[New aspects of idiopathic cardiomyopathy--from gene diagnosis to gene therapy].
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Animals↗
Biomedical subjects
I Shin
Publications and source records attributed to I Shin.
Phosphatidic acid-induced elevation of intracellular Ca2+ is mediated by RhoA and H2O2 in Rat-2 fibroblasts.
We have investigated possible roles of RhoA and H2O2 in the elevation of intracellular Ca2+ ([Ca2+]i) by phosphatidic acid (PA) in Rat-2 fibroblasts. PA induced a transient elevation of [Ca2+]i in the presence or absence of EGTA. Lysophosphatidic acid (LPA) also increased [Ca2+]i, but the sustained Ca2+ response was inhibited by EGTA. LPA stimulated the production of inositol phosphates, but PA did not. In the presence of EGTA, preincubation with LPA completely blocked the subsequent elevation of [Ca2+]i by PA, but not vice versa. PA stimulated the translocation of RhoA to the particulate fraction as did LPA. Scrape loading of C3 transferase inhibited the transient Ca2+ response to PA, but not to LPA, suggesting an essential role of RhoA in the elevation of [Ca2+]i by PA. H2O2 also induced a transient increase of [Ca2+]i as did PA. H2O2 scavengers, catalase and N-acetyl-L-cysteine, completely blocked the rise of [Ca2+]i stimulated by PA, but not by LPA. Furthermore, preincubation with PA blocked the subsequent Ca2+ response to H2O2, and the incubation with H2O2 also blocked the PA-induced rise of [Ca2+]i. Thus, it was suggested that PA stimulated Ca2+ release from PA-sensitive, but not inositol 1,4,5-trisphosphate-sensitive, Ca2+ stores by the activation of RhoA and intracellular H2O2.
Animals↗
Liposome-catalyzed unfolding of acetylcholinesterase from Bungarus fasciatus.
The kinetics of thermal inactivation of acetylcholinesterase from the venom of the snake, Bungarus fasciatus, were studied at 45-54 degrees C. An Arrhenius plot reveals an activation energy of 113 kcal/mol. The thermally denatured enzyme displays the spectroscopic characteristics of a partially unfolded 'molten globule' state. The rate of thermal denaturation is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, the energy barrier for the transition being lowered from 113 to 52 kcal/mol. In contrast to our findings for partially unfolded Torpedo californica acetylcholinesterase [Shin et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2848-2852], the thermally denatured snake enzyme does not remain bound to the liposomes but is released after unfolding and subsequently aggregates. The liposomes thus serve as catalysts for unfolding of the snake enzyme, and its rate of unfolding in the presence of liposomes can be described by the Michaelis-Menten equation (Km = 8 x 10(-7) M). The phospholipid vesicles display a catalytic turnover number of kcat approximately 4 min-1, assuming 15 binding sites per vesicle for the snake acetylcholinesterase.
Acetylcholinesterase↗
Changes of phospholipase D activity in TNF-alpha and anti-Fas/Apo1 monoclonal antibody induced apoptosis in HL-60 and A20 cells.
The changes of phospholipase D (PLD) activity were investigated during the courses of apoptotic process induced by tumor necrosis factor (TNF)-alpha or anti-Fas/Apo1 antibody in human premyelocyte HL-60 and murine B cell lymphoma A20 cells. The treatment of recombinant TNF-alpha to HL-60 cells resulted in the increased PLD activity as determined by the phosphatidylethanol formation in the presence of 1% ethanol. The enhancement of PLD activity was also observed in the anti-Fas/Apo1 monoclonal antibody-treated A20 cells. However, the activity of PLD was maximized when HL-60 and A20 cells were treated with either TNF-alpha or anti-Fas/Apo1 monoclonal antibody for 6 h. Both TNF-alpha and anti-Fas/Apo1 monoclonal antibody increased PLD activity in a dose-dependent manner up to 200 U/ml and 200 ng/ml, respectively. When the intracellular activity of protein kinase C (PKC) was interrupted by treatment of calphostin-C, both the PLD activation and the apoptosis induced by TNF-alpha and anti-Fas/Apo1 monoclonal antibody appeared to be inhibited. Since PKC is reported to activate PLD, the results indicate that the intracellular signaling cascade via PLD may play a role in the induction of apoptosis induced by TNF-alpha and anti-Fas/Apo1 monoclonal antibody.
Animals↗
Colchicine activates actin polymerization by microtubule depolymerization.
Swiss 3T3 fibroblasts were treated with the microtubule-disrupting agent colchicine to study any interaction between microtubule dynamics and actin polymerization. Colchicine increased the amount of filamentous actin (F-actin), in a dose- and time-dependent manner with a significant increase at 1 h by about 130% over control level. Confocal microscopic observation showed that colchicine increased F-actin contents by stress fiber formation without inducing membrane ruffling. Colchicine did not activate phospholipase C and phospholipase D, whereas lysophosphatidic acid did, indicating that colchicine may have a different mechanism of actin polymerization regulation from LPA. A variety of microtubule-disrupting agents stimulated actin polymerization in Swiss 3T3 and Rat-2 fibroblasts as did colchicine, but the microtubule-stabilizing agent taxol inhibited actin polymerization induced by the above microtubule-disrupting agents. In addition, colchicine-induced actin polymerization was blocked by two protein phosphatase inhibitors, okadaic acid and calyculin A. These results suggest that microtubule depolymerization activates stress fiber formation by serine/threonine dephosphorylation in fibroblasts.
3T3 Cells↗
Membrane-promoted unfolding of acetylcholinesterase: a possible mechanism for insertion into the lipid bilayer.
Acetylcholinesterase from Torpedo californica partially unfolds to a state with the physicochemical characteristics of a "molten globule" upon mild thermal denaturation or upon chemical modification of a single non-conserved buried cysteine residue, Cys231. The protein in this state binds tightly to liposomes. It is here shown that the rate of unfolding is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, with concomitant incorporation of the protein into the lipid bilayer. Arrhenius plots reveal that in the presence of the liposomes the energy barrier for transition from the native to the molten globule state is lowered from 145 to 47 kcal/mol. Chemical modification of Cys231 by mercuric chloride produces initially a quasinative state of Torpedo acetylcholinesterase which, at room temperature, undergoes spontaneous transition to a molten globule state with a half-life of 1-2 hr. This permitted temporal resolution of interaction of the quasi-native state with the membrane from the transition of the membrane-bound protein to the molten globule state. The data presented here suggest that either the native enzyme, or a quasi-native state with which it is in equilibrium, interacts with the liposome, which then promotes a fast transition to the membrane-bound molten globule state by lowering the energy barrier for the transition. These findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active posttranslational role in insertion and translocation of proteins in situ.
Acetylcholinesterase↗
Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking.
Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway. Amino acids with benzophenone side chains of various lengths [DL-2-amino-3-(p-benzoylphenyl)propanoic acid (1) and DL-2-amino-5-(p-benzoylphenyl)pentanoic acid (2)] were incorporated at specific sites throughout the cytochrome b2-dihydrofolate reductase fusion proteins, pb2(220)-DHFR and pb2 delta 19(220)-DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively. In vitro import of pb2(220)-DHFR and pb2 delta 19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface. The matrix-targeted pb2 delta 19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels. On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted pb2(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40. The N-terminal portion of pb2(220)-DHFR, but not pb2 delta 19(220)-DHFR, was photocrosslinked to an as-yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.
Biological Transport↗
Interhead distances in myosin attached to F-actin estimated by fluorescence energy transfer spectroscopy.
Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine distances between probes attached to the most reactive sulfhydryl (SH1) group on individual myosin "heads." We measured intramolecular and intermolecular interhead distances as well as the distance between one head of heavy meromyosin (HMM) mixed with subfragment-1 (S1) heads attached to F-actin under rigor conditions. The SH1 cysteine was specifically labeled with either a donor (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid) or an acceptor probe (5-iodoacetamidofluorescein). In free solution, the distance between these probes was too large to allow significant FRET, but in the rigor complex with F-actin, intermolecular interhead distances between S1 molecules, HMM molecules, or S1 and HMM were determined to be 6.0-6.3 nm. The radial coordinate of the labels relative to F-actin was 5.0-6.4 nm. However, the intramolecular interhead distance in HMMs in which the two heads were labeled with D and A probes was estimated to be larger. The binding affinity of the second head of HMM(D/A) to F-actin may be reduced because of heterogeneous modification of the SH1 groups, such that the probability of single-head binding is increased.
Actins↗
Redesigning the active-site of an acyl-CoA dehydrogenase: new evidence supporting a one-base mechanism.
The acyl-CoA dehydrogenases are a family of related enzymes that share high structural homology and a common catalytic mechanism which involves abstraction of an alpha-proton from the substrate by an active site glutamate residue. Several lines of investigation have shown that the position of the catalytic glutamate is conserved in most of these dehydrogenases (the E2 site), but is in a different location in two other family members (the E1 site). Using site specific in vitro mutagenesis, a double mutant rat short chain acyl-CoA dehydrogenase (rSCAD) has been constructed in which the catalytic glutamate is moved from the E2 to the E1 site (Glu368Gly/Gly247Glu). This mutant enzyme is catalytically active, but utilizes substrate less efficiently than the native enzyme (K(m) = 0.6 and 2.0 microM, and Vmax = 2.8 and 0.3 s-1 for native and mutant enzyme respectively). In this study we show that both the wild-type and mutant rSCADs display identical stereochemical preference for catalysis--abstraction of the alpha-HR from the substrate followed by transfer of the beta-HR to the FAD coenzyme. These results, in conjunction with molecular modeling of the native and double mutant SCAD indicate that the catalytic base in the E1 and E2 sites are topologically similar and catalytically competent. However, analysis of the 1H NMR spectra of the incubation products of these two enzymes revealed that, in contrast to the wild-type rSCAD, the Gly368Glu/Gly247Glu rSCAD could not perform gamma-proton exchange of the product with the solvent, a property inherent to most acyl-CoA dehydrogenases. It is evident that the base in the mutant enzyme has access to the alpha-HR but is far removed from the gamma-Hs. These findings provide further support for a one base mechanism of alpha- and gamma-reprotonation/deprotonation catalysis by acyl-CoA dehydrogenases.
Acyl-CoA Dehydrogenases↗
Modulation of protein kinase C activity in NIH 3T3 cells by plant glycosides from Panax ginseng.
The involvement of ginsenosides in the signal cascade that stimulates cellular growth was investigated. It was found that ginsenosides Rh1 and Rh2 extracted from the root of Panax ginseng inhibited cellular proliferation in NIH 3T3 fibroblasts. Both ginsenosides Rh1 and Rh2 effectively reduced phospholipase C activity resulting in a decrease in the intracellular level of diacylglycerol, an endogenous activator of protein kinase C. The treatment of cells with Rh1 or Rh2 was thus found to reduce intracellular protein kinase C activity. We also observed that the phosphorylation of myristoylated alanine-rich C kinase substrate, one of the major substrates of protein kinase C in cells, was inhibited by the ginsenosides. Data suggest that the ginsenoside Rh1 or Rh2 exerts antiproliferative effects by inhibiting phospholipase C, which produces second messengers necessary for the activation of protein kinase C.
3T3 Cells↗
Inhibition of the phosphorylation of a myristoylated alanine-rich C kinase substrate by methyl methanesulfonate in cultured NIH 3T3 cells.
The effect of methyl methanesulfonate (MMS) on the phosphorylation of an acidic 80-kDa myristoylated alanine-rich C kinase substrate (MARCKS) protein was investigated in NIH 3T3 fibroblasts. An alkylating agent, MMS inhibited protein kinase C activity and the phosphorylation of MARCKS. MMS treatment also lowered the cellular amounts of second messengers of inositol-1,4,5-trisphosphate and diacylglycerol. Data suggest that MMS decreased the phosphorylation of phospholipase C, a protein whose activity is influenced by its phosphorylation state. We present here the first report that MMS intervenes in a signal cascade by inhibiting the phosphorylation of phospholipase C, which in turn leads to the inactivation of protein kinase C and the subsequent inhibition of MARCKS phosphorylation.
3T3 Cells↗
Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes.
A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.
Acetylcholinesterase↗
Two partially unfolded states of Torpedo californica acetylcholinesterase.
Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.
Acetylcholinesterase↗
[Mechanism of anti-leishmanial drugs on the basis of the nucleoside structures].
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Animals↗
Inhibition of 80 kDa protein phosphorylation by short-wavelength UV light in NIH 3T3 cells.
The exposure of NIH 3T3 fibroblast cells to 254 nm UV radiation resulted in a temporary depression of DNA synthesis and inhibition of 80 kDa protein phosphorylation. This inhibition of protein phosphorylation was correlated with decreased protein kinase C activity in the membrane fractions of UV-damaged cells. The inositol triphosphate contents measured, by the competitive binding assay using bovine adrenal binding protein, showed 80% reduction in the fibroblasts treated with 15 J/m2 of UV light. The intracellular diacylglycerol concentration was also markedly reduced in UV-damaged cells. The results suggest that UV light causes acute reductions of inositol triphosphate and diacylglycerol contents in cells along with decreases in membrane protein kinase C activity, which leads to the inhibition of phosphorylation of an acidic protein of 80 kDa.
3T3 Cells↗
Effect of cytoskeletal geometry on intracellular diffusion.
A method is presented for determining the retardation of diffusion of particles inside cells owing to cytoskeletal barriers. The cytoskeletal meshwork is treated as a repeating periodic two-dimensional or three-dimensional lattice composed of elements of given size, shape, and spacing. We derive an analytic expression for the diffusion coefficient relative to that of the cytosol. This expression is evaluated by solving numerically an appropriate boundary-value problem for the Laplace equation. For the two-dimensional case, e.g., diffusion in a membrane, the results are quantitatively similar to those obtained by Saxton (1987. Biophys. J. 52:989-997) using Monte Carlo methods. The three-dimensional results are quantitatively similar to experimental results reported by Luby-Phelps et al. (1987. Proc. Natl. Acad. Sci. USA. 84:4910-4913) for the diffusion of dextran and Ficoll particles in Swiss 3T3 cells. By accounting for geometrical factors, these results allow one to assess the relative contributions of geometrical hindrance and of binding to the cytoskeletal lattice from measurements of intracellular diffusion coefficients of proteins.
Cytoskeleton↗
The value of electroencephalographic recordings in dialysis encephalopathy: case-reports and review of literature.
The electroencephalographic abnormalities with dialysis dementia are described in five patients with this disorder. The tracings of all patients showed a mild to moderate slowing of the background rhythm, intermixed with paroxysmal and rhythmic waves, most prominent on the frontal areas. Epileptiform discharges were seen in only one record. Clinical features, hypotheses regarding pathogenesis, and treatment of this usually fatal encephalopathy are briefly discussed.
Adult↗
Effect of shear rates on protein adsorption in the total artificial heart.
Plasma protein adsorption is the first event in blood-material interaction and influences subsequent platelet adhesion and thrombus formation. Thromboembolic events are strongly influenced by surface characteristics of materials and fluid dynamics inside the blood pump. In vitro flow visualization, and an animal experiment with a moving actuator total artificial heart (TAH), were performed to investigate fluid dynamic effects on protein adsorption. The different levels of shear rate inside the ventricle were determined by considering the direction of opening of the four heart valves in the implanted TAH, and the visualized flow patterns as well. Each ventricle of the explanted TAH was cut into 12 segments according to the shear rate level. The adsorbed protein on each segment was quantified using an ELISA method after soaking in 2% (w/v) SDS/PBS for 2 days. Adsorbed protein layer thicknesses were measured by the immunogold method under transmission electron microscopy. Scanning electron microscopic observation showed that the right ventricle, immobilized with albumin, displayed different degrees of platelet adhesion on each segment, whereas the left ventricle, coated with polythyleneoxide-sulfonate, indicated nearly the same platelet adhesion behavior, regardless of shear rates. The surface concentrations of adsorbed proteins in the low shear rate region are higher than those in the high shear region, which was confirmed statistically.
Adsorption↗