needed to be an L-type Ca2+ channel and a skeletal-type voltage sensor. Currents expressed in tsA201 cells had rapid activation (5-10 ms half-time) and Ca2+-dependent inactivation. Although functional expression of the full Fgalpha1S was difficult, the chimera consisting of Fgalpha1S domain I in the rabbit cardiac Ca channel had high expression and a rapidly activating current. The slow native activation is therefore not determined solely by the alpha1 subunit sequence. Its Ca2+-dependent inactivation strengthens the notion that in rabbit skeletal muscle this capability is inhibited by a C-terminal stretch (Adams, B., and Tanabe, T. (1997) J. Gen. Physiol. 110, 379-389). This molecule constitutes a new tool for studies of excitation-contraction coupling, gating, modulation, and gene expression.

Amino Acid Sequence↗
PubMed1998

Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin diphosphate.Mg(II) - evidence for tertiary and quaternary interactions.

The regulatory mechanism of substrate activation in yeast pyruvate decarboxylase is triggered by the interaction of pyruvic acid with C221 located on the beta domain at >20 A from the thiamin diphosphate (ThDP). To trace the putative information transfer pathway, substitutions were made at H92 on the alpha domain, across the domain divide from C221, at E91, next to H92 and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substitutions were made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kinetic parameters, including the Hill coefficient, provide useful insight to this effort. In addition to C221, the residues H92, E91, E51 and H114 and H115 together appear to have a critical impact on the Hill coefficient, providing a pathway for information transfer. To study the activation by ThDP.Mg(II), variants at G231 (of the conserved GDG triplet) and at N258 and C259 (all three being part of the putative ThDP fold) of the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex were studied. Kinetic and spectroscopic evidence suggests that the Mg(II) ligands are very important to activation of the enzymes by cofactors.

Binding Sites↗
PubMed1998

The human TRIP6 gene encodes a LIM domain protein and maps to chromosome 7q22, a region associated with tumorigenesis.

The thyroid receptor interacting protein-6 (TRIP6) was first identified as a ligand-dependent binding partner for the thyroid hormone receptor in a yeast two-hybrid screen. A partial TRIP6 cDNA clone that was isolated in the initial screen encodes two copies of the LIM domain. The LIM domain is a double zinc-finger structure that mediates protein-protein interactions. Here we report the complete amino acid sequence of human TRIP6. The TRIP6 protein displays a proline-rich N-terminal region linked to three tandemly arrayed C-terminal LIM domains. The global molecular architecture and sequence of TRIP6 place it in the same family as the adhesion plaque protein, zyxin, and the lipoma preferred partner (LPP). Zyxin and LPP are implicated in cellular signaling and tumorigenesis, respectively. By radiation hybrid mapping, the human TRIP6 gene was assigned to a segment of chromosome 7q22 that is commonly deleted in malignant myeloid diseases and uterine leiomyoma.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Systematic study of the six cysteines of the E1 subunit of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: none is essential for activity.

Variants of the Escherichia coli 1-lip pyruvate dehydrogenase multienzyme complex (1-lip PDHc) with the C259N and C259S substitutions in the putative thiamin diphosphate-(ThDP-) binding motif of the pyruvate dehydrogenase component (E1, EC 1.2.4.1) were characterized. Single substitutions were made at the five remaining cysteines of the E1 component, creating the C120A, C575A, C610A, C654A, and C770S variants to test the hypothesis that the activity loss that accompanies exposure of the enzyme to fluoropyruvate, bromopyruvate, and 2-oxo-3-butynoic acid is the result of the modification of approximately one cysteine residue per E1 monomer. Surprisingly, all single cysteine E1 variants could be reconstituted with E2-E3 subcomplex and showed PDHc activity ranging from 74% to 96% that of the parental enzyme. The specific activities of C259N and C259S variants of 1-lip PDHc were 58% and 27% relative to that of the parental 1-lip PDHc. All five single cysteine E1 variants, along with the C259N and C259S variants of 1-lip PDHc, could also (1) be inactivated with fluoropyruvate and 2-oxo-3-butynoic acid, (2) were subject to inactivation by the monoclonal antibody 18A9 reported from one of our laboratories, and (3) were subject to regulation by pyruvate and acetyl-CoA. It was therefore concluded that none of the six cysteine residues is essential for the activity of the E1 component or of the complex. When tested with the putative transition-state analogue, thiamin 2-thiothiazolone diphosphate, all but the C259S and C259N variants were very potently inhibited, the stoichiometry for parental E1 being about 1.6 mol of inhibitor/mol of E1 subunit. The C259S and C259N E1 variants required at least 25-fold greater inhibitor concentration to achieve the same level of inhibition. C259 is located in the putative thiamin diphosphate-binding motif of the enzyme [more exactly, it is adjacent to a ligand to the Mg(II) ion]. It is therefore concluded that thiamin 2-thiothiazolone diphosphate is not a transition-state analogue; rather, it is a potent inhibitor of the complex because of a specific interaction with the C259 residue.

Acetyl Coenzyme A↗
PubMed1998

A high-level mammalian expression system based on the Madin-Darby canine kidney cell line.

In this report, we demonstrate that a combination of the Madin-Darby canine kidney (MDCK) cell line and a cytomegalovirus promoter-based expression vector can achieve high-level expression of secretory recombinant proteins in mammalian cells. The matrix metalloproteinase MMP13, a secretory protein, was expressed in MDCK cells at a level high enough to be detectable in crude supernatants without concentration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis stained with Coomassie brilliant blue R-250. The secreted MMP-13 accounts for about 15 to 20% of the total secreted proteins and reaches a concentration of at least 10 mg/liter of unconcentrated conditioned medium harvested from confluent monolayer culture. Furthermore, the recombinant protein appears to be properly folded and modified posttranslationally. This system may be employed for the production of human proteins of special interests, such as those for structural determination or therapeutical development.

Animals↗
PubMed1998

Inactivation of gating currents of L-type calcium channels. Specific role of the alpha 2 delta subunit.

In studies of gating currents of rabbit cardiac Ca channels expressed as alpha 1C/beta 2a or alpha 1C/beta 2a/alpha 2 delta subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489-517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863-895). Charge 1 (voltage of half-maximal transfer, V1/2 approximately 0 mV) gates noninactivated channels, while charge 2 (V1/2 approximately -90 mV) is generated in inactivated channels. In alpha 1C/beta 2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant tau approximately 8, while the available charge 2 decreased upon recovery from inactivation (at -200 mV) with tau approximately 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of alpha 2 delta, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the alpha 2 delta subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to tau approximately 2 s), while slowing the reduction of charge 2 upon recovery (tau approximately 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489-498) and inactivation as discrete modal change. The effects of alpha 2 delta are reproduced assuming that the subunit lowers the free energy of the inactivated mode.

Algorithms↗
PubMed1998

Visualization of sulfated glycoconjugates in the chicken embryonic heart by whole-mount alcian blue staining.

In the atrioventricular canal (AVC) and outflow tract (OT) of the developing heart, endothelial cells transform specifically to mesenchymal cells. The mesenchymal cells migrate into the underlying acellular matrix termed cardiac jelly and form endocardial cushion tissue. It is believed the that the highly hydrated nature of cardiac jelly is ascribed to sulfated glycoconjugates in the components of jelly matrix. In the present study, we have visualized the distribution and its temporal changes of sulfated glycoconjugates in the embryonic heart from stage 12 to 26 using whole mount alcian blue (AB) histochemistry. Atrial matrix was AB-negative in all the stages examined. Cardiac jelly in the AVC and OT were positive and the staining intensity increased as heart development proceeded, while AB-positive staining in the matrix of the ventricle became negative by stage 19. At stages later than 19, AB-positive matrix was localized in only the AVC and OT where endothelially-derived mesenchymal cells populated.

Alcian Blue↗
PubMed1998

Endothelial outgrowth and the subsequent cell transition in the culture of embryonic atrioventricular canal placed in an inverted polarity.

Endothelial cells in the atrioventricular (AV) segment of the developing chicken heart undergo a transition into mesenchymal cells. When the AV segment is explanted onto a hydrated collagen gel, endothelial cells grow out and reproduce in vivo cell transition regardless of the precise orientation of the explant on a gel. Our results showed that when the luminal side of an explant was placed towards a gel surface, the inverted polarity of endothelium was not adjusted by direct reorganization of polarity, but that the endothelium crawled down so as to settle on a gel surface in the correct original cell polarity. Subsequently, endothelial cells showed cytoplasmic hypertrophy, formed microvillous projections and then extended filopodial migratory appendages. These cellular changes were quite similar to those in vivo. However, the continuity of the endothelial layer was specifically disrupted in AV explant cultures. Such disruption was never observed in ventricle explant cultures in which endothelial-mesenchymal cell transition did not occur. The disintegration of AV endothelial outgrowth must be closely related to its capability to transform into mesenchymal cells and mitotic activity to keep a depository of endothelial cells.

Animals↗
PubMed1998

[Indigenous hydroxyapatite as a successful anophthalmic orbital implant].

OBJECTIVE: The aim of the work was to improve the operative result of eye socket reconstruction after enucleation. METHODS: The hydroxyapa tite implant of eyeball shape was buried in the scleral shell, which was stitched to the extraocular muscles at the points of muscle attachment so as to create a rotatable eye seat connector. RESULTS: One hundred and two patients have received eye socket reconstruction of this method with improved results. The prosthetic eyes move freely. Follow-up for 6 to 38 months reveals that the results are stable and satisfactory. CONCLUSION: The indigenous hydroxyapa tite implant is a good biomaterial for socket reconstruction.

Adolescent↗
PubMed1997

2-Oxo-3-alkynoic acids, universal mechanism-based inactivators of thiamin diphosphate-dependent decarboxylases: synthesis and evidence for potent inactivation of the pyruvate dehydrogenase multienzyme complex.

A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.

Bacterial Proteins↗
PubMed1997

Ion-dependent inactivation of barium current through L-type calcium channels.

It is widely believed that Ba2+ currents carried through L-type Ca2+ channels inactivate by a voltage-dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca2+ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba2+ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, IBa decayed as the sum of two exponentials. The slow phase (tau approximately 6 s, 21 degrees C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (tau approximately 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na+ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba2+ current. The reduction of Ba2+ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba2+ entry during the pulse did not change the reversal potential for Ba2+. A simple model of Ca(2+) -dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and F., Ríos. 1993. J. Gen. Physiol. 102:1005-1030) robustly accounts for fast Ba2+ current decay assuming the affinity of the inactivation site on the alpha 1 subunit to be 100 times lower for Ba2+ than Ca2+.

Animals↗
PubMed1997

Biotin and digoxigenin as labels for light and electron microscopy in situ hybridization probes: where do we stand?

Biotin was recently applied to detect cellular DNA or RNA. In combination with avidin, streptavidin or antibody, it can be conjugated with fluorescent dye, enzyme, ferritin, or gold. However, emphasis has recently been placed on the false-positive results that are obtained when this probe is used, because endogenous biotin may sometimes interfere with specific signals. Digoxigenin appears to be an interesting alternative because it is present exclusively in Digitalis plants as a secondary metabolite. We discuss in this review the efficiency and the respective advantages and disavantages of these two probes for in situ hybridization, mainly at the electron microscopic level.

Biotin↗
PubMed1996

Effect of substitutions in the thiamin diphosphate-magnesium fold on the activation of the pyruvate dehydrogenase complex from Escherichia coli by cofactors and substrate.

The homotropic regulation of the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) by its coenzyme thiamin diphosphate and its substrate pyruvate was re-examined with complexes containing three and one lipoyl domains per E2 chain, and several variants of the latter, containing substitutions in the putative thiamin diphosphate fold of E1 (G231A, G231S, C259S, C259N, and N258Q). It was found that all of the E1 variants had significantly reduced specific activities, as reported elsewhere (Russell, G. C., Machado, R. S., and Guest, J. R. (1992) Biochem. J. 287, 611-619). In addition, extensive kinetic studies were performed in an attempt to determine the effects of the amino acid substitutions on the Hill coefficients with respect to thiamin diphosphate and pyruvate. All but one of the variants were incapable of being saturated with thiamin diphosphate, even at concentrations > 5 mM. Most importantly, the striking activation lag phase lasting for many seconds in the parental complexes containing three and one lipoyl domains per E2 chain was totally eliminated in the variants. Furthermore, activation by the coenzyme was localized to the E1 subunit, because resolved E1 exhibits virtually the same behavior during the activation lag phase as does the complex. In the parental complexes two distinct lag phases could be resolved, the duration of both decreases with increasing ThDP concentration. A mechanism that is consistent with all of the kinetic data on the parental complexes involves rapid equilibration of the first ThDP with the E1 dimer, followed by a slow conformational equilibration, that in turn is followed by slow addition of the second ThDP to form the fully activated dimer. When the diphosphate site is badly impaired, the binding affinity is very much reduced, this perhaps eliminates the slow step leading to the activated dimer form of the E1.

Acetyltransferases↗
PubMed1996

Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH in mammalian cells.

Recently our laboratory has cloned both the rat canalicular and sinusoidal GSH transporters (RcGshT and RsGshT, respectively; Yi, J., S. Lu, J. Fernandez-Checa, and N. Kaplowitz. 1994. J. Clin. Invest. 93:1841-1845; and 1995. Proc. Natl. Acad. Sci. USA. 92:1495-1499). The current work characterized GSH transport and the expression of these two GSH transporters in various mammalian cell lines. The average cell GSH levels (nmol/10(6) cells) were 25, 22, 32, 13, and 13 in HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. GSH efflux was temperature dependent and averaged 0.018, 0.018, 0.012, 0.007, and 0.019 nmol/10(6) cells/min from HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. Dithiothreitol (DTT), which stimulates rat sinusoidal GSH efflux, stimulated GSH efflux only in HepG2 and HeLa cells which was partially reversed by subsequent cystine treatment. GSH uptake (1 mM plus 35S-GSH) was temperature dependent, linear up to 45 min, and Na+-independent with average rates of 1.12, 0.91, 0.45, and 0.45 nmol/10(6) cells/30 min for HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. BSP-GSH (2mM), which cis-inhibits sinusoidal GSH uptake in rat liver and HepG2 cells, inhibited GSH uptake only in HeLa cells. mRNA and polypeptide of RcGshT are expressed in all cells whereas those of RsGshT are expressed only in HepG2 and HeLa cells. In conclusion, bidirectional GSH transport, mediated by the "canalicular" GSH transporter, is ubiquitous in mammalian cells. Sinusoidal GSH transporter expression is more restricted, being present in HepG2 and HeLa cells. DTT and BSP-GSH affect GSH transport only in cells expressing the sinusoidal transporter confirming their selective action on this transporter.

Animals↗
PubMed1996

Alterations in glutathione homeostasis in mutant Eisai hyperbilirubinemic rats.

Eisai hyperbilirubinemic rats (EHBR) are mutant Sprague-Dawley rats that exhibit impaired biliary organic anion and reduced glutathione (GSH) secretion. In addition, liver GSH levels are twice that of age-matched controls. The mechanisms for the defect in biliary GSH secretion and the increase in cell GSH are not fully understood. We previously showed that canalicular membrane-enriched vesicles isolated from EHBR livers exhibited normal GSH transport. In the present study, we examined the steady-state rat canalicular reduced glutathione transporter (RcGshT) messenger RNA (mRNA) and protein levels, as well as the mechanisms for the increase in cell GSH. Both Northern and Western blot analyses of EHBR livers showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared with controls. Treatment with phenobarbital, which increased steady-state RcGshT mRNA by five- to sixfold, RcGshT polypeptide, and biliary GSH secretion by onefold in controls, had a smaller effect on steady-state RcGshT-mRNA level in EHBR (by 1.5-fold) and did not increase RcGshT polypeptide or biliary GSH secretion. In examining possible mechanisms for increased liver GSH, both cysteine level and gamma-glutamylcysteine synthetase (GCS) activity were significantly higher than controls, while the activity of GSH synthetase was unchanged. Northern and Western blot analyses also showed increased steady-state GCS heavy subunit (GCS-HS) mRNA and polypeptide levels, respectively. In addition to liver, GSH levels in kidney, duodenal, jejunal, and ileal mucosa of EHBR were 200% to 300% of age-matched control rats. GCS activity was also increased in kidney cytosol of EHBR. Thus, the defect in biliary GSH secretion in EHBR most likely is either at the posttranslational level of RcGshT or in the inhibition exerted by retained endogenous organic anions. In addition, there is a widespread up-regulation of GSH synthesis capacity in the tissues of EHBR.

Analysis of Variance↗
PubMed1996

A neural network model forecasting for prediction of daily maximum ozone concentration in an industrialized urban area.

Prediction of ambient ozone concentrations in urban areas would allow evaluation of such factors as compliance and noncompliance with EPA requirements. Though ozone prediction models exist, there is still a need for more accurate models. Development of these models is difficult because the meteorological variables and photochemical reactions involved in ozone formation are complex. In this study, we developed a neural network model for forecasting daily maximum ozone levels. We then compared the neural network's performance with those of two traditional statistical models, regression, and Box-Jenkins ARIMA. The neural network model for forecasting daily maximum ozone levels is different from the two statistical models because it employs a pattern recognition approach. Such an approach does not require specification of the structural form of the model. The results show that the neural network model is superior to the regression and Box-Jenkins ARIMA models we tested.

Journal Article↗
PubMed1996

[Clinical application of a new mobile integrated orbital implant].

OBJECTIVE: To improve the socket reconstruction after enucleation. METHODS: Hydroxyapatite (HA, made in China) was made into a porous HA sphere and wrapped up in the scleral shell as a mobile integrated orbital implant. It was applied to 43 patients with enucleation and anophthalmia, The surgical technique is described. RESULTS: All sockets have been corrected very well and the prosthetic mobility and the cosmetic appearance are excellent. After a follow-up of 6 to 24 months, no migration or extrusion of the implant or infection occurred. CONCLUSION: It is considered that at present this new mobile integrated orbital implant is made of one of the best materials and this method of socket reconstruction is one of the best method for selection.

Adolescent↗