Biomedical subjects

H Higuchi

Publications and source records attributed to H Higuchi.

At least 19 recordsLinked to original sources

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↗

PubMed1992

HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri.

An aqueous extract of Phyllanthus niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 reverse transcriptase (HIV-1-RT). The inhibitor against HIV-1-RT in this plant was purified by combination of three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus repandus. The 50% inhibitory doses (ID50) of RA on HIV-1-RT and DNA polymerase alpha (from HeLa cells) were 0.05 microM and 0.6 microM, respectively, representing approximately a 10-fold more sensitivity of HIV-1-RT compared with DNA polymerase alpha. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 microM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 microM of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 microM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system.

Antigens, Viral↗

PubMed1992

Biochemical characterization of 25 distinct carcinoembryonic antigen (CEA) epitopes recognized by 57 monoclonal antibodies and categorized into seven groups in terms of domain structure of the CEA molecule.

The chemical nature of 25 distinct carcinoembryonic antigen (CEA) epitopes, which were recognized by 57 different monoclonal antibodies and categorized into 7 groups (Groups A to G) in terms of domain structure of the CEA molecule, was analyzed and the findings obtained were compared with the results of our previous studies using recombinant CEA proteins. All 21 epitopes of Groups A to F defined by 48 MAbs were resistant to periodate oxidation and were to a greater or lesser extent retained after deglycosylation of CEA, indicating that they are all protein in nature. The 21 epitopes were detected in recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Seven of the 21 epitopes of protein nature were partially or completely sensitive to reduction and alkylation of CEA and not detected or only slightly revealed in the recombinant CEA proteins expressed in E. coli, indicating that those epitopes are dependent on the tertiary structure of the peptide chain, which is formed by disulfide bonds. All 4 epitopes of Group G defined by 9 MAbs were sensitive to mild periodate oxidation and deglycosylation, but resistant to reduction and alkylation and to digestion with pepsin or pronase, indicating that those 4 epitopes are carbohydrate in nature. Although none of the 4 epitopes of Group G were detected in the recombinant CEA proteins expressed in E. coli, two were detected in those expressed in CHO cells. The biochemical studies reported here thus provide information as to the nature of the epitopes on the CEA molecule and help form the basis for selecting the anti-CEA MAbs for use in biological study and potential clinical applications of CEA.

Animals↗

PubMed1992

Changes in contractile properties with selective digestion of connectin (titin) in skinned fibers of frog skeletal muscle.

Changes in contractile properties of mechanically skinned fibers were examined when connectin in the fibers was selectively digested by a low concentration (0.25 microgram/ml) of trypsin. Resting tension and isometric active tension were reduced as the digestion of the connectin progressed; the rate of reduction of active tension was larger than that of resting tension. Maximum shortening speed and calcium ion sensitivity of active tension were not changed by the digestion. Electron micrographs showed that A-bands in the fibers treated with trypsin are dislocated from I-bands. These results suggest that the digestion of connectin does not directly influence the reaction of actin-myosin-regulatory proteins, and thus the resultant reduction in the active tension is mainly due to disordering of the regular structure in a sarcomere.

Animals↗

PubMed1992

Subjective side effects of mianserin in relation to plasma concentrations of mianserin and desmethylmianserin.

The main purpose of the present study was to examine the possibility that plasma concentrations of mianserin and its metabolite, desmethylmianserin, might be predicted by recording subjective side effects. In 44 depressed patients, subjective side effects during 3 weeks of treatment with 30 mg of mianserin were evaluated by the UKU Side Effect Rating Scale, and their relationships to plasma concentrations of mianserin and desmethylmianserin were analyzed. There was no significant relationship between plasma concentrations of these compounds and the occurrence of mianserin-induced side effects, except for dryness of mouth during week one. Our study, therefore, suggests that it is difficult to predict plasma concentrations of mianserin and desmethylmianserin based on the occurrence of subjective side effects.

Adult↗

PubMed1992

Effect of interleukin-1 beta on gene expressions and functions of fibroblastic cells derived from human periodontal ligament.

The present study shows the effect of interleukin-1 beta (IL-1 beta) on some gene expressions and functions of fibroblastic cells (HPLF) derived from human periodontal ligament. HPLF were used at passages number 5 to 10. IL-1 beta increased DNA synthesis in both a dose- and an incubation time-dependent manner. IL-1 beta in combination with tumor-necrosis factor alpha or transforming growth factor beta synergistically stimulated the DNA synthesis in the cells. Since many studies have shown that the c-myc oncogene is involved in cell proliferation and differentiation, the effect of IL-1 beta on c-myc messenger RNA (mRNA) level in HPLF was examined. IL-1 beta induced a marked c-myc mRNA level in the cells at 90 minutes after initiation of the cytokine treatment. On the other hand, IL-1 beta significantly inhibited alkaline phosphatase (ALP) activity of the cells in a dose-dependent manner. Also an inhibitory effect was observed on the liver/bone/kidney ALP mRNA level of the cells, and this inhibition by IL-1 beta was dose- and incubation time-dependent. These results suggest that IL-1 beta is a regulatory cytokine involved in the regeneration of the human periodontal ligament.

Alkaline Phosphatase↗

PubMed1992

Six new triterpenoidal glycosides including two new sapogenols from Albizziae Cortex. V.

Six new triterpenoid glycosides called julibrosides A1-A4, B1 and C1 were isolated from Albizziae Cortex, the dried stem bark of Albizzia julibrissin Durazz. Their structures were determined based on spectral and chemical evidence. Julibrosides B1 and C1 had new sapogenols, designated julibrogenin B and C, respectively, while julibrosides A3 included N-acetyl-D-glucosamine as a sugar component.

Carbohydrate Sequence↗

PubMed1992

An arrhythmic-inducing glycoside from Albizzia julibrissin Durazz, IV.

Three pyridoxine derivatives have been isolated from the fresh stem bark of Albizzia julibrissin DURAZZ.. One of them, named julibrin II, was found to exhibit arrhythmic-inducing action. However, neither the others having the same aglycone nor some glycosides having the same sugar unit showed the action.

Animals↗

PubMed1992

Determination of epitope specificities of a large number of monoclonal antibodies by solid-phase mutual inhibition assays using biotinylated antigen.

A generally applicable method for the determination of the epitope specificities of a large number of monoclonal antibodies (MAbs) is presented. The method is based on the solid-phase mutual inhibition assay using 96-well plates coated with the respective MAbs, competitor MAbs, biotinylated antigen and avidin-peroxidase conjugate. Using carcinoembryonic antigen (CEA) as a model antigen the method was applied to the determination of epitope specificities of anti-CEA MAbs. A constant amount of biotinylated CEA was incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated CEA bound to the immobilized antibody were then reacted with avidin-peroxidase conjugate and the activity of the bound peroxidase was determined by the use of o-phenylenediamine and hydrogen peroxidase. The method used here alleviates the laborious procedures of labeling all antibodies to be tested and the confusion caused by differential labeling among different MAbs, and is convenient for mapping analysis of many MAbs if the corresponding purified antigen is available.

Animals↗

PubMed1992

Contractile actions of endothelin-1 in isolated helical strips from rat pulmonary artery: potentiation of serotonin-induced contraction.

Endothelin-1 (ET-1) caused a concentration-dependent contraction of helical strips from rat pulmonary arteries. Removal of endothelial cells did not change the response to ET-1. ET-1 (10(-8) M) induced a small contraction of the arterial strips in the absence of extracellular Ca2+ (31.0% of the contraction in the presence of extracellular Ca2+). Pretreatment of pulmonary arterial strips with 6 x 10(-11) M ET-1 potentiated the serotonin-induced contraction (10(-7)-3 x 10(-6) M), but showed no significant effect on KCl-induced contraction. The ET-1-induced potentiation of the response to serotonin was prevented by nordihydroguaiaretic acid (3 x 10(-6) M), quinacrine (10(-6) M), and AA861 (10(-6) M), but not by indomethacin (10(-7) M) and baicalein (10(-6) M). These results suggest that ET-1 may cause pulmonary hypertension through a direct vasoconstrictor action and potentiation of serotonin-induced contraction, which may involve the 5-lipoxygenase pathway.

Animals↗

PubMed1992

[Experimental study on possibility of corneal injury by electromagnetic wave].

The possibility of epithelial injury of BALB/C mice cornea exposed to a cathode ray tube (CRT) of a commercially available television was investigated. Epithelial injury of the cornea was confirmed by fluorescein-positive staining after 24 hours of CRT exposure approximately 3-9 cm from the surface of the CRT. The corneal epithelial injury was shown to be mainly limited to the most superficial layer by transmission electron microscope finding showing slight deterioration of the basal cell layer. The corneal epithelial defect was further measured by a scanning electron microscope in relation to the time of exposure to the CRT. The impaired area increased in dose-dependent manner to the some extent. The epithelial defect increased at 5 hours of CRT exposure and reached a maximum, that is 46.6% of the entire area of the cornea, by 12 hours of exposure, thereafter maintaining an equilibrium. The mechanism of corneal epithelial injury by CRT was discussed.

Animals↗

PubMed1991

Sliding distance between actin and myosin filaments per ATP molecule hydrolysed in skinned muscle fibres.

Muscle contraction is generally thought to be driven by tilting of the 19-nm-long myosin head, part of the thick filament, while attached to actin, part of the thin filament. This motion would produce about 12 nm of filament sliding. Recent estimates of the sliding distance per ATP molecule hydrolysed by actomyosin in vitro vary widely from 8 nm to greater than or equal to 200 nm. The latter value is incompatible with a power stroke incorporating a single tilting motion of the head. We have measured the isotonic sliding distance per ATP molecule hydrolysed during the interaction between myosin and actin in skinned muscle fibres. We directly estimated the proportion of simultaneously attached actomyosin complexes and their ATP use. We report here that at low loads the interaction distance is at least 40 nm. This distance corresponds to the length of the power stroke plus the filament sliding while actomyosin crossbridges bear negative drag forces. If the power stroke is 12 nm, then our results indicate the drag distance to be at least 28 nm. Our results could also be explained by multiple power strokes per ATP molecule hydrolysed.

Actins↗

PubMed1991

Myosin heads contact with thin filaments in compressed relaxed skinned fibres of frog skeletal muscle.

When skinned skeletal muscle fibres with rest sarcomere length (L = 2.5 microns) are compressed by the addition of various concentrations ([PVP]) of polyvinylpyrrolidine, the relation between the 1,0 spacing (d) of thick filament lattice and [PVP] has been known to break at d of around 35 nm, resulting in a steeper slope of the relationship at d greater than 35 nm. To clarify the cause of this, X-ray diffraction and crosslinking experiments were carried out. The d versus [PVP] relationship of stretched fibres (L = 3.5 microns) breaks at a d of around 29 nm. The difference between these characteristic d values, 35-29 = 6 nm, is close to the diameter of thin filaments (8 nm). The crosslinking efficiency of formaldehyde between myosin heads and thin filament surface, measured by radial stiffness increase, was found to begin to markedly increase when the relaxed fibre with rest L was compressed to a d of nearly 35 nm. In addition to these results, the d versus [PVP] relationship obtained in rigor and in high [Mg2+] (30 mM) relaxing solutions, and the crosslinking efficiency seen in high [Mg2+] solutions supported our previous hypothesis that in normal relaxing solution (containing 1 mM Mg2+) the probability of myosin heads coming into contact with the thin filament surface abruptly increases at d near 35 nm in fibres with rest L.

Adenosine Triphosphate↗

PubMed1991

Time-resolved synchrotron X-ray diffraction studies of a single frog skeletal muscle fiber. Time courses of intensity changes of the equatorial reflections and intracellular Ca2+ transients.

Time-resolved X-ray equatorial diffraction studies on a single frog skeletal muscle fiber were performed with a 10 ms time resolution using synchrotron radiation in order to compare the time courses of the molecular changes of contractile proteins and the intracellular Ca2+ transient during an isometric twitch contraction at 2.7 degrees C. Measurements of the Ca2+ transient using aequorin as an intracellular Ca2+ indicator were conducted separately just before and after the X-ray experiments under very similar experimental conditions. The results, which showed a similar time course of tension to that observed in the X-ray experiment, were compared with the aequorin light signal, tension and the intensity changes of the 1,0 and 1,1 equatorial reflections. No appreciable change in both reflection spacings indicated that the effect of internal shortening of the muscle was minimized during contraction. The intensity change of the equatorial reflections generally occurred after the aequorin light signal. In the rising phase, the time course of increase in the 1,1 intensity paralleled that of the rise of the light signal and the intensity peak occurred 20-30 ms after the peak of the light signal. The decrease in the 1,0 intensity showed a time course similar to that of tension and the intensity minimum roughly coincided with the tension peak, coming at 80-90 ms and about 60 ms after the peaks of the light signal and the 1,1 intensity change, respectively. In the relaxation phase, the 1,1 intensity seemed to fall rapidly just before the tension peak and then returned to the original level in parallel with the decay of tension. The 1,0 intensity returned more slowly than the tension relaxation. Thus, the change of the 1,1 intensity was faster than that of the 1,0 intensity in both the rising and relaxation phases. When the measured aequorin light signal was corrected for the kinetic delay of the aequorin reaction with a first-order rate constant of either 50 or 17 s-1, the peak of the corrected light signal preceded that of the measured one by approx. 30 ms. Thus, the peak of the Ca2+ transient appeared earlier than the peaks of the 1,1 and 1,0 intensity changes by 50-60 and 110-120 ms, respectively. The time lag between the extent of structural change and the Ca2+ transient is discussed in relation to the double-headed attachment of a cross-bridge to actin.

Aequorin↗

PubMed1991

Immunological and biochemical characterization of the nonspecific cross-reacting antigen epitopes using twenty-three monoclonal antibodies.

Twenty-three monoclonal antibodies (MAbs) reactive with nonspecific cross-reacting antigen (NCA) were prepared and used for constructing a serological map of the NCA molecule. The MAbs were generated using purified NCA or carcinoembryonic antigen (CEA) as immunogen. The MAbs could be divided into two groups: Group X, 10 clones reactive with NCA and CEA; and Group Y, 13 clones specific for NCA. Cross-competition enzyme immunoassays between MAbs of the individual groups revealed that at least 8 different subgroups can be defined i.e., 5 and 3 subgroups in Groups X and Y, respectively. The chemical nature of the epitopes recognized by those MAbs was tested using chemically or enzymatically treated antigens; all MAbs reacted with periodate-treated NCA and deglycosylated NCA, indicating that all the epitopes identified appeared to be protein in nature. Reduction and alkylation, pepsin digestion or pronase treatment of NCA, however, gave some differential results with respect to MAb binding. The serologic mapping and biochemical studies reported here thus provide information as to the range and nature of the epitopes on the NCA molecule and help form the basis for selecting the anti-NCA MAbs for use in biological and immunological study of NCA.

Animals↗