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Biomedical subjects

E Kim

Publications and source records attributed to E Kim.

At least 199 records · Page 11Linked to original sources
PubMed1996

Effects of volatile anesthetic isoflurane on ATP-sensitive K+ channels in rabbit ventricular myocytes.

It has been suggested that volatile anesthetic, isoflurane mediates cardioprotective effects via activation of the ATP-sensitive K+ (KATP) channels. However, no direct evidence has been provided to define whether isoflurane activates cardiac KATP channels using patch-clamp technique. We examined the effects of isoflurane on the KATP channels in rabbit ventricular myocytes by use of patch-clamp technique. Contrary to the results of the in vivo experiments, isoflurane inhibited the channel activity without a change in the single-channel conductance. Isoflurane decreased the channel activity by a decrease in burst duration and an increase in the inter-burst duration. On the other hand, isoflurane diminished the ATP sensitivity of KATP channels, indicating an increased probability of KATP channel opening for a given concentration of ATP after isoflurane anesthesia. The result supports, at least in part, the hypothesis that isoflurane mediates cardioprotective effects via KATP channel activation.

Anesthetics, Inhalation↗
PubMed1996

Polymerization and in vitro motility properties of yeast actin: a comparison with rabbit skeletal alpha-actin.

Actin purified from the yeast (Saccharomyces cerevisae) was polymerized faster than rabbit skeletal alpha-actin by MgCl2. The two actins polymerized at similar rates in the presence of CaCl2. Yeast actin, up to 25 microM, was not polymerized by KCl (100-300 mM); the monovalent salt also inhibited the MgCl2-induced polymerization of actin. The local structure of the subdomain-2 region in yeast actin filaments was probed by subtilisin and trypsin digestions. Loop 38-52 appeared more flexible and accessible to subtilisin in yeast than in rabbit actin. In contrast, tryptic digestions at Lys-61 and -68 occurred at the same rate for yeast and alpha-actin filaments. Modification of yeast actin by a sulfhydryl reagent CPM [7-(diethylamino)-3-(4'-maleimidophenyl)-4-methylcoumain] was specific to the Cys-374 residue; no labeling of a yeast actin mutant containing an alanine substitution for cysteine 374 was observed. The rates of Cys-374 labeling by CPM were similar for yeast and muscle actin, suggesting a similar environment for the C terminus in both polymers. In the in vitro motility assays, yeast actin required higher concentrations of heavy meromyosin (HMM) for its sliding than did the rabbit actin. At saturating concentrations of HMM, the sliding velocities of both actins were the same (3.0 microns/s). Relative forces generated by HMM with yeast and muscle actin were assessed by monitoring their in vitro motility in the presence of NEM-HMM load. The sliding of yeast actin was stopped at a level of external load (molar ratio NEM-HMM/HMM = 0.25) lower than that of muscle actin (NEM-HMM/HMM = 0.43), suggesting lower force production with yeast actin. These results are discussed in terms of the myosin cross-bridge cycle and actomyosin interactions.

Actins↗
PubMed1996

Structure of the pcbC gene encoding 2,3-dihydroxybiphenyl dioxygenase of Pseudomonas sp. P20.

Pseudomonas sp. P20 is a soil bacterium growing in biphenyl or 4-chlorobiphenyl as the sole carbon and energy source, where the initial catabolism of biphenyl to form benzoate is catalyzed by four enzymes encoded in the pcbABCD gene cluster. The nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene corresponding to the pcbC gene was determined. The pcbC gene was composed of 921 base pairs with an ATG initiation codon and a TGA termination codon, which can encode a polypeptide of molecular mass 34 kDa containing 306 amino acids. A promoter-like sequence and ribosome-binding sequence were identified upstream of the initiation codon. The deduced amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase exhibited 47% identity with that of corresponding enzyme of Pseudomonas sp. DJ-12, and less than 35% identity with those of other extradiol-type dioxygenases.

Amino Acid Sequence↗
PubMed1996

Complex Optical Surfaces Formed by Replica Molding Against Elastomeric Masters

Complex, optically functional surfaces in organic polymers can be fabricated by replicating relief structures present on the surface of an elastomeric master with an ultraviolet or thermally curable organic polymer, while the master is deformed by compression, bending, or stretching. The versatility of this procedure for fabricating surfaces with complex, micrometer- and submicrometer-scale patterns was demonstrated by the production of (i) diffraction gratings with periods smaller than the original grating; (ii) chirped, blazed diffraction gratings (where the period of a chirped grating changes continuously with position) on planar and curved surfaces; (iii) patterned microfeatures on the surfaces of approximately hemispherical objects (for example, an optical surface similar to a fly's eye); and (iv) arrays of rhombic microlenses. These topologically complex, micropatterned surfaces are difficult to fabricate with other techniques.

Journal Article↗
PubMed1996

Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ.

Modular PDZ domains, found in many cell junction-associated proteins, mediate the clustering of membrane ion channels by binding to their C-terminus. The X-ray crystallographic structures of the third PDZ domain from the synaptic protein PSD-95 in complex with and in the absence of its peptide ligand have been determined at 1.8 angstroms and 2.3 angstroms resolution, respectively. The structures reveal that a four-residue C-terminal stretch (X-Thr/Ser-X-Val-COO(-)) engages the PDZ domain through antiparallel main chain interactions with a beta sheet of the domain. Recognition of the terminal carboxylate group of the peptide is conferred by a cradle of main chain amides provided by a Gly-Leu-Gly-Phe loop as well as by an arginine side chain. Specific side chain interactions and a prominent hydrophobic pocket explain the selective recognition of the C-terminal consensus sequence.

Crystallography↗
PubMed1996

Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases.

Selective concentration and anchoring of ionotropic receptors at the synapse is essential for neuronal signaling. Little is known about the molecules that mediate receptor clustering in the CNS. With use of the yeast two-hybrid system to screen a rat brain cDNA library and by in vitro binding assays, we have identified an interaction between NMDA receptor subunits 2A and 2B (NR2A and NR2B) and three distinct members of the PSD-95/SAP90 family of membrane-associated putative guanylate kinases. The interaction is mediated by binding of the C terminus of the NMDA receptor subunits to the first two PDZ (also known as GLGF or DHR) domains of PSD-95/SAP90, an abundant synaptic protein associated with the membrane cytoskeleton. PSD-95 is also known to bind and cluster Shaker-type voltage-gated K+ channels. Similarities between the C-termini of NR2 subunits and K+ channels suggest a common C-terminal binding motif for PDZ domains. These data suggest that PDZ domains can function as modules for protein-protein interactions. Members of the PSD-95 family might serve to anchor NMDA receptors to the submembrane cytoskeleton and aid in the assembly of signal transduction complexes at postsynaptic sites.

Animals↗
PubMed1996

Sulfhydryl redox modulates ATP-sensitive K+ channels in rabbit ventricular myocytes.

The properties of sulfhydryl redox modulation of the ATP-sensitive K+ K(ATP) channel have been examined in rabbit ventricular myocytes, using the patch-clamp technique. The sulfhydryl oxidizing agent 5.5'-dithio bis-(2-nitro-benzoic acid) (DTNB) induced an inhibition of the channel activity without change in the single channel conductance. DTNB had no effect on the inhibitory action by ATP. Analysis of the open and closed time distributions showed that DTNB decreased the life time of bursts and increased the interburst interval without changes in open and closed time distributions shorter than 5 ms. N-ethylmaleimide (NEM), a substance that reacts with sulfhydryl groups of cysteine residues in proteins, induced an irreversible closure of the channel. The results suggested that changes in the sulfhydryl redox also modulate K(ATP) channel activity of the K(ATP) channel in rabbit ventricular myocytes.

Adenosine Triphosphate↗
PubMed1996

Blockade of the ATP-sensitive potassium channel by taurine in rabbit ventricular myocytes.

Using patch-clamp techniques, the effects of taurine on properties of ATP-sensitive K+ (KATP) channel of rabbit ventricular myocytes were examined. Intracellular taurine (20 mM) markedly depressed the KATP channel activity. The taurine concentration for half-inhibition (apparent Kd) was 13.5 mM with a Hill coefficient, n, of 1.3. Intracellular taurine caused channel inhibition without affecting channel inhibition by ATP. In control conditions, the ATP concentration for half-inhibition (Ki) and n were 73 microM and 1.2 (n = 6), respectively. In the presence of taurine, Ki and n were 81 microM and 1.3 (n = 6), respectively. Analysis of the open and closed time distributions showed that taurine decreased the life time of bursts and increased the inter-burst interval and/or reduced the number of functional channels. 2,4-Dinitrophenol (DNP) activated KATP channel after a lag period. This lag period was much longer after pretreatment with taurine (6.6 +/- 1.2 min, n = 5) than in the absence of taurine (2.8 +/- 1.5 min, n = 12). When DNP was removed in the bath solution, channel activity showed a gradual reduction with time and this process was facilitated by the presence of external taurine (20 mM). From these results it is suggested that taurine blocks KATP channel activity in dose-dependent manner and the depletion of taurine during myocardial ischemia contribute to the early activation of the KATP channel.

2,4-Dinitrophenol↗
PubMed1996

Preparation of a cell-free translation system from PC12 cell.

The postmitochondrial fraction (S10) contains the cellular components essential for translation, and a high-salt wash (HSW) of the ribosomes is enriched in eukaryotic initiation factors. This report describes the preparation of a cell-free translation system utilizing an S10 extract from PC12 cells. The products synthesized from either firefly luciferase mRNA or PC12 cell poly(A) RNAs in the PC12-S10 extract were increased by the addition of the HSW from PC12 cells. Increases in the translation of luciferase mRNA by the addition of PC12-HSW were dose-dependent and also dependent on the time of incubation. The translation of human epidermal growth factor receptor (hEGFR) mRNA could also be detected in the PC12-S10 extract translation system by immuno-precipitation. N-linked glycosylation of the translation products also was observed. The efficiency of translation was altered by the addition of Mg2- or K+, and optimization of the concentrations of these ions was necessary for each mRNA. The translation system made from PC12 cells, then, is capable of the synthesis of proteins of relatively high molecular weight and should be useful for analyzing mechanisms of translational control during proliferation and differentiation of cells from a neuronal lineage.

Animals↗
PubMed1996

Differential K+ channel clustering activity of PSD-95 and SAP97, two related membrane-associated putative guanylate kinases.

The molecular mechanisms underlying the clustering and localization of K+ channels in specific microdomains on the neuronal surface are largely unknown. The Shaker subclass of voltage-gated K+ channel alpha-subunits interact through their cytoplasmic C-terminus with a family of membrane-associated putative guanylate kinases, including PSD-95 and SAP97. We show here that heterologous coexpression of either sap97 or PSD-95 with various Shaker-type subunits results in the coclustering of these proteins with the K+ channels. Mutation of the C-terminal sequence (-ETDV) of the Shaker subunit Kv1.4 abolishes its binding to, and prevents its clustering with, SAP97 and PSD-95. Whereas PSD-95 induces plaque-like clusters of K+ channels at the cell surface; however, SAP97 coexpression results in the formation of large round intracellular aggregates into which both SAP97 and the K+ channel proteins are colocalized. The efficiency of surface clustering by PSD-95 varies with different Shaker subunits: striking Kv1.4 clustering occurs in > 60% of cotransfected cells, whereas Kv1.1 and Kv1.2 form convincing clusters with PSD-95 only in approximately 10% of cells.

Animals↗
PubMed1996

Intermolecular coupling between loop 38-52 and the C-terminus in actin filaments.

The recently reported structural connectivity in F-actin between the DNase I binding loop on actin (residues 38-52) and the C-terminus region was investigated by fluorescence and proteolytic digestion methods. The binding of copper to Cys-374 on F- but not G-actin quenched the fluorescence of dansyl ethylenediamine (DED) attached to Gin-41 by more than 50%. The blocking of copper binding to DED-actin by N-ethylmaleimide labeling of Cys-374 on actin abolished the fluorescence quenching. The quenching of DED-actin fluorescence was restored in copolymers (1:9) of N-ethylmaleimide-DED-actin with unlabeled actin. The quenching of DED-actin fluorescence by copper was also abolished in copolymers (1:4) of DED-actin and N-ethylmaleimide-actin. These results show intermolecular coupling between loop 38-52 and the C-terminus in F-actin. Consistent with this, the rate of subtilisin cleavage of actin at loop 38-52 was increased by the bound copper by more than 10-fold in F-actin but not in G-actin. Neither acto-myosin subfragment-1 (S1) ATPase activity nor the tryptic digestion of G-actin and F-actin at the Lys-61 and Lys-69 sites were affected by the bound copper. These observations suggest that copper binding to Cys-374 does not induce extensive changes in actin structure and that the perturbation of loop 38-52 environment results from changes in the intermolecular contacts in F-actin.

Actins↗
PubMed1996

Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.

Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.

Actins↗
PubMed1996

In vitro evaluation of neonatal human immunity against the pig.

The critical shortage of organ donors has greatly limited the number of clinical allotransplantations. This is particularly true for neonatal patients, for whom xenotransplantation could provide an alternative therapeutic option to allotransplantation. The role of neonatal infant immunity in xenotransplantation is not, however, clearly understood. We examined both the proliferative responses of human neonatal lymphocytes to pig aortic endothelial cells and serum levels of neonatal natural antipig xenoantibody. Neonatal human lymphocytes and serum were isolated from umbilical cord blood. Adult human lymphocytes and serum were used as controls. A one-way xenogeneic mixed lymphocyte-endothelial cell reaction was performed, and lymphocyte proliferation was measured by tritiated thymidine uptake. Neonatal human lymphocytes recognized and proliferated in response to pig aortic endothelial cells (mean 63,926 +/- 26,054 counts per minute). The level of xenogeneic mixed lymphocyte-endothelial cell reaction of neonatal lymphocytes was significantly lower (p < 0.004) than that of adult human lymphocytes (mean 122,444 +/- 33,132 counts per minute). An enzyme-linked immunosorbent assay was performed to determine the binding of natural immunoglobulin M and G antibodies to pig endothelial cells. Whole-cell enzyme-linked immunosorbent assay demonstrated neonatal human serum to contain very low binding levels of natural antipig immunoglobulin M xenoantibody compared with adult serum. Like adult serum, neonatal human serum contained natural antipig immunoglobulin G xenoantibody. Neonatal serum was not cytotoxic to pig endothelial cells, suggesting that immunoglobulin G was not the predominant xenoreactive antibody. To assess whether neonatal pig endothelial cells also expressed xenoantigens, adult and neonatal cultured pig endothelial cells were examined by enzyme-linked immunosorbent assay with adult human serum. Adult human natural immunoglobulin M xenoantibody recognized not only adult pig endothelial cell xenoantigens but also neonatal pig endothelial cell xenoantigens. The binding levels of adult natural antipig immunoglobulin M xenoantibodies to adult and neonatal pig endothelial cells were similar, suggesting that neonatal pig aortic endothelial cells express xenoantigens. The findings of low binding levels of cytotoxic antipig immunoglobulin M xenoantibody and low levels of lymphocyte xenoreactivity to pig endothelial cells in human neonates suggests that pig organs may eventually be a suitable source of xenografts for human neonates.

Adult↗
PubMed1996

Heteromultimerization and NMDA receptor-clustering activity of Chapsyn-110, a member of the PSD-95 family of proteins.

Chapsyn-110, a novel membrane-associated putative guanylate kinase (MAGUK) that binds directly to N-methyl-D-aspartate (NMDA) receptor and Shaker K+ channel subunits, is 70%-80% identical to, and shares an identical domain organization with, PSD-95/SAP90 and SAP97. In rat brain, chapsyn-110 protein shows a somatodendritic expression pattern that overlaps partly with PSD-95 but that contrasts with the axonal distribution of SAP97. Chapsyn-110 associates tightly with the postsynaptic density in brain, and mediates the clustering of both NMDA receptors and K+ channels in heterologous cells. Indeed, chapsyn-110 and PSD-95 can heteromultimerize with each other and are recruited into the same NMDA receptor and K+ channel clusters. Thus, chapsyn-110 and PSD-95 may interact at postsynaptic sites to form a multimeric scaffold for the clustering of receptors, ion channels, and associated signalling proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Ion channel associated proteins.

Neuronal ion channels are directly associated in vivo with a wide variety of proteins. New classes of channel-associated proteins have been identified recently, including the PSD-95/SAP90 family of channel-clustering molecules and components of the synaptic vesicle release machinery. Recent findings suggest that non-pore-forming subunits of ion channels may also have cell biological functions independent of their effects on channel electrophysiological properties.

Animals↗
PubMed1996

Sequence analysis of the florfenicol resistance gene encoded in the transferable R-plasmid of a fish pathogen, Pasteurella piscicida.

The florfenicol resistance gene (pp-flo) derived from a transferable R-plasmid of Pasteurella piscicida consisted of 1,122 nucleotides, and the predicted amino acid sequence showed 47.4% identity to that of a non-enzymatic chloramphenicol resistance gene (cmlA). The pp-flo gene was located in the downstream region of the sulfonamide resistance gene of the transferable R-plasmid.

Amino Acid Sequence↗