Biomedical subjects

M Sakai

Publications and source records attributed to M Sakai.

At least 19 recordsLinked to original sources

PubMed1992

Circular dichroic evidence for regulation of enzymatic activity by nonsubstrate hydrophobic ligand on glutathione S-transferase P.

1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand.

Anilino Naphthalenesulfonates↗

PubMed1992

Cross-linking of apolipoproteins is involved in a loss of the ligand activity of high density lipoprotein upon Cu(2+)-mediated oxidation.

A recent study demonstrated that Cu(2+)-mediated oxidation of high density lipoprotein (HDL) resulted in a loss of the capacity to reduce cholesterol from macrophage foam cells [(1991) Proc. Natl. Acad. Sci. USA 88, 6457-6461]. In the present study we characterized the physicochemical properties of oxidized HDL and correlated them with the ligand activity toward the HDL receptor. Among them, the cross-linking of apolipoproteins and an increase in lipid peroxides were characteristic and closely similar to those of tetranitromethane-treated HDL, an abortive ligand for the HDL receptor. Cellular experiments with murine peritoneal macrophages revealed that both the cellular binding activity of HDL and its capacity to enhance cholesterol efflux from macrophage foam cells were markedly reduced upon oxidation. These results suggest that cross-linking of HDL apolipoproteins is involved in the loss of the ligand activity of oxidized HDL.

Animals↗

PubMed1992

Identification of the fatty acid binding site on glutathione S-transferase P.

Glutathione S-transferase P (GST-P) bound a series of endogenous fatty acids (C12-C18). To clarify the function and the binding site of the fatty acids, interaction between fatty acids and GST-P was investigated by using 12-(9-anthroyloxy) stearic acid conjugated with Woodward's reagent K. The fluorescence-conjugated fatty acid noncompetitively inhibited GST activity. After GST-P was covalently labeled with the fatty acid, the enzyme was digested with Lysyl Endopeptidase. From the peptide mapping, a single fluorescence-labeled peptide was obtained. By the sequence analysis, the peptide binding fatty acid was determined as the residues of 141-188 from the amino terminus.

Affinity Labels↗

PubMed1992

Characterization of cysteine residues of glutathione S-transferase P: evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47.

Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F. On carbon 13 NMR experiments, a NMR signal of S-[13C]CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione. These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.

Amino Acid Sequence↗

PubMed1992

Suppression of glutathione transferase P expression by glucocorticoid.

A strong enhancer element, GPEI, of the glutathione transferase P gene (GST-P) gene is composed of two phorbol 12-O-tetradecanoate 13-acetate (TPA) responsive element (TRE)-like sequences at opposite orientation. Unlike TRE sequences of other genes, GPEI exhibits a strong enhancer activity in F9 cells, which contains little AP-1. GPEI bound to AP-1 In vitro and GST-P expression was activated by TPA and exogenously introduced c-jun gene in a rat fibroblast cell line. Both the stimulated expression of GST-P gene by TPA and that by over-expressed c-Jun were suppressed to the basal level by dexamethasone, an inhibitor of AP-1. Basal expression of GST-P gene, however, was not inhibited by dexamethasone. Transfected chloramphenicol acetyltransferase (CAT) gene having GPEI also behaved as the endogenous GST-P gene. These results indicate that the GPEI is activated by AP-1 but constitutive activity of this enhancer in a rat fibroblast cell line 3Y1 cells is due to some unknown mechanism other than AP-1.

Animals↗

PubMed1992

Evidence for the involvement of tryptophan 38 in the active site of glutathione S-transferase P.

Glutathione S-transferase P (GST-P) exists as a homodimeric form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modification by N-bromosuccinimide (NBS). The quenching of intrinsic fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost when single tryptophan residue was oxidized by NBS. To identify which tryptophan residue is involved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activity with that of the wild type one. Trp38His mutant neither was bound to S-hexylglutathione-linked Sepharose nor exhibited any GST activity. These findings indicate that Trp38 is important for the catalytic function and substrate binding of GST-P.

Amino Acid Sequence↗

PubMed1992

Genotoxicity of fungi evaluated by SOS microplate assay.

By an introduction of sodium dodecylsulfate for cell lysis and immunomicroplate for mass assay, the modified SOS microplate assay method was established and applied for the evaluation of genotoxicity of mycotoxins and fungal cultures. Among 20 mycotoxins, the carcinogenic dihydrobisfuranoids such as aflatoxin B1, sterigmatocystin, and versicolorin A were positive in the presence of the activation system. While, the carcinogenic anthraquinones and lactones such as luteoskyrin, rugulosin, ochratoxin A, patulin, and citrinin were negative. The survey on genotoxic fungi revealed that, among 15 fungal isolates Aspergillus versicolor, Emericella acristata, and others were positive. Additional survey on 265 fungal isolates have revealed that various Aspergillus genera such as A. flavus, A. parasiticus, A. ustus, A. nidulans, and others were positive for SOS induction, along with several isolates of Fusarium moniliforme. The chemical analysis revealed that the dihydrobisfuranoids such as aflatoxin B1, and sterigmatocystin were the major genotoxic metabolites of several Aspergillus species. The SOS microplate assay system is a simple and rapid procedure for the mass screening of genotoxic fungi, particularly of the dihydrobisfuranoids-producing strains.

Animals↗

PubMed1992

Detection of tyrosine hydroxylase and phenylethanolamine-N-methyltransferase messenger RNAs in the mouse adrenal gland and the brain by in situ hybridization.

To study the expression of tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) genes in the mouse adrenal gland and the brain, we performed in situ hybridization studies by using several types of complementary DNA probes recognizing coding regions of human TH (THc), the 3'-end of the human region of TH (TH3'), and the coding region of the human PNMT (PNMTc). THc mRNA was detected in the chromaffin cells of the mouse adrenal medulla and brain catecholaminergic neurons including the substantia nigra and ventral tegmental area. TH-immunopositive neurons were located in a similar pattern in adjacent sections. However no positive signals were detected by the TH3' probe. Using the PNMTc probe, the majority of cells in the adrenal medulla demonstrated positive labelling. Although the mouse TH and PNMT genes have not been fully isolated and sequenced, the present study strongly suggests that the sequence of the coding regions of TH and PNMT are similar in human and mouse. The THc and PNMTc probes are particularly useful in investigating the loci of gene transcription in mouse tissues.

Adrenal Glands↗

PubMed1992

Long-term callosal lesions and learning of a black-white discrimination by one-eyed rats.

We know from our previous studies that mature rats with monocular enucleation at birth (OEBs), as well as animals enucleated at maturity (OETs), were unable to learn a black-white discrimination when they were trained after lesions of the visual cortex contralateral to the remaining eye. Since it is well known that synaptic reorganization takes place in the adult rat brain through reactive synaptogenesis following deafferentation, we wondered if long-term callosal lesions in OEBs and OETs would bring out such synaptic reorganization in the visual cortex and, consequently, affect the outcome of the discrimination mentioned above. In the present study, two experiments were carried out: in Experiment 1 the previous experiment was replicated in that OEBs and OETs of 3 months of age were trained on the discrimination 10 days following unilateral visual cortex lesions; in Experiment 2, effects of callosal lesions made 10 weeks earlier either at 3 weeks of age or 13 weeks of age were investigated. The results were: 1) the findings of the previous experiment were confirmed; 2) the long-term callosal lesions facilitated the acquisition of discrimination in OEBs but not in OETs; 3) the facilitative effects were more prominent in OEBs with callosal lesions at 3 weeks of age than in those at 13 weeks of age. The findings were discussed in relation to possible synaptic reorganization produced in the visual cortex ipsilateral to the remaining eye following callosal lesions made 10 weeks earlier and also in relation to reorganization of the uncrossed visual pathways resulting from monocular enucleation at birth.

Aging↗

PubMed1992

Change in Fos-like immunoreactivity in the suprachiasmatic nucleus in the adult male rat.

The circadian change in the number of the Fos-like immunoreactive (IR) cells in the suprachiasmatic nucleus (SCN) was examined in the adult male rats, whose eyeballs were enucleated two months before sacrifice. Their circadian rhythms were determined by their locomotor activity. They were sacrificed in the middle of the active phase or inactive phase. Then brain sections were cut for immunocytochemistry for Fos. A marked increase in the number of the Fos-like IR cells was observed in the inactive phase in the SCN, whereas no such increase was observed in the supraoptic nucleus. These results indicate that, in the SCN, Fos expression was changed with endogenous circadian rhythm in the free-running rat.

Animals↗

PubMed1992

The cardiac functional reserve in elderly hypertensive patients with abnormal diurnal change in blood pressure.

OBJECTIVE: To evaluate the left ventricular function of hypertensive patients with abnormal diurnal change in blood pressure. DESIGN: We compared left ventricular structural and functional characteristics between hypertensive patients with a normal diurnal change in blood pressure (H2 group) and those with a nocturnal blood pressure increment (H1 group) using echocardiography. METHODS: The study group consisted of 36 hypertensives and 16 normotensives whose 24-h ambulatory blood pressure monitorings were measured non-invasively. The hypertensive group was subdivided into the H1 group, consisting of 11 patients (76 +/- 7 years), and the H2 group with 25 patients (73 +/- 7 years). The normotensive control group had a mean age of 73 +/- 6 years. Echocardiographic examinations were performed before and at the end of isometric exercise (handgrip for 3 min) and isoproterenol infusion (0.02 micrograms/kg per min for 5 min). RESULTS: The left ventricular mass index in the H1 group was significantly greater than in the H2 or control group. Left ventricular fractional shortening (LVFS) at rest in the H1 group was also significantly greater than in the other two groups. However, the peak late: early diastolic filling ratio, which indicated diastolic function, significantly deteriorated in the H1 group compared with the H2 and control groups. Furthermore, changes in LVFS after isometric exercise in the H1 group were more suppressed than in the H2 or control group. In addition, a significantly lower increment in LVFS after isoproterenol was observed in the H1 group compared with the H2 or control group. CONCLUSION: The H1 group had greater left ventricular mass and impaired left ventricular functional reserve than the H2 group.

Aged↗

PubMed1992

Interferon-alpha and dexamethasone inhibit adhesion of T cells to endothelial cells and synovial cells.

We investigated whether interferon-gamma (IFN-gamma), interferon-alpha (IFN-alpha) and glucocorticoids affected the adhesion of T cells to human umbilical endothelial cells or human synovial cells. About 30% of peripheral blood T cells could bind to unstimulated endothelial cells, but only a few T cells could bind to unstimulated synovial cells. When both endothelial cells and synovial cells were cultured with recombinant IFN-gamma (rIFN-gamma), the percentage of T cell binding to both types of cells increased in a dose-dependent manner. rIFN-alpha and dexamethasone blocked the T cell binding to unstimulated endothelial cells. Furthermore, rIFN-alpha and dexamethasone suppressed T cell binding to both endothelial cells and synovial cells stimulated by IFN-gamma, and also inhibited intercellular adhesion molecule-1 (ICAM-1) expression on both endothelial cells and synovial cells stimulated by IFN-gamma. These results suggest that IFN-alpha and glucocorticoids may inhibit T cell binding to endothelial cells or synovial cells by modulating adhesion molecule expression on these cells.

Arthritis, Rheumatoid↗

PubMed1992

Production of basic fetoprotein in human peripheral lymphocytes during blastic transformation.

To clarify the significance of basic fetoprotein (BFP) in lymphocytes, we investigated whether BFP is produced in lymphocytes during blastic transformation. Peripheral blood lymphocytes obtained from 14 adults were cultured under the stimulation of lectins. The concentration of BFP in the culture medium (extracellular BFP) was estimated serially. The incorporation of [6-3H]-thymidine was assayed simultaneously. The intracellular BFP was measured by dual flow cytometry for DNA and BFP. A lymph node was studied immunohistochemically. Serum BFP was measured in four cases of lymphocytic leukaemia. In two cases, dual staining was performed. The intracellular BFP of the mitogen-stimulated lymphocytes was increased within 24 h. The extracellular BFP was increased exponentially from 72 h. The extracellular BFP at 96 h did not correlate with the [3H]-thymidine incorporation. The intracellular BFP increase began in G1 phase. Immunostaining showed that the B cells also produced BFP. The serum BFP level in leukaemia was high in 1 of 4 cases and the leukaemic cells in two cases showed high intracellular BFP content. These observations indicate that BFP is produced in activated human lymphocytes and in lymphocytic leukaemic cells. The production of BFP during blastic transformation will be a useful new in vitro model for studying the biological role of BFP, and BFP labelling may offer some new possibilities for the study of lymphocytes.

Adult↗

PubMed1992

Primary Sjögren's syndrome with antibodies to HTLV-I: clinical and laboratory features.

The prevalence of antibodies to human T lymphotropic virus type I (HTLV-I) was studied in patients with primary Sjögren's syndrome. Thirteen of 36 serum samples were positive by enzyme linked immunosorbent assay (ELISA) and particle agglutination assay for antibodies to HTLV-I and were confirmed by western blotting. The presence of antibodies to HTLV-I may signify an HTLV-I carrier state. These patients had a high occurrence of extraglandular manifestations such as uveitis, myopathy, and recurrent high fever compared with patients who did not have antibodies to HTLV-I. Patients with antibodies to HTLV-I had an increased spontaneous proliferation of peripheral blood mononuclear cells compared with those without the antibodies. The proportions of activated and memory T cells (HLA-DR+ CD3+, CD25+ CD3+, and CD29+ CD4+ cells) were higher in HTLV-I carriers than in non-carriers. The presence of antibodies to HTLV-I in some patients with primary Sjögren's syndrome suggests that HTLV-I may cause primary Sjögren's syndrome or its extraglandular manifestations, or both.

Adult↗

PubMed1992

Contractile response of individual cardiac myocytes to norepinephrine declines with senescence.

The present study utilized individual isolated left ventricular cardiac myocytes from hearts of animals of a broad age range to evaluate the response to norepinephrine and to other stimuli that augment myocardial cell contractile performance. During electrical stimulation before drugs neither the amplitude nor the velocity of shortening normalized for resting cell length differed among cells isolated from 2-, 6- to 8-, or 24-mo-old animals. Norepinephrine augmented twitch amplitude and velocity about fourfold in cells from 2-mo-old hearts but only by 2.5-fold in cells from 24-mo-old hearts (age effect, P less than 0.001). In contrast, the contractile response to increases in bathing [Ca2+] or to the addition of the calcium channel agonist BAY K 8644 or of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT cAMP) did not vary with age. These results indicate that the age-associated contractile deficit during beta-adrenergic stimulation is specific to the beta-adrenergic pathway and an age-associated deficit in the net production of cAMP. This can be attributed to a diminished cardiac myocyte response to beta-adrenergic agonists, in contrast to modulation of the beta-adrenergic response by other receptor agonists, which are present in intact tissue but absent under the conditions of the present study.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Giant air cell of the petrous apex: a possible cause of facial hypalgesia.

A giant air cell of the left petrous apex was found in a 23-year-old man with ipsilateral facial hypalgesia. The size of the giant air cell depicted on computed tomography was 1.5 x 2.0 x 2.0 cm. A coronal T1-weighted magnetic resonance image showed that the trigeminal nerve was compressed superomedially by a large signal void area that was probably a result of excessive pneumatization of the petrous apex. It is suggested that the facial hypalgesia was caused by the compression by the giant air cell of the petrous apex on the trigeminal nerve.

Adult↗

PubMed1992

[Effect of seasonal and intermittent work at high altitude on health status].

Health status of 7 men was examined in order to study the effect of duty work cycle of 3-d stay at the altitude of 2,000 m and one-day holiday at 600 m for a period of 6 months from May to October. Their usual works were desk work and road patrol. Total hemoglobin concentration in blood increased significantly after work at high altitude, which was considered to be a compensatory response to the hypoxic state. Subjective symptoms were obtained by a self-administered questionnaire before (April), during (June and October) and after (November) work at high altitude. Fatigue was observed in three out of the seven subjects in June, which subsequently disappeared in October. Dyspnea was observed also in June in three of the subjects, which disappeared in two, persisted in one, and newly appeared in another subject in November. In contrast, a tendency of favorable effects of the work environment on nausea and lumbago was observed. Blood pressure and pulse rate were measured for 24 h. The mean systolic blood pressure during 24 h decreased in two of five subjects in October and November, and the mean values during the working hours decreased in two in October from 145 to 131 mmHg and from 147 to 129 mmHg, respectively. The blood pressure measured at health examination correlated well with the mean blood pressure during 24 h and working hours. The mean pulse rate during 24 h and working hours increased in one in June, October and November. These results indicate that adaptive responses to low barometric pressure environment developed during a work period of 5 months at high altitude.(ABSTRACT TRUNCATED AT 250 WORDS)

Altitude↗

PubMed1992

A three-dimensional observation of acid phosphatase-positive structures in rat testis by high voltage electron microscopy.

Using a fixative of low concentrated glutaraldehyde in PIPES, we examined three-dimensional structures of acid phosphatase (ACPase) activity in the cells of testes of normal adult and 30 min heat-treated rats by high voltage electron microscopy. There were less ACPase-positive structures in germ cells, but more in Sertoli cells, particularly in residual bodies, and also in macrophages lying between seminiferous tubules. Reaction products of ACPase were distributed in the trans-most cisternae of Golgi apparatus, many spherical lysosomes, and acrosomes. The elongated thread-like lysosomes (nematolysosomes) were absent on the testicular cells. After heat-treatment of rat testis, though ACPase-positive lysosomes increased in number, nematolysosomes were never seen in Sertoli cells and most of the other testicular cells; a number of rod-like lysosomes, however, emerged in macrophages. The results indicate that (1) Sertoli cells usually have more ACPase-positive lysosomal contents than germ cells; (2) Sertoli cells are less affected by stress which may largely hurt germ cells; (3) nematolysosome is not a ubiquitous structure in all the types of cells; and (4) the function of nematolysosomes in macrophages may involve in the disposition of disintegrated cells in rat testis.

Acid Phosphatase↗