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S Sugiura

Publications and source records attributed to S Sugiura.

At least 19 recordsLinked to original sources
PubMed1996

Open strands adjacent to iterons promote the binding of the replication initiator protein (Rep) of pSC101 to the unit sequence of the iterons in vitro.

The purified dimeric form of the Rep protein, a replication initiator protein of the plasmid pSC101, has a low affinity for repeated sequences, iterons, in the replication origin of the plasmid, and higher affinities for two inverted repeats in the operator region of the rep gene resulting in its functioning as an autorepressor. Studies of binding to various synthetic DNA have established that Rep can bind to duplex iteron-sequence carrying open (non-complementary) strands at one end proximal to the rep gene. Open strands at the opposite end of the iteron have no effect on Rep-binding. One open strand seems to be required in a sequence-specific fashion. A randomly sequenced duplex DNA with the open strands cannot bind to Rep but can function as a significant competitor. This suggests that Rep has some affinity for the open strands and forms a stable complex with the adjacent iteron. The mutated Rep protein, Rep1, which causes an increase in the plasmid copy number in vivo, has equally high affinity for the iteron with the open strands as wild type Rep, though it has a lower affinity for the inverted repeats than the wild type. The Rep dimer might bind to these DNA sequences with different modes.

Base Sequence↗
PubMed1996

Different cardiac myosin isoforms exhibit equal force-generating ability in vitro.

We measured forces generated by myosin molecules and a single actin filament using an optical trap system. The force per unit length of actin filament did not differ significantly between cardiac myosin isoforms. V1 and V3. This indicates that the ability to generate force is equal between V1 and V3, despite their difference in the unloaded sliding velocity past actin.

Actins↗
PubMed1996

Coupling between myosin ATPase cycle and creatinine kinase cycle facilitates cardiac actomyosin sliding in vitro. A clue to mechanical dysfunction during myocardial ischemia.

BACKGROUND: There is much evidence to support the favorable effects of the phosphocreatine shuttle on myocardial contraction and relaxation. However, experiments in which cardiac muscle fiber or myofibril was used have not elucidated its precise mechanism. METHODS AND RESULTS: Active movements of fluorescently labeled actin filaments on a cardiac myosin layer coimmobilized with creatinine kinase (CK) onto a nitrocellulose-coated glass coverslip were studied under various concentrations of adenine nucleotides. At a constant phosphocreatine concentration (5 mmol/L, pH 7.1), the relation of sliding velocity to MgATP concentration followed Michaelis-Menten kinetics. The apparent Km was significantly smaller in the presence of CK (0.041 +/- 0.001 mmol/L) than in the absence of CK (0.080 +/- 0.001 mmol/L), indicating that coattached CK facilitated the propelling of actin filaments by the myosin ATPase. This phenomenon was also seen under acidic conditions (pH 6.7) as well as in the presence of inorganic phosphate (10 mmol/L. At a constant MgATP concentration (1 mmol/L), the inhibitory effect of MgADP on the actin-myosin interaction was weaker in the presence of CK than in the absence of CK. Another ATP-regenerating system, pyruvate kinase and phospho(enol)pyruvate, while maintaining a low ratio of [MgADP] to [MgATP], did not reduce the Km value (0.156 +/- 0.001 mmol/L), suggesting that the effect of coattached CK was not achieved only by prevention of MgADP accumulation. CONCLUSIONS: Coupling between the ATPase cycle and the CK cycle may serve not only to maintain the ATP concentration within the myofibril but also to provide optimal conditions for cardiac actomyosin interaction. Consideration of this coupling will offer a clue to elucidating the systolic or diastolic dysfunction during myocardial ischemia or reperfusion.

Actomyosin↗
PubMed1995

Force-velocity relations of rat cardiac myosin isozymes sliding on algal cell actin cables in vitro.

The difference in kinetic properties between two myosin isozymes (V1 and V3) in rat ventricular myocardium was studied by determining the steady-state force-velocity (P-V) relations in the ATP-dependent movement of V1 and V3-coated polystyrene beads on actin cables of giant algal cells mounted on a centrifuge microscope. The maximum unloaded velocity of bead movement was larger for V1 than for V3. The velocity of bead movement decreased with increasing external load applied by the centrifuge microscope, and eventually reached zero when the load was equal to the maximum isometric force (P0) generated by the myosin heads. The maximum isometric force P0 was less than 10 pN, and did not differ significantly between V1 and V3. The P-V curves consisted of a hyperbolic part in the low force range and a non-hyperbolic part in the high force range. The critical force above which the curve deviated from the hyperbola was much smaller for V1 than for V3. An analysis using a model with an extremely small number of myosin heads involved in the bead movement suggested a marked difference in kinetic properties between V1 and V3.

Actins↗
PubMed1995

Pimobendan directly sensitizes reconstituted thin filament to slide on cardiac myosin.

To elucidate the mechanism of the Ca(2+)-sensitizing action of pimobendan, cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex and made to slide on a myosin layer. Although filaments showed Brownian movement with a low Ca2+ concentration, they slid at a constant velocity above a certain level of Ca2+ concentration, showing that the sliding was regulated by Ca2+ within a narrow pCa range. Acidosis, addition of inorganic phosphate, and phosphorylation of troponin I increased the threshold Ca2+ concentration. Addition of pimobendan reversed these desensitization effects. These results clearly demonstrated that pimobendan directly increases the Ca2+ sensitivity of thin filament.

Animals↗
PubMed1995

A new in vitro motility assay technique to evaluate calcium sensitivity of the cardiac contractile proteins.

We attempted to introduce calcium regulation into in vitro motility assay. Cardiac thin filament was reconstituted from actin and tropomyosin-troponin complex purified from rat myocardium separately. Double staining of the filaments showed tropomyosin-troponin complex was integrated along actin filaments homogeneously. The reconstituted thin filaments were made to slide on cardiac myosin fixed on a glass coverslip in the presence of MgATP while varying free Ca2+ concentration of the medium ([Ca2+]). Filaments showed only Brownian motion when [Ca2+] was below 10(-6.4) M. However, filaments slid at a constant velocity when [Ca2+] exceeded 10(-6.4) M, showing that the sliding was regulated in an on-off manner. The threshold [Ca2+] increased to 10(-5.0) M under acidic conditions, indicating a decrease in Ca2+ sensitivity of the contractile proteins. Simple actin filaments slid at a constant velocity independently of [Ca2+], demonstrating that the regulatory proteins were responsible for this on-off manner regulation. This new assay technique may be a powerful tool to directly evaluate the Ca2+ sensitivity of the contractile apparatus and to investigate how cardiac contraction is regulated by Ca2+.

Animals↗
PubMed1995

MCI-154 increases Ca2+ sensitivity of reconstituted thin filament. A study using a novel in vitro motility assay technique.

MCI-154 (6-[4-(4'-pyridylamino)phenyl]-4,5-dihydro-3(2H)pyridazinone hydrochloride trihydrate) is a potent novel cardiotonic agent whose positive inotropism is shown to be mainly based on an increase in Ca2+ sensitivity of the contractile apparatus. To elucidate the exact mechanism through which this drug acts, we investigated the movement of the reconstituted thin filament on a myosin layer in vitro. Cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex purified from rat cardiac acetone powder separately. Double staining of the filament showed that tropomyosin-troponin complex was integrated along actin filament homogeneously. Thin filaments thus prepared were fluorescently labeled and made to slide on rat cardiac myosin fixed on a glass coverslip while varying the [Ca2+] of the medium (control, pH 7.2 at 25 degrees C). When [Ca2+] was low, the filaments showed only brownian motion. However, above a certain level of [Ca2+] (the threshold [Ca2+]), the filaments started to slide, and the velocity increased, reaching the maximum velocity within a very narrow range of [Ca2+]. The regulation was completely abolished by using simple actin filaments without tropomyosin-troponin complex, demonstrating that the regulatory proteins are responsible for this Ca2+ regulation of the movement of the reconstituted thin filament. Under the control condition, addition of MCI-154 shifted the threshold [Ca2+] to a lower level (sensitization) in a concentration-related manner. And 10(-4) mol/L of MCI-154 reversed the desensitization effect induced by either acidosis (pH 6.8), low temperature (15 degrees C), or the addition of inorganic phosphate (10 mmol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1995

Immediate-early gene induction and MAP kinase activation during recovery from metabolic inhibition in cultured cardiac myocytes.

To investigate how cardiac myocytes recover from a brief period of ischemia, we used a metabolic inhibition (MI) model, one of the in vitro ischemic models, of chick embryo ventricular myocytes, and examined the induction of immediate-early (IE) genes mRNAs and the activity of mitogen-activated protein (MAP) kinase. We performed Northern blot analysis to study the expression of c-jun, c-fos, and c-myc mRNAs during MI using 1 mM NaCN and 20 mM 2-deoxy-d-glucose, and also during the recovery from MI of 30 min. The c-fos mRNA was induced transiently at 30 and 60 min during the recovery. The expression of c-jun mRNA was significantly augmented at 30, 60, 90, and 120 min during the recovery (3.0-, 4.7-, 2.4-, and 1.9-fold induction, respectively) and so did the expression of c-myc mRNA (1.4-, 1.7-, 1.8-, and 2.0-fold induction, respectively). In contrast, the levels of these mRNAs remained unchanged during MI. The electrophoretic mobility shift assay revealed that AP-1 DNA binding activity markedly increased at 120 min during the recovery. When the cells were pretreated with protein kinase C (PKC) inhibitors, 100 microM H-7 or 1 microM staurosporine, the induction of c-jun mRNA at 60 min during the recovery was markedly suppressed (95 or 82% reduction, respectively). The c-jun induction was partially inhibited when the cells were treated with 2 mM EGTA during MI and the recovery (42% reduction). MAP kinase activity quantified with in-gel kinase assay was unchanged during MI, but significantly increased at 5, 10, and 15 min during the recovery (3.0-, 4.1-, and 3.4-fold increase, respectively). S6 kinase activity was also augmented significantly at 15 min during the recovery. Thus, these data suggest that IE genes as well as MAP kinase may play roles in the recovery process of cardiac myocytes from MI, and that the augmentation of c-jun expression needs the activation of PKC and to some extent, [Ca2+]i.

Animals↗
PubMed1995

Effect of lateral forces on the movement of myosin-coated beads on actin cables studied using a centrifuge microscope.

We developed an in vitro motility assay system, in which myosin-coated polystyrene beads were made to slide on actin filament arrays (actin cables) in giant algal cells and subjected to centrifugal forces, which were parallel to the direction of bead movement to serve as external loads on actin-myosin sliding (Oiwa et al. (1990) Proc Natl Acad Sci USA 87: 7893-7897), and succeeded in determining the steady-state force-velocity relation of ATP-dependent actin-myosin sliding. To give further information about the properties of actin-myosin sliding, we have applied centrifugal forces, in parallel with the plane of actin-myosin sliding but at right angles with the direction of bead movement, and have found that such "lateral" centrifugal forces reduced the velocity of bead movement. In addition, we have also found that the velocity of bead movement is reduced more markedly with lateral forces applied from the left side of the bead ("left" lateral forces) than those applied from the right side of the bead ("right" lateral forces). These results are discussed in connection with the direction of sliding force generated by the myosin heads on the bead which interact with the right-handed double helix of actin monomers constituting actin filaments.

Actins↗
PubMed1995

The effects of hemodialysis (HD) membranes on interleukin 1-beta (IL-1 beta) production from peripheral blood mononuclear cells (PBMC).

Dialysis-related symptoms are thought to be mediated by monocyte/macrophage-derived inflammatory cytokines, including interleukin 1-beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha). We investigated the effect of hemodialysis (HD) membranes on IL-1 beta production using cultured peripheral blood mononuclear cells (PBMC). PBMC were stimulated with lipopolysaccharide (LPS) and the cell content and production of IL-1 beta were measured by ELISA. PBMC from a single healthy donor incubated with 5% of untreated plasma, and we found that HD patients plasma enhanced IL-1 beta production less than normal control plasma. The plasma obtained from the venous side of HD patients 15 minutes after starting a single HD (15-min HD plasma) did not enhance IL-1 beta production as much as the plasma from the arterial side, either before or upon completion of a single HD (pre-HD plasma, post-HD plasma). We studied IL-1 beta productivity when pre-HD plasma (arterial side) and 15-min HD plasma (venous side) were added to autologous PBMC. The IL-1 beta production by PBMC was less when the 15-min HD plasma was added to PBMC as compared to when the pre-HD plasma was added. This reduction tended to be greater when a dialyzer membrane used was a large-pore type made of regenerated cellulose (RC) or polymethylmetacrylate (PMMA) than with a small-pore RC membrane. After HD for 2 weeks using a small-pore RC membrane, the same HD patient was then treated with a large-pore RC membrane. The PBMC IL-1 beta production level decreased with the use of the large-pore RC as compared to the case with the small-pore RC membranes. The present study suggests that large-pore dialyzer membranes remove middle molecules and low molecular weight proteins which enhance IL-1 beta production, and that this production may be regulated by some mechanism unrelated to complement activation.

Biocompatible Materials↗
PubMed1994

ADP inhibits the sliding velocity of fluorescent actin filaments on cardiac and skeletal myosins.

We studied the effect of MgADP on the mechanical interaction of actomyosin in cardiac and skeletal muscles using an in vitro motility assay. The sliding velocities of fluorescently labeled actin filaments on rat cardiac and skeletal myosins were measured at various MgATP and MgADP concentrations. The filament velocity depended on MgATP concentration according to classic Michaelis-Menten kinetics with apparent Michaelis constants (Km) of 43 and 137 mumol/L and maximum velocity of 5.6 and 8.6 microns/s for cardiac and skeletal myosins, respectively. The presence of 2 mmol/L MgADP decreased the filament velocity and shifted the substrate concentration dependence of the velocity toward higher MgATP concentrations, yielding the inhibition constants of 194 and 478 mumol/L for cardiac and skeletal myosins, respectively. The activation energies determined by the temperature dependence of the velocity were 61 and 83 kJ/mol for rat V1 and rabbit cardiac myosins, which were similar to those of the dissociation rate constant of actomyosin-ADP complex reported in a solution study. The inhibition of the velocity by MgADP can be explained by the crossbridge scheme in which MgADP competes with MgATP for the substrate site on myosin molecules. In cardiac myosin, addition of a concentration of MgADP as low as 25 mumol/L significantly inhibited the velocity in the presence of 2 mmol/L MgATP, suggesting that increased intracellular MgADP may reduce the rate of crossbridge detachment, resulting in a decreased ATP consumption and an increased economy of force production under ischemic conditions. The present results support the idea that MgADP may be a physiologically important modulator of contraction in cardiac muscle.

Actins↗
PubMed1993

Depressed sliding velocity of isolated cardiac myosin from cardiomyopathic hamsters: evidence for an alteration in mechanical interaction of actomyosin.

We measured the relative sliding velocity of cardiomyopathic hamster cardiac myosin on actin cables by using an in vitro motility assay system. We also investigated the relationship between the velocity and both myosin isozyme content and ATPase activity. Cardiac myosin was obtained from cardiomyopathic hamsters (BIO 14.6; B) aged 3, 6, 9, and 18 months and age-matched controls (F1B; F). Long well-organized actin cables of an alga, Nitellopsis, were used for the motility assay. Small latex beads (2 microns in diameter) were coated with purified cardiac myosin. When myosin-coated beads were introduced into an algal cell in the presence of Mg-ATP, myosin interacted with actin and dragged the beads. Active movement of the beads along the actin cables was observed under a photomicroscope and the velocity was measured. The velocity was significantly lower in B than in F for each age group (0.47 vs. 0.71 microns/s at the age of 3 months, p < 0.05; 0.44 vs. 0.88 microns/s at 6 months, p < 0.01; 0.44 vs. 0.67 microns/s at 9 months, p < 0.01; 0.35 vs. 0.52 microns/s at 18 months, p < 0.05). Both Ca(2+)-activated ATPase activity and the percentage of alpha-myosin heavy chain were also lower in B than in F for each age group. When examined for individual specimens, there was a positive correlation between the velocity and both myosin Ca(2+)-activated ATPase activity (r = 0.84) and percentage of alpha-myosin heavy chain (r = 0.83). These data points of both control and cardiomyopathic hamsters were distributed near the regression line obtained from control and thyroxine-treated rabbits reported previously. The present results indicate that the difference in mechanical properties between control and cardiomyopathic cardiac myosin is attributed to isozyme redistribution and not to a qualitative change in each myosin molecule.

Actomyosin↗
PubMed1993

Thermodynamic and kinetic analyses of DNA triplex formation: application of filter-binding assay.

We have developed a simple and efficient method for studying equilibrium thermodynamics and kinetics of DNA triplex formation, which utilizes a filter-binding procedure. The application of this method to the triplex formation between a double-stranded homopurine-homopyrimidine and a single-stranded homopyrimidine oligonucleotides has demonstrated its ability in the quantitative estimation of equilibrium binding constants and rate constants under various conditions. Thus, this simple method can serve as a powerful tool for the systematic analysis of sequence and environmental effects on the equilibrium and kinetic quantities in the triplex formation.

Base Sequence↗
PubMed1993

Analysis of cardiac assistance by latissimus dorsi cardiomyoplasty with a time varying elastance model.

OBJECTIVE: The clinical use of skeletal muscle cardiomyoplasty is limited because of its inadequate haemodynamic benefits. To facilitate experimental and clinical efforts to improve the efficacy of this technique, a mathematical model was proposed and its validity was tested in acute experiments. METHODS: The model was based on the assumption that the skeletal muscle wrapped around the heart behaves as a time varying elastance that is connected in series with another time varying elastance representing the native heart. From this model two predictions were made: (1) Skeletal muscle augments the contractility of the heart by increasing the slope (Ees) of the end systolic pressure-volume relation; (2) time varying elastance of the skeletal muscle chamber (Es(t)) can be estimated from that of the assisted heart. These predictions were examined in experiments. In nine anaesthetised, open chest dogs, preconditioned latissimus dorsi muscle was transposed to wrap the heart. Left ventricular pressure (catheter tipped micromanometer), and volume (conductance catheter) were measured while reducing the preload by vena caval occlusion to evaluate Ees with 1:2 (stimulation:heart beat ratio) stimulation of the skeletal muscle. RESULTS: With the stimulation of latissimus muscle, the end systolic pressure-volume relation was linear and Ees increased from 8.6(SEM 2.4) to 11.9(SEM 3.4) mm Hg.ml-1. Estimated Es(t) reflected the stimulation pattern and could account for the mechanism of the cardiac assistance. CONCLUSIONS: Skeletal muscle cardiomyoplasty improved the haemodynamic variable (Ees) as predicted by a mathematical model.

Animals↗
PubMed1993

A pseudo-symmetric DNA binding motif of purified Rep dimer of plasmid pSC101.

The Rep protein, a replication initiator of plasmid pSC101, also functions as an autorepressor for its own structure gene, rep, through binding to the operator which consists of imperfectly dyad-symmetric (pseudo-symmetric) sequences, IR-1 and IR-2. In order to define the DNA binding motif of Rep, we have analyzed its binding affinity for each half repeat of the IR sequences and found that the right half of IR-2 was the preferred sequence for Rep, although its affinity was much lower than those of the IR sequences. Next, dimeric sequences of each half repeat with different configurations, head to head (the same configuration as natural IRs), head to tail, and tail to tail, were constructed and their affinities for Rep were examined. Almost all of sequences with the head-to-head configuration had binding affinity for Rep but these, including even the symmetric sequence of the right half of IR-2, exhibited lower Rep-binding abilities than natural IR sequences. We presume a pseudo-symmetric sequence, 5'GGNNTAGNNATTNNNATNN(N)CTAGNCC3', to be the Rep-binding motif from a sequence comparison. Some single-base substitution experiments confirmed the motif and suggested that each subunit of the Rep dimer recognizes each half repeat of IR asymmetrically.

Bacterial Proteins↗
PubMed1993

Minimal essential origin of plasmid pSC101 replication: requirement of a region downstream of iterons.

The minimal replication origin (ori) of the plasmid pSC101 was defined as an about 220-bp region under the condition that the Rep (or RepA) protein, a plasmid-encoded initiator protein, was supplied in trans. The DnaA box is located at one end of ori, as in other plasmids, like mini-F and P1. The other border is a strong binding site (IR-1) of Rep which is palindromic sequence and lies in an about 50-bp region beyond the repeated sequences (iterons) in ori. This IR-1 is located just upstream of another strong Rep binding site (IR-2), the operator site of the structure gene of Rep (rep), but its function has not been determined. The present study shows that the IR-1 sequence capable of binding to Rep is essential for plasmid replication with a nearly normal copy number. Furthermore, a region between the third iteron and IR-1 is also required in a sequence-specific fashion, since some one-base substitution in this region inactivate the origin function. It is likely that the region also is a recognition site of an unknown protein. Three copy number mutations of rep can suppress any one-base substitution mutation. On the other hand, the sequence of a spacer region between the second and the third iterons, which is similar to that of the downstream region of the third iteron, can be changed without loss of the origin function. The requirement of the region downstream of iterons in pSC101 seems to be unique among iteron-driven plasmid replicons.

Bacterial Proteins↗
PubMed1993

Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.

Amino Acid Sequence↗