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H Higuchi

Publications and source records attributed to H Higuchi.

At least 199 records · Page 11Linked to original sources
PubMed1994

Single-molecule analysis of the actomyosin motor using nano-manipulation.

The elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules using a recently developed technique for nano-manipulation of single actin filaments by a microneedle. In order to avoid the effects of random orientation of myosin and association of myosin with an artificial substrate in the surface motility assay, we used single myosin-rod cofilaments with various ratios. Distinct actomyosin attachment, force generation (the power stroke) and detachment events were detected at a very low myosin: rod ratio. At high load, one power stroke generated 5-6 pN peak force and 2.3 pN force averaged over the cycle, which were compatible with those deduced from noise analysis of force fluctuations caused by multiple molecules. As the load was reduced, the length of the power stroke increased. At near zero load, the length of a power stroke was approximately 17 nm. The results suggested that an ATPase cycle produces one power stroke at high load and many ones at low load.

Actins↗
PubMed1994

Urine concentrating ability after prolonged sevoflurane anaesthesia.

Few studies have used the vasopressin test to evaluate urine concentrating ability after sevoflurane anaesthesia. We performed a vasopressin test on the first day after operation to compare the effect of prolonged sevoflurane anaesthesia for orthopaedic procedures (n = 11) with that of isoflurane (n = 10). Mean doses of sevoflurane and isoflurane were 10.6 (SE 0.9) and 8.5 (1.5) MAC-h, respectively. Mean peak serum fluoride concentration in patients anaesthetized with sevoflurane was 41.9 (2.5 mumol litre-1 and exceeded 20 mumol litre-1 for approximately 20 h. Each group showed similar responses to vasopressin. There was no evidence of subclinical nephrotoxicity in patients given prolonged sevoflurane anaesthesia.

Adult↗
PubMed1994

High levels of expression of neuropeptide Y mRNA in human phaeochromocytomas.

1. Neuropeptide Y (NPY) gene expression in human phaeochromocytomas was investigated by measuring the levels of NPY mRNA and NPY-immunoreactivity (NPY-IR) in human phaeochromocytoma tissues in comparison with those in normal human adrenal tissues. 2. The amounts of NPY mRNA and NPY-IR in human phaeochromocytomas were 18 and 93 times higher, respectively, than those in normal adrenal glands. In contrast, beta-actin gene expression was similar in human phaeochromocytomas to that in normal adrenal glands. 3. The amount of NPY mRNA relative to total cellular RNA was 6-fold higher in phaeochromocytoma tissues than in normal human adrenal medulla, suggesting increased NPY gene expression in the tumour cells. 4. Induction of differentiation of PC12 rat phaeochromocytoma cells by compounds, such as dexamethasone and nerve growth factor, resulted in a marked increase in the NPY mRNA level. 5. These findings suggest that NPY gene expression is increased in well-differentiated human phaeochromocytoma cells. Its high level of expression could be responsible for the marked overproduction of NPY by this tumour.

Actins↗
PubMed1994

Growth hormone induces expression of the c-fos gene on hypothalamic neuropeptide-Y and somatostatin neurons in hypophysectomized rats.

The neuronal expression of the protooncogene c-fos may serve as a marker of neural activity. We previously examined brain sites upon which GH exerts an immediate early influence in rats and determined that the c-fos gene was transiently expressed in the hypothalamic periventricular nucleus (PeV) and arcuate nucleus (ARC) after recombinant human GH (rhGH) administration. As the distribution of c-fos messenger RNA (mRNA)-containing cells appeared to overlap with that of somatostatin (SS) neurons in both the PeV and ARC, we hypothesized that GH exerts a feedback effect on hypothalamic SS neurons. To extend this hypothesis, we characterized the neurons expressing the c-fos gene in response to rhGH administration in hypophysectomized rats. Adult male Wistar rats were hypophysectomized 10 days before use. After hypophysectomy, rats received daily sc injections of cortisone acetate (0.5 mg/kg BW) and L-T4 (20 micrograms/kg BW). Four international units (1.33 mg) of rhGH were given iv through an indwelling right atrial cannula. The vehicle was given to the control animals. Coronal sections of the hypothalamus were processed for in situ hybridization after rhGH or vehicle administration. To estimate the localization of neurons expressing the c-fos gene, the adjacent hypothalamic sections, 30 microns in thickness, were processed for hybridization histochemistry for SS, neuropeptide-Y (NPY), or GRF mRNA. In the ARC, the distribution of c-fos mRNA-containing cells appeared to overlap with that of NPY and partially with that of SS mRNA-containing cells, but it clearly differed from the distribution of GRF mRNA-containing cells. In the PeV, distribution of the cells expressing the c-fos gene was comparable to that of SS mRNA-containing cells. To further ascertain the distribution, hypothalamic sections, 6 microns in thickness, were processed by double label in situ hybridization using a 35S-labeled c-fos cRNA probe and a digoxigenin-labeled NPY or SS cRNA probe. In the ARC, 65% of the c-fos gene-expressing cells were NPY neurons. In the PeV, 60% of the c-fos gene-expressing cells were SS neurons. NPY is known to act within the hypothalamus and inhibit GH secretion via SS in rats, and the NPY neurons in the ARC have been shown to project to SS neurons in the PeV. Our findings suggest that the feedback effect of GH on the hypothalamus is mediated not only by SS neurons in the PeV, but also by NPY neurons in the ARC.

Animals↗
PubMed1994

Dexamethasone-induced haptoglobin release by calf liver parenchymal cells.

Parenchymal cells were isolated from the liver of male calves, and monolayer cultures formed were treated with glucocorticoids to examine whether haptoglobin, appearance of which is associated with hepatic lipidosis (fatty liver) in cattle, is induced by steroid hormones. Without addition of dexamethasone, only trace amounts of haptoglobin were detected in culture medium. With addition of dexamethasone (10(-12) to 10(-4) M), considerable amounts of haptoglobin were released into the medium. Maximal release was observed at concentrations of 10(-8) to 10(-6) M dexamethasone. Haptoglobin release was similarly induced by cortisol, although the effect was less potent than that of dexamethasone. Actinomycin D (a known protein synthesis inhibitor) dose-dependently reduced amounts of haptoglobin released in response to 10(-8) M dexamethasone. Dexamethasone also induced annexin I, which is known to be synthesized in response to glucocorticoids. Dexamethasone treatment resulted in reduced protein kinase C activity in the cell cytosol, which has been shown to be an early event in dexamethasone-treated cells. Other than glucocorticoids, estradiol induced haptoglobin release, whereas progesterone was less effective. The association of haptoglobin with hepatic lipidosis can be reasonably explained by the fact that haptoglobin production by the liver is induced by glucocorticoids and estradiol, and these steroid hormones are triggers for development of hepatic lipidosis in cattle.

Animals↗
PubMed1993

Connectin, giant elastic protein, in giant sarcomeres of crayfish claw muscle.

In the giant sarcomeres (sarcomere length, 10 microns at rest) of crayfish claw muscle, 3000 kDa connectin-like protein but not projectin (mini-titin) appears to be responsible for passive tension generation. Proteolysis of crayfish connectin in skinned fibres was parallel with disappearance of resting tension. Immunofluorescence observations using the antiserum to crayfish connectin showed that crayfish connectin linked the A band to the Z line in a giant sarcomere. It appears that crayfish connectin exerts a centering force on the A band in a sarcomere. Very thin filaments in the I band were visualized after the actin filaments had been removed by the treatment with plasma gelsolin. Crayfish connectin was partially purified and its rotary shadowed image was a very long filament. Projectin was localized on the A band of crayfish giant sarcomeres and remained unmoved during stretch or contraction. However, on dissolution of myosin filaments, projectin moved to the Z line together with crayfish connectin. It seems that projectin binds to connectin on the myosin filament. In regular size of sarcomeres (sarcomere lengths, 3-4 microns at rest) of crayfish stretcher muscle, projectin linked the A band to the Z line, as in insect flight muscle.

Animals↗
PubMed1993

Decrease in prepro-neuropeptide Y gene expression in the adrenal gland and cerebral cortex of spontaneously hypertensive rats.

Changes in the mRNA and peptide levels of neuropeptide Y (NPY) in the adrenal gland and brain areas of spontaneously hypertensive rats (SHR) and of age-matched normotensive rats were investigated. The level of adrenal NPY mRNA of SHR decreased during progression of hypertension and was lower than that of Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats in the early stage of hypertension, while the level of adrenal NPY was unchanged or even higher than that of control rats. These findings suggest that both the biosynthesis and release of vasoconstrictive NPY peptide in the adrenal glands of SHR might decrease during progression of hypertension. In contrast, the NPY mRNA level neither decreased remarkably until week 17 in the cerebral cortex of SHR, nor changed at all in the other brain areas. This late decrease in cerebral NPY gene expression might be related to the disturbance in behavior and memory of SHR.

Adrenal Glands↗
PubMed1993

Characterization of beta-connectin (titin 2) from striated muscle by dynamic light scattering.

Connectin (titin) is a large filamentous protein (single peptide) with a molecular mass of approximately 3 MDa, contour length approximately 900 nm, and diameter approximately 4 nm, and resides in striated muscle. Connectin links the thick filaments to the Z-lines in a sarcomere and produces a passive elastic force when muscle fiber is stretched. The aim of this study is to elucidate some aspects of physical properties of isolated beta-connectin (titin 2), a proteolytic fragment of connectin, by means of dynamic light-scattering (DLS) spectroscopy. The analysis of DLS spectra for beta-connectin gave the translational diffusion coefficient of 3.60 x 10(-8) cm2/s at 10 degrees C (or the hydrodynamic radius of 44.1 nm), molecular mass little smaller than 3.0 MDa (for a literature value of sedimentation coefficient), the root-mean-square end-to-end distance of 163 nm (or the radius of gyration of 66.6 nm), and the Kuhn segment number of 30 and segment length of 30 nm (or the persistence length of 15 nm). These results permitted to estimate the flexural rigidity of 6.0 x 10(-20) dyn x cm2 for filament bending, and the elastic constant of 7 dyn/cm for extension of one persistence length. Based on a simple model, implications of the present results in muscle physiology are discussed.

Animals↗
PubMed1993

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments.

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.

Actin Cytoskeleton↗
PubMed1993

N-acetoxy-N-acetyl-2-aminofluorene-induced mutation spectrum in a human hprt cDNA shuttle vector integrated into mammalian cells.

The spectrum of mutations induced by N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) was examined by the pZipHprtNeo shuttle vector in mammalian cells. The vector carries a cDNA of the human hypoxanthine phosphoribosyl transferase (hprt) gene, which is stably integrated into chromosomal DNA of a mouse cell line, VH12. After treatment of the cell with N-AcO-AAF, 48 independent 6-thioguanine-resistant clones were obtained and altered sequences of the mutated cDNA hprt genes were determined. Frameshifts and deletions were the predominant mutational events (68%) induced by N-AcO-AAF and the remainder were base substitutions (32%) of various types. Analysis of sequence alterations at all the sites of mutation revealed that: (i) > 65% of mutations occurred at G:C sites, suggesting C8G adducts are responsible premutagenic lesions for these mutations; and (ii) short sequence repeats were frequently found at the sites of frameshift and deletion, and slippage--misalignment is the suggested mechanism for the induction of mutations at these sites. Implied significance of slippage--misalignment as a fundamental mechanism for mutagenesis is discussed.

3T3 Cells↗
PubMed1993

Serum inorganic fluoride levels in mildly obese patients during and after sevoflurane anesthesia.

Serum inorganic fluoride levels in obese versus control patients were compared during and after sevoflurane anesthesia. Mean serum inorganic fluoride levels in the obese group increased more rapidly and were significantly higher than in the control group at each sampling time (P < 0.01). The area under the curve of fluoride concentration, versus time up to 24 h and 48 h in the obese patients, was significantly greater than that in the nonobese patients (P < 0.001). Peak serum fluoride level in the obese patients was 51.7 +/- 2.5 mumol/L and exceeded 50 mumol/L for nearly 2 h. Our study showed that serum fluoride concentrations between mildly obese and nonobese patients differed during and after sevoflurane anesthesia.

Adult↗
PubMed1993

[Computerized recording system during cardiopulmonary bypass utilizing the "GEM 6 PLUS analyzer"].

We developed a new computerized recording system to reduce artificial errors by perfusionists during cardiopulmonary bypass. This system included the efficient functions to record the progress of perfusion flow, perfusion pressure, urine output and temperatures from several sources. Furthermore, it could automatically record considerable data of the "GEM 6 PLUS analyzer" which was developed for monitoring of pH, PCO2, PO2, BE, Ca2+, K+ and hematocrit during cardiopulmonary bypass. We considered it very important that the errors of hematocrits associated with electrolytic concentrations. Therefore, we corrected the hematocrit according to a formula based on our experiments, i.e., HCT = GEMHCT + 0.28 x ([Na+]+[K+]) -44. And also we expect that the "GEM 6 PLUS analyzer" will be improved to measure Na+ value in near future.

Body Temperature↗
PubMed1993

Sequence analysis of X-ray-induced mutations occurring in a cDNA of the human hprt gene integrated into mammalian chromosomal DNA.

Sequences of 38 independent X-ray-induced mutations in a cDNA of the human hypoxanthine phosphoribosyltransferase (hprt) gene were analyzed after it was integrated into mammalian chromosomes as part of a shuttle vector. There were 15 base substitutions (39%), 9 frameshifts (24%), 11 deletions (29%), and 3 other mutations (8%) of two kinds. Radiation-induced mutations were found throughout the coding region of the gene. Base substitution mutations occurred more often at A:T sites than at G:C sites, and there were more transversions than transitions. Most of the frameshifts and deletions had short direct repeats or a run of several identical bases at the site of the mutation. A slippage misalignment mechanism, suggested previously, can account for the generation of these classes of mutations. The kinds, distribution, and possible mechanism of X-ray-induced mutations were similar to those of spontaneous mutations.

Animals↗
PubMed1992

Identification of NGF-response element in the rat neuropeptide Y gene and induction of the binding proteins.

Gene expression of the rat neuropeptide Y (NPY) increases by 100 times, as the PC12 cells differentiate into sympathetic neuron-like cells with NGF treatment and this increase is partly due to transcriptional activation of the NPY gene (Sabol and Higuchi, Mol. Endocrinol. 4, 384, 1990). To identify the NGF-response element, a transient expression assay was carried out by using the CAT reporter genes containing various lengths of the 5' upstream region of the NPY gene in the PC12 cells. The 48-base element (-80/-33 upstream of the Cap site) was identified as a NGF-response element (NGFRE). Gel shift assay indicated the existence of at least two DNA-binding proteins to NGFRE. The binding activity of the protein(s) (NDF1) to the upper region (-80/-63) was increased by 3-fold with NGF treatment for 24 h. These findings suggest that these nuclear proteins are involved in the enhanced transcription of the NPY gene by NGF.

Animals↗
PubMed1992

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments.

The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 microns remained at about 0.8 microns from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 microns. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 microns of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.

Actin Cytoskeleton↗
PubMed1992

G-protein mediating the slow depolarization induced by FMRFamide in the ganglion cells of Aplysia.

Application of FMRFamide (Phe-Met-Arg-Phe-NH2) induced a slow depolarization in neurons of the Aplysia abdominal ganglion. In voltage-clamped cells, FMRFamide induced a slow inward current that increased when the membrane was depolarized beyond -85 mV, showing a negative slope conductance. However, this inward current never reversed to outward current when hyperpolarized beyond the equilibrium potential for K+. The FMRFamide-induced response was markedly augmented in Ca(2+)-free media, but depressed in Na(+)-free media. It was unaffected by a change in external potassium. Intracellular injection of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) significantly depressed the FMRFamide response in a dose-dependent way. Injection of cholera toxin (CTX) which did not cause any current response, selectively and irreversibly blocked the FMRFamide response. Neither 3'-deoxyadenosine, an inhibitor of adenylate cyclase, nor H-8, an inhibitor of cyclic adenosine 3',5'-monophosphate (cyclic AMP)-dependent kinase, depressed the FMRFamide response. 3-Isobutyl-1-methylxanthine (IBMX) did not augment the FMRFamide response appreciably. The FMRFamide response was not occluded at all by a relatively large injection of 8-bromo-cyclic AMP. It was concluded that the FMRFamide response is produced by the opening of the voltage-dependent Na(+)-channels via activation of a certain CTX-sensitive G-protein which is different from conventional "Gs" that activates adenylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Identification of glucocorticoid responsive elements (GREs) at far upstream of rat NPY gene.

The location of three glucocorticoid responsive elements (GREs) in rat neuropeptide Y (NPY) gene was determined by chloramphenicol acetyltransferase (CAT) assay and nucleotide sequencing. We have reported that mRNA content of rat prepro-NPY is increased by 1.7-fold in NG108-15 cells by 1 microM dexamethasone, suggesting the presence of GRE in the gene. To identify the element, the 5'-flanking DNA of 3.3 kilobases (kb) was isolated from rat NPY gene. When chimeric chloramphenicol CAT plasmids containing various deletions of the NPY upstream sequence were transfected into NG108-15 cells, the region between -2.9 and -2.1 kb relative to the cap site was found to potentiate the transcription of CAT gene in the presence of 1 microM dexamethasone. The nucleotide sequencing of this region revealed three GRE consensus sequences at -2.5, -2.2 and -2.1 kb. The results indicate that these elements present in the far upstream region of the NPY gene confer induction by glucocorticoids.

Animals↗