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H Higuchi

Publications and source records attributed to H Higuchi.

At least 253 records · Page 14Linked to original sources
PubMed1987

Antigenic heterogeneity of carcinoembryonic antigen in the circulation defined by monoclonal antibodies against the carbohydrate moiety of carcinoembryonic antigen and closely related antigens.

Six mouse monoclonal antibodies reactive with carcinoembryonic antigen (CEA) were prepared and used for the analysis of the antigenic heterogeneity of CEA in patient sera. Their reaction specificity and the chemical nature of antigenic epitopes recognized by them were analyzed by radioimmunoassay on the basis of reactivities with different preparations of CEA, normal fecal antigen 2, and nonspecific cross-reacting antigen 2 before and after chemical and/or enzymatic treatment. Two antibodies, F3-30 and F4-82, raised with CEA were reactive with different peptide epitopes on the antigen molecules and revealed a quite universal reactivity with all CEA, normal fecal antigen 2, or nonspecific cross-reacting antigen 2 preparations tested. The serum CEA values obtained with these antibodies were highly correlated with those obtained with conventional radioimmunoassays for CEA. The other four antibodies (F4-11 and F33-37 raised with CEA, F8-52 with normal fecal antigen 2, and F48-60 with nonspecific cross-reacting antigen 2) were found to recognize carbohydrate epitopes with different specificities and revealed very heterogeneous reactivities. The serum CEA values estimated with these four antibodies were highly variable depending on the antibody used, suggesting that the expression of carbohydrate epitopes on the CEA molecules in patient sera was quite heterogeneous. The antigenic heterogeneity of the carbohydrate epitopes was detected even in a single patient serum by affinity chromatography. The causes that give rise to the difference in CEA values between the Roche and the Daiichi kits were analyzed on the basis of reactivities of three groups of patient sera, which showed extremely different ratios for the Roche and Daiichi kits, with monoclonal anti-carbohydrate antibodies. The results obtained suggest that, at least in part, the diversity of antigenic expression on carbohydrate chains on the CEA molecules in patient sera and the variation in specificity or quantity of anti-carbohydrate antibodies in the polyclonal antibody preparations used for the respective assay systems may result in the differences in the estimated CEA values.

Antibodies, Monoclonal↗
PubMed1987

Lattice swelling with the selective digestion of elastic components in single-skinned fibers of frog muscle.

Changes in the 1.0 lattice spacing during trypsin (0.25 micrograms/ml) treatment in mechanically skinned single fibers of frog muscle was examined by an x-ray diffraction method at various sarcomere lengths. The resting tension of a relaxed fiber was decreased by trypsin treatment but the stiffness of a rigor fiber was not, suggesting that elastic components were selectively digested. With progression of the digestion, the lattice spacing increased remarkably at longer sarcomere lengths and finally became independent of the sarcomere length. The increase in the lattice spacing was proportional to the decrease in the resting tension. These results support our previous suggestion (Higuchi, H., and Y. Umazume, 1986, Biophys. J., 50:385-389) that the lattice spacing decreases at long lengths due to compressive force exerted by a lateral elastic component that connects thick filaments to an axial elastic component. Consequently, it is unlikely that the decrease in the lattice spacing is determined by a decrease in the repulsive force between thick and thin filaments with stretching a fiber.

Animals↗
PubMed1987

Elastic properties and beta-sheet structure of connectin threads.

Connectin is a very long and flexible protein of striated muscle, linking myosin filaments to z discs in a sarcomere. Isolated native connectin in solution frequently forms elastic threads upon concentration of the solution, by side-by-side association of molecules. An X-ray diffraction study was performed to examine the presence of beta-sheet structure in artificially prepared threads. The elastic properties of such threads were measured at various temperatures. Negative temperature dependence of the elastic coefficient suggests that the elasticity of connectin threads is due to deformation of the three-dimensional structure and not to rubber-like behavior.

Animals↗
PubMed1987

Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system.

A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radioimmunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels.

Antibodies, Monoclonal↗
PubMed1986

CD spectra and some properties of deoxyoligonucleotide duplexes having a C:G terminus (nucleosides and nucleotides. Part 69).

In the course of an investigation of the mode of recognition of nucleotide sequences with restriction endonucleases, several deoxyoligonucleotide duplexes having G:C terminal base pairs were synthesized. The oligonucleotides having a 5'-C (3'-G) terminus showed unusual CD spectra with a negative band at the longer wavelength region, when compared to those of the same internal sequences but a 5'-G (3'-C) terminus, which showed a positive band like the B- or A-DNA type. The nature of these CD spectra was compared with those of the Z-DNAs on the effect of salt concentrations, intercalation with ethidium bromide, or 31P-NMR spectra. These unusual spectra may be attributed to the terminal effect of the 5'-C:3'-G pairs.

Base Sequence↗
PubMed1986

Width and lattice spacing in radially compressed frog skinned muscle fibres at various pH values, magnesium ion concentrations and ionic strengths.

The width (D) and the 1,0 lattice spacing (d1,0) at various ionic compositions of mechanically skinned single fibres (from semitendinosus muscle of Rana catesbeiana) were measured at various concentrations of polyvinyl pyrrolidone (PVP K-30, Mn = 40 000) from 0 to 6% at 20 degrees C. In a standard relaxing solution (4 mM MgATP2-, 1 mM Mg2+, 4 mM EGTA, ionic strength 150 mM and pH 7), d1,0 decreased exponentially as the PVP concentration increased: d1,0 was 41.3 +/- 0.4 (mean +/- S.D.) nm at 0% PVP and 32.9 +/- 0.4 nm at 6% PVP. D was proportional to d1,0 except at very low PVP concentrations, i.e. at 1% PVP, D decreased by 7%, whereas d1,0 decreased by only 3%. At 0% PVP, D and d1,0 decreased when either pH or ionic strength (gamma/2) was lowered. At 6% PVP, D and d1,0 decreased with lowered pH or increased [Mg2+], but was independent of gamma/2. The radial stiffness, or degree of resistance to the changes of D against the compressing force, increased considerably at d1,0 less than or equal to 35 nm in a standard relaxing solution, but not at pH 5.5 or 30 mM [Mg2+]. These effects of pH, [Mg2+] and gamma/2 on D or d1,0 and on the radial stiffness can be explained by the modification of the properties of the elastic element and the hinge between subfragment-1 and -2 and/or the hinge between subfragment-2 and light meromyosin.

Animals↗
PubMed1986

Accumulated strain mechanism for length determination of thick filaments in skeletal muscle. I. Experimental bases.

The kinetics of dissociation of myosin from both ends of thick filaments in glycerinated skeletal muscle fibres and myofibrils was studied in the presence of MgATP by use of an optical diffraction method and phase-contrast microscopy. The dissociation velocity, v (identical to -dL/dt where L is the length of thick filaments at time t), increased with increasing KCl concentration (0.225 to 0.5 M), or increasing pH (6.5 to 8.0) but hardly changed with temperature (5 and 25 degrees C), micromolar concentrations of Ca2+ or sarcomere length (2.4 and 2.75 micron). Over a wide range of filament length, the dissociation velocity could be expressed by v0exp(alpha L), where v0 and alpha are positive constants depending upon the dissociation condition. When the effects of crossbridge formation are minimized it was thus shown that the structural stability of thick filaments in a muscle fibre and a myofibril gradually decreases from the central part to the tips of the filaments. On the basis of these results we propose that the length of thick filaments is largely regulated by an accumulated strain mechanism in which the free energy of association of myosin molecules increases with filament length.

Animals↗
PubMed1986

Lattice shrinkage with increasing resting tension in stretched, single skinned fibers of frog muscle.

The 1,0 lattice spacing d1,0 in chemically and mechanically skinned single fibers of frog muscle was measured at various sarcomere lengths, L, in the range from L = 2.1 to 6.0 microns by an x-ray diffraction method. In chemically skinned fibers, d1,0 decreased with a similar slope to that of mechanically skinned fibers up to L congruent to 3 microns, but beyond this point d1,0 steeply decreased with further stretching. This steep decrease in d1,0 could be ascribed mainly to an increase in the compressing force associated with the longitudinal extension of a remnant of the sarcolemma. In mechanically skinned fibers, the gradual decrease in d1,0 continued beyond filament overlap (L greater than or equal to 3.5 microns) and was highly proportional to a resting tension. This decrease in d1,0 at L greater than or equal to 3.5 microns could be ascribed to an increase in the force exerted by lateral elastic components, which is proportional to the longitudinal resting tension. A conceptual model is proposed of a network structure of elastic components in a sarcomere.

Animals↗
PubMed1986

Splanchnic nerve transection abolishes the age-dependent increase of neuropeptide Y-like immunoreactivity in rat adrenal gland.

Using an antiserum directed against porcine neuropeptide Y (NPY), a high concentration of NPY immunoreactivity (NPY-IR) was detected in rat adrenal gland. The level of NPY-IR in the adrenal gland was found to increase considerably with age. Biochemical characterization by reverse-phase HPLC indicated that this increase was due to accumulations of NPY and another molecular form of NPY-like immunoreactive peptide. Chronic denervation of the splanchnic nerve abolished this age-dependent increase of NPY-IR rat adrenal gland.

Adrenal Glands↗
PubMed1985

Multiple agonist binding sites of muscarinic acetylcholine receptors and their relation to the negative inotropic action of agonists in guinea-pig heart.

The Kd values of the multiple agonist binding sites in cardiac muscarinic receptors (mAChR) and pD2 values for negative inotropic actions were determined independently and their relation was examined. The guinea-pig cardiac mAChR is known to have three agonist binding sites (super-high (SH), high (H) and low (L) affinity agonist binding sites) for carbachol (CCh). Pilocarpine (Pilo) and oxotremorine (Oxo) distinguished two sites (higher (Ho/p) with pKd of 5.88 and 8.20, respectively, and lower (Lo/p) affinity agonist binding sites with pKd of 5.08 and 6.17, respectively). The effects of guanine nucleotide and sulfhydryl reagent indicated that the Ho/p site corresponded with the SH site for carbachol, and the Lo/p site with the H + L sites for carbachol. The pD2 values of CCh, Pilo and Oxo for negative inotropic actions on autocontraction of right atria were 5.38, 5.30 and 6.80, respectively. The pD2 values of CCh and Oxo on electrically stimulated contraction of left atria in the presence of isoproterenol were 5.80 and 6.46, respectively, thus being closely related to H or Lo/p agonist binding sites of mAChR.

Animals↗
PubMed1985

Discrepancy between alpha 1-adrenoceptor-mediated contraction and the occupation theory in rat vas deferens--possible existence of a 'silent' receptor.

The relation of the amount of alpha 1-adrenoceptors (alpha 1-R) with contraction to norepinephrine (NE) through alpha 1-R in rat vas deferens was examined by means of radiobinding assays. Treatment with dibenamine decreased the maximal contraction to NE with a decrease in the amount of alpha 1-R but the relation was not linear. The contractile response disappeared completely when 20% of the alpha 1-R still remained. Moreover, culture of dibenamine-pretreated muscle restored the contraction to NE without a significant increase in the amount of alpha 1-R in the muscle. These findings suggest that some alpha 1-R are 'silent' in the contraction of rat vas deferens in response to NE under physiological conditions.

Animals↗
PubMed1985

Recovery of the muscarinic cholinergic receptor from its down-regulation in cultured smooth muscle.

The recovery of the muscarinic cholinergic receptor (mAChR) from its down-regulation by long-term exposure to ACh was investigated. This was done to obtain information about regulation of the mAChR. Exposure of guinea-pig vas deferens to 30 microM ACh for 24 h decreased the amount of mAChR to 30% of the initial level, as measured with L-[3H]quinuclidinyl benzilate (QNB). The amount of mAChR was restored to 190% of its prewithdrawal level within 48 h of removal of ACh, without change in the KD value for L-[3H]QNB. This restoration was entirely dependent on protein synthesis. The half-life of the receptor was calculated to be 69 h. The recovery of mAChR was blocked by treatment with antimicrotubular agents, carboxylic ionophores, or 5 mM EGTA, which affect membrane protein synthesis. However, cytochalasin B and cyclic nucleotide derivatives had no effect. These data indicate that the recovery of mAChR was due to new synthesis of mAChR. The findings suggested that microtubules and the Golgi apparatus were involved in the biosynthesis of mAChR and that extracellular Ca2+ was necessary for the synthesis. Unlike the case with nicotinic AChR, the synthesis did not seem to be increased by cAMP or high extracellular Ca2+.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Localization of the parallel elastic components in frog skinned muscle fibers studied by the dissociation of the A- and I-bands.

Localization of the parallel elastic components (PECs) in skinned muscle fibers was investigated by analyzing the change of the resting tension, which accompanies the dissociation of the A- and I-bands. The A-band was dissociated from both ends by increasing the concentration of KCl under relaxing conditions (0.09-0.54 M KCl, 4.0 mM MgATP, 1.0 mM Mg2+, 4.0 mM EGTA, pH 6.0-9.0, 20 degrees C). At sarcomere lengths greater than or equal to 3.5 microns, the length of the A-band was estimated by comparing the intensity of the first-order optical diffraction line with the results of model calculations. These results were supported by differential-interference microscopy and sodium dodecyl sulfate gel electrophoresis. It was shown that the resting tension decreased nearly in proportion to the residual length of the A-band. At sarcomere lengths less than or equal to 4.0 microns, the resting tension after the dissociation of the A-band was lowered to less than 10% of the initial value. On the other hand, at sarcomere lengths greater than or equal to 5.0 microns the resting tension after the dissociation of the A-band still showed approximately 35% of the initial value and did not change even after the I-band was dissociated by a solution containing KI. From these results, we propose that most of the PECs contributing to resting tension bind almost uniformly to the A-band and there are also PECs connecting Z-lines.

Actins↗
PubMed1985

Disassembly from both ends of thick filaments in rabbit skeletal muscle fibers. An optical diffraction study.

We show in this paper that the change of the internal structure of a sarcomere in a rabbit glycerinated psoas muscle fiber can be examined by analyzing the intensity change of the first- and the second-order optical diffraction lines. A unit-cell (sarcomere)-structure model has been applied to the estimation of the length of thick filaments in a muscle fiber while they undergo dissociation. The optical factors, except for the unit-cell-structure factor, hardly changed during the dissociation of the filaments. Our results show that thick filaments dissociate from both ends on increasing the KCl concentration in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed to some extent the dissociation of thick filaments. The disassembly of thick filaments occurred at higher KCl concentrations in the absence of pyrophosphate. There was a correlation between the stability of the thick filament structure and cross-bridge formation, which was induced either by the addition of micromolar concentrations of Ca2+ in the presence of Mg-pyrophosphate or by removal of Mg-pyrophosphate.

Animals↗
PubMed1985

Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure.

The kinetics of dissociation from both ends of thick filaments in a muscle fiber was investigated by an optical diffraction method. The dissociation velocity of thick filaments at a sarcomere length of 2.75 microns increased with increasing the KCl concentration (from 60 mM to 0.5 M), increasing the pH value (from 6.2 to 8.0) or decreasing the temperature (from 25 to 5 degrees C) in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed the dissociation velocity markedly at shorter sarcomere lengths. The dissociation velocity, v, decreased as thick filaments became shorter, and v = -db/dt = vo exp (alpha b), where b is the length of the thick filament at time t and vo and alpha are constants. The vo value was largely dependent on the KCl concentration but the alpha value was not. The stiffness of a muscle fiber decreased nearly in proportion to the decrease of overlap between thick and thin filaments induced by the dissociation of thick filaments. This indicates that cross-bridges are uniformly distributed and contribute independently to the stiffness of a muscle fiber during the dissociation of thick filaments.

Animals↗