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Z Qu

Publications and source records attributed to Z Qu.

At least 37 records · Page 2Linked to original sources
PubMed2001

[Mapping the interaction site of Rpb2 and Rpb3 subunit of fission yeast RNA polymerase II].

To map the interacting site of subunit Rpb2 to subunit Rpb3 of RNA polymerase II in fission yeast Schizosaccharomyces pombe, the yeast two-hybrid system was employed in this paper to screen the interacting clones between Rpb2 and Rpb3.4 fragments of Rpb2 cDNA were cloned into the Ga14 BD vector pAS2. The 4 clones were named as pAS2 Rpb2-1, 2-2, 2-3 and 2-4, respectively. The complete cDNA of Rpb3 was cloned into the Gal 4 AD vector pGADGH. The clone was named as pGADGH Rpb3. The two-hybrid plasmids pGADGH Rpb3 and pAS2Rpb2-1, 2-2, 2-3 or 2-4 respectively were cotransformed into host cell yeast Y190. The interaction positive cotransformants were identified by beta-gal activity assay. The beta-gal positive cotransformants were selected from pGADGH Rpb3 and pAS2Rpb2-4 two-hybrid system. DNA sequencing and alignment results showed that the interacting site of Rpb2 to Rpb3 located within the fragment from base 2701 to 2966 of Rpb2 cDNA, or within the C-termini polypeptide from amino acid 902 to 989 of Rpb2 protein.

Amino Acid Sequence↗
PubMed2000

Ventricular fibrillation: how do we stop the waves from breaking?

Combined experimental and theoretical developments have demonstrated that in addition to preexisting electrophysiological heterogeneities, cardiac electrical restitution properties contribute to breakup of reentrant wavefronts during cardiac fibrillation. Developing therapies that favorably alter electrical restitution properties have promise as a new paradigm for preventing fibrillation.

Action Potentials↗
PubMed2000

Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains.

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO(2)/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO(2) and pH(i). These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K(+) channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator.

Amino Acid Motifs↗
PubMed2000

Mechanisms of discordant alternans and induction of reentry in simulated cardiac tissue.

BACKGROUND: T-wave alternans, which is associated with the genesis of cardiac fibrillation, has recently been related to discordant action potential duration (APD) alternans. However, the cellular electrophysiological mechanisms responsible for discordant alternans are poorly understood. METHODS AND RESULTS: We simulated a 2D sheet of cardiac tissue using phase 1 of the Luo-Rudy cardiac action potential model. A steep (slope >1) APD restitution curve promoted concordant APD alternans and T-wave alternans without QRS alternans. When pacing was from a single site, discordant APD alternans occurred only when the pacing rate was fast enough to engage conduction velocity (CV) restitution, producing both QRS and T-wave alternans. Tissue heterogeneity was not required for this effect. Discordant alternans markedly increases dispersion of refractoriness and increases the ability of a premature stimulus to cause localized wavebreak and induce reentry. In the absence of steep APD restitution and of CV restitution, sustained discordant alternans did not occur, but reentry could be induced if there was marked electrophysiological heterogeneity. Both discordant APD alternans and preexisting APD heterogeneity facilitate reentry by causing the waveback to propagate slowly. CONCLUSION: Discordant alternans arises dynamically from APD and CV restitution properties and markedly increases dispersion of refractoriness. Preexisting and dynamically induced (via restitution) dispersion of refractoriness independently increase vulnerability to reentrant arrhythmias. Reduction of dynamically induced dispersion by appropriate alteration of electrical restitution has promise as an antiarrhythmic strategy.

Action Potentials↗
PubMed2000

Mechanisms of ventricular fibrillation induction by 60-Hz alternating current in isolated swine right ventricle.

BACKGROUND: The mechanisms by which 60-Hz alternating current (AC) can induce ventricular fibrillation (VF) are unknown. METHODS AND RESULTS: We studied 7 isolated perfused swine right ventricles in vitro. The action potential duration restitution curve was determined. Optical mapping techniques were used to determine the patterns of activation on the epicardium during 5-second 60-Hz AC stimulation (10 to 999 microA). AC captured the right ventricles at 100+/-65 microA, which is significantly lower than the direct current pacing threshold (0.77+/-0.45 mA, P:<0.05). AC induced ventricular tachycardia or VF at 477+/-266 microA, when the stimulated responses to AC had (1) short activation CLs (128+/-14 ms), (2) short diastolic intervals (16+/-9 ms), and (3) short diastolic intervals associated with a steep action potential duration restitution curve. Optical mapping studies showed that during rapid ventricular stimulation by AC, a wave front might encounter the refractory tail of an earlier wave front, resulting in the formation of a wave break and VF. Computer simulations reproduced these results. CONCLUSIONS: AC at strengths less than the regular pacing threshold can capture the ventricle at fast rates. Accidental AC leak to the ventricles could precipitate VF and sudden death if AC results in a fast ventricular rate coupled with a steep restitution curve and a nonuniform recovery of excitability of the myocardium.

Animals↗
PubMed2000

Ascorbate-dependent protection of human erythrocytes against oxidant stress generated by extracellular diazobenzene sulfonate.

Diazobenzene sulfonic acid (DABS) has been used to label thiols and amino groups on cell-surface proteins. However, we found that in addition to inhibiting an ascorbate-dependent trans-plasma membrane oxidoreductase in human erythrocytes, it also depleted alpha-tocopherol severely in the cell membrane. When erythrocytes were loaded with ascorbate, DABS-dependent loss of alpha-tocopherol was decreased, despite little change in intracellular ascorbate content. Sparing of alpha-tocopherol also was seen in erythrocyte ghosts resealed to contain ascorbate, although this was accompanied by loss of intravesicular ascorbate, probably due to the inability of ghosts to recycle ascorbate. A transmembrane transfer of electrons from ascorbate was confirmed by electron paramagnetic resonance spectroscopy, in which extracellular DABS was found to generate the ascorbate free radical within cells. When the membrane content of alpha-tocopherol was decreased to 20% of the initial value by DABS treatment, lipid peroxidation ensued, manifest by generation of F(2)-isoprostanes in the cell membranes. Intracellular ascorbate also strongly protected against F(2)-isoprostane formation. These results show that DABS causes an oxidant stress at the membrane surface that is transmitted within the cell, in part by an alpha-tocopherol-dependent mechanism, and that ascorbate recycling of alpha-tocopherol can protect against loss of alpha-tocopherol and the ensuing lipid peroxidation.

Ascorbic Acid↗
PubMed2000

Biophysical and molecular mechanisms underlying the modulation of heteromeric Kir4.1-Kir5.1 channels by CO2 and pH.

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.

Animals↗
PubMed2000

Role of EGR1 in hippocampal synaptic enhancement induced by tetanic stimulation and amputation.

Hippocampal neurons fire spikes when an animal is at a particular location or performs certain behaviors in a particular place, providing a cellular basis for hippocampal involvement in spatial learning and memory. In a natural environment, spatial memory is often associated with potentially dangerous sensory experiences such as noxious or painful stimuli. The central sites for such pain-associated memory or plasticity have not been identified. Here we present evidence that excitatory glutamatergic synapses within the CA1 region of the hippocampus may play a role in storing pain-related information. Peripheral noxious stimulation induced excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal cells in anesthetized animals. Tissue/nerve injury caused a rapid increase in the level of the immediate-early gene product Egr1 (also called NGFI-A, Krox24, or zif/268) in hippocampal CA1 neurons. In parallel, synaptic potentiation induced by a single tetanic stimulation (100 Hz for 1 s) was enhanced after the injury. This enhancement of synaptic potentiation was absent in mice lacking Egr1. Our data suggest that Egr1 may act as an important regulator of pain-related synaptic plasticity within the hippocampus.

Amputation, Surgical↗
PubMed2000

Preventing ventricular fibrillation by flattening cardiac restitution.

Ventricular fibrillation is the leading cause of sudden cardiac death. In fibrillation, fragmented electrical waves meander erratically through the heart muscle, creating disordered and ineffective contraction. Theoretical and computer studies, as well as recent experimental evidence, have suggested that fibrillation is created and sustained by the property of restitution of the cardiac action potential duration (that is, its dependence on the previous diastolic interval). The restitution hypothesis states that steeply sloped restitution curves create unstable wave propagation that results in wave break, the event that is necessary for fibrillation. Here we present experimental evidence supporting this idea. In particular, we identify the action of the drug bretylium as a prototype for the future development of effective restitution-based antifibrillatory agents. We show that bretylium acts in accord with the restitution hypothesis: by flattening restitution curves, it prevents wave break and thus prevents fibrillation. It even converts existing fibrillation, either to a periodic state (ventricular tachycardia, which is much more easily controlled) or to quiescent healthy tissue.

Action Potentials↗
PubMed2000

Modulation of kir4.1 and kir5.1 by hypercapnia and intracellular acidosis.

CO2 chemoreception may be mediated by the modulation of certain ion channels in neurons. Kir4.1 and Kir5.1, two members of the inward rectifier K+ channel family, are expressed in several brain regions including the brainstem. To test the hypothesis that Kir4.1 and Kir5. 1 are modulated by CO2 and pH, we carried out experiments by expressing Kir4.1 and coexpressing Kir4.1 with Kir5.1 (Kir4.1-Kir5. 1) in Xenopus oocytes. K+ currents were then studied using two-electrode voltage clamp and excised patches. Exposure of the oocytes to CO2 (5, 10 and 15 %) produced a concentration-dependent inhibition of the whole-cell K+ currents. This inhibition was fast and reversible. Exposure to 15 % CO2 suppressed Kir4.1 currents by approximately 20 % and Kir4.1-Kir5.1 currents by approximately 60 %. The effect of CO2 was likely to be mediated by intracellular acidification, because selective intracellular, but not extracellular, acidification to the measured hypercapnic pH levels lowered the currents as effectively as hypercapnia. In excised inside-out patches, exposure of the cytosolic side of membranes to solutions with various pH levels brought about a dose-dependent inhibition of the macroscopic K+ currents. The pK value (-log of dissociation constant) for the inhibition was 6.03 in the Kir4.1 channels, while it was 7.45 in Kir4.1-Kir5.1 channels, an increase in pH sensitivity of 1.4 pH units. Hypercapnia without changing pH did not inhibit the Kir4.1 and Kir4.1-Kir5.1 currents, suggesting that these channels are inhibited by protons rather than molecular CO2. A lysine residue in the N terminus of Kir4.1 is critical. Mutation of this lysine at position 67 to methionine (K67M) completely eliminated the CO2 sensitivity of both the homomeric Kir4. 1 and heteromeric Kir4.1-Kir5.1. These results therefore indicate that the Kir4.1 channel is inhibited during hypercapnia by a decrease in intracellular pH, and the coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivity to CO2/pH and may enable cells to detect both increases and decreases in PCO2 and intracellular pH at physiological levels.

Acidosis↗
PubMed2000

Involvement of histidine residues in proton sensing of ROMK1 channel.

ROMK channels are inhibited by intracellular acidification. This pH sensitivity is related to several amino acid residues in the channel proteins such as Lys-61, Thr-51, and His-206 (in ROMK2). Unlike all other amino acids, histidine is titratable at pH 6-7 carrying a positive charge below pH 6. To test the hypothesis that certain histidine residues are engaged in CO(2) and pH sensing of ROMK1, we performed experiments by systematic mutations of all histidine residues in the channel using the site-directed mutagenesis. There are two histidine residues in the N terminus. Mutations of His-23, His-31, or both together did not affect channel sensitivity to CO(2). Six histidine residues are located in the C terminus. His-225, His-274, His-342, and His-354 were critical in CO(2) and pH sensing. Mutation of either of them reduced CO(2) and pH sensitivities by 20-50% and approximately 0.2 pH units, respectively. Simultaneous mutations of all of them eliminated the CO(2) sensitivity and caused this mutant channel to respond to only extremely acidic pH. Similar mutations of His-280 had no effect. The role of His-270 in CO(2) and pH sensing is unclear, because substitutions of this residue with either a neutral, negative, or positive amino acid did not produce any functional channel. These results therefore indicate that histidine residues contribute to the sensitivity of the ROMK1 channel to hypercapnia and intracellular acidosis.

Carbon Dioxide↗
PubMed2000

CO(2) inhibits specific inward rectifier K(+) channels by decreases in intra- and extracellular pH.

Hypercapnia has been shown to affect cellular excitability by modulating K(+) channels. To understand the mechanisms for this modulation, four cloned K(+) channels were studied by expressing them in Xenopus oocytes. Exposures of the oocytes to CO(2) for 4-6 min produced reversible and concentration-dependent inhibitions of Kir1.1 and Kir2.3 currents, but had no effect on Kir2.1 and Kir6.1 currents. Intra- and extracellular pH (pH(i), pH(o)) dropped during CO(2) exposures. The inhibition of Kir2.3 currents was mediated by reductions in both intra- and extracellular pH, whereas the suppression of Kir1.1 resulted from intracellular acidification. In cell-free excised inside-out patches with cytosolic-soluble factors washed out, a decrease in pH(i) produced a fast and reversible inhibition of macroscopic Kir2.3 currents. The degree of this inhibition was similar to that produced by hypercapnia when compared at the same pH(i) level. Exposure of cytosolic surface of patch membranes to a perfusate bubbled with 15% CO(2) without changing pH failed to inhibit the Kir2.3 currents. These results therefore indicate that (1) hypercapnia inhibits specific K(+) channels, (2) these inhibitions are caused by intra- and extracellular protons rather than molecular CO(2), and (3) these effects are independent of cytosol-soluble factors.

Animals↗
PubMed2000

Scroll wave dynamics in a three-dimensional cardiac tissue model: roles of restitution, thickness, and fiber rotation.

Scroll wave (vortex) breakup is hypothesized to underlie ventricular fibrillation, the leading cause of sudden cardiac death. We simulated scroll wave behaviors in a three-dimensional cardiac tissue model, using phase I of the Luo-Rudy (LR1) action potential model. The effects of action potential duration (APD) restitution, tissue thickness, filament twist, and fiber rotation were studied. We found that APD restitution is the major determinant of scroll wave behavior and that instabilities arising from APD restitution are the main determinants of scroll wave breakup in this cardiac model. We did not see a "thickness-induced instability" in the LR1 model, but a minimum thickness is required for scroll breakup in the presence of fiber rotation. The major effect of fiber rotation is to maintain twist in a scroll wave, promoting filament bending and thus scroll breakup. In addition, fiber rotation induces curvature in the scroll wave, which weakens conduction and further facilitates wave break.

Action Potentials↗
PubMed2000

Metabolic effects of insulin and insulin-like growth factor-I in endotoxemic rats during total parenteral nutrition feeding.

The effects of insulin and insulin-like growth factor-I (IGF-I) on protein, energy, and glucose metabolism were examined in endotoxemic rats receiving total parenteral nutrition (TPN) for 3 days. The endotoxemic model was induced by constant infusion of lipopolysaccharide (1 mg/kg x d) for 3 days. The TPN regimen provided 200 kcal/kg x d and 1.5 g protein/kg x d. The dosage of insulin (5 mU/kg x h) and IGF-I (20 microg/kg x h), either alone or in combination, was chosen to maintain normal levels of leucine and glucose in plasma during feeding. One normal control and 4 endotoxemic groups with different treatments (saline, IGF-I, insulin, or IGF-I and insulin) were included. The effects of endotoxin were compared between the group receiving endotoxin alone and normal controls, and the effects of insulin and IGF-I were compared within the endotoxemic groups. The results show that endotoxin significantly increased the mortality and induced a hypermetabolic state, and nutrition alone could not overcome the catabolism induced by endotoxin. However, administration of insulin and IGF-I enhanced protein preservation in muscle tissue in endotoxemic rats during TPN. This effect was greater for insulin either alone or in combination with IGF-I. Insulin also significantly reduced the mortality. There were no additive effects of these two anabolic hormones on any measured parameter in these experimental conditions.

Animals↗
PubMed2000

Matching host muscle and donor myoblasts for myosin heavy chain improves myoblast transfer therapy.

Intensive efforts have been made to develop an effective therapy for Duchenne muscular dystrophy (DMD). Although myoblast transplantation has been found capable of transiently delivering dystrophin and improving the strength of the injected dystrophic muscle, this approach has been hindered by the immune rejection problems as well as the poor survival and limited spread of the injected cells. In the present study, we have investigated whether the careful selection of donor myoblasts and host muscle for the myosin heavy chain expression (MyHCs) plays a role in the success of myoblast transfer. Highly purified normal myoblasts derived from the m. soleus and m. gastrocnemius white of normal mice were transplanted into the m. soleus (containing 70% of type I fibers) and gastrocnemius white (100% of type II fibers) of dystrophin deficient mdx mice. At several time-points after injection (10, 20 and 30 days), the number of dystrophin-positive fibers was monitored and compared among the different groups. A significantly higher number and better persistence of dystrophin-positive myofibers were observed when the injected muscle and donor myoblasts expressed a similar MyHC in comparison with myoblast transfer between host muscle and donor myoblasts that were not matched for MyHC. These results suggest that careful matching between the injected myoblasts and injected muscle for the MyHC expression can improve the efficiency of myoblast-mediated gene transfer to skeletal muscle. Gene Therapy (2000) 7, 428-437.

Animals↗
PubMed2000

Anion permeation in Ca(2+)-activated Cl(-) channels.

Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore.

Animals↗
PubMed2000

From local to global spatiotemporal chaos in a cardiac tissue model.

Two kinds of chaos can occur in cardiac tissue, chaotic meander of a single intact spiral wave and chaotic spiral wave breakup. We studied these behaviors in a model of two-dimensional cardiac tissue based on the Luo-Rudy I action potential model. In the chaotic meander regime, chaos is spatially localized to the core of the spiral wave. When persistent spiral wave breakup occurs, there is a transition from local to global spatiotemporal chaos.

Action Potentials↗