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Biomedical subjects

M Sakai

Publications and source records attributed to M Sakai.

At least 433 records · Page 24Linked to original sources
PubMed1993

Identification of ionizable groups essential for the enzyme catalysis on glutathione S-transferase P.

The pH-Vmax/KmGSH plot of glutathione S-transferase P (GST-P) showed a bell-shaped profile, indicating bifunctional catalysis for glutathione (GSH) conjugation. The ionization constant (Ke) and the heat of ionization (delta He) of the essential ionizable group in the GSH binding site were measured and the value of pKe1 was 5.9 and that of pKe2, 8.4, while the delta He1 and delta He2 were -0.2 and 7.9 kcal/mole, respectively. In a solvent containing 25% ethanol, pKe1 and pKe2 shifted to the alkaline side by 0.47 and 0.2, respectively. These kinetic results indicated that carboxyl and phenolic groups were ionizable groups essential for the GSH conjugation. Chemical modifications using aminomethane sulfonic acid and N-acetylimidazole supported the results of the kinetic studies.

Animals↗
PubMed1993

Alteration of carnosine expression in olfactory system of mouse after unilateral naris closure and partial bulbectomy.

To investigate the function of the dipeptide carnosine in the olfactory system, alterations of carnosine in the primary olfactory nerve after unilateral naris closure or olfactory bulb semilesion were studied by means of immunocytochemistry. After unilateral naris closure, carnosine staining showed no alteration, but immunoreactivity of tyrosine hydroxylase (TH), the rate-limiting enzyme for biosynthesis of dopamine in dopaminergic neurons, decreased dramatically in periglomerular neurons. This behavior of carnosine is consistent with that reported for the olfactory marker protein (OMP). On the other hand, following partial lesion of the olfactory bulb, ordinary to strong carnosine expression was seen in newly innervated glomeruli that exceeded that seen in the conventional targets. Ectopic, TH-positive, periglomerular cell-like neurons appeared around the newly formed glomeruli. These data suggest that carnosine may have some role in regulating TH expression in post-synaptic neurons.

Animals↗
PubMed1993

High level expression of human alpha-fetoprotein in transgenic mice.

Transgenic mice were produced by the microinjection of the cDNA of human alpha-fetoprotein (AFP) under control of the enhancer/promoter of the human beta-actin gene. Among the 4 mouse lines where the transgene were stable transmitted to the progeny, 3 produced human AFP. The expression was not developmental stage-specific nor tissue-specific as predicted from the properties of enhancer/promoter. The serum human AFP levels of adult mice were 30-600-fold higher than those of mouse AFP. These mice could be useful for the studies of possible biological functions of AFP during development as well as in malignancies.

Actins↗
PubMed1993

Identification of the fatty acid binding site on glutathione S-transferase P by immobilization to fatty acid-linked sepharose.

The association of glutathione S-transferase P (GST-P) with various fatty acids was confirmed by gas-liquid chromatography and mass-spectrometry. Palmitic and stearic acids were the major fatty acids bound to the enzyme in which the molar ratio was 1:0.8 (GST-P:fatty acid). To evaluate the association with respect to the fatty acid carbon chain length and identify the binding site, we prepared a series of fatty acid-linked Sepharoses. GST-P tightly bound the fatty acid-linked Sepharoses (CH3(CH2)nCOOH, n = 4 approximately 16), and the site was determined to be residues 121-156 from the amino terminus by tryptic digestion of GST-P bound to a fatty acid-linked Sepharose. By fatty acid affinity labeling, we identified the binding site as residues 141-188 (Biochem. Biophys. Res. Commun., in press). The overlapping hydrophobic residues (residue 141-156) is expected to be essential for the ligand binding site.

Amino Acid Sequence↗
PubMed1993

Effects of intrathymic injection of organ-specific autoantigens, parietal cells, at the neonatal stage on autoreactive effector and suppressor T cell precursors.

Thymectomy on day 3 after birth (3d-Tx) induces autoimmune gastritis (AIG) in 81%, and oophoritis (AIO) in 25% of BALB/c mice at the age of 2 to 3 months. Intrathymic, but not intraperitoneal injection of syngeneic parietal cells into sex-matched BALB/c mice within 24 h of birth resulted in almost complete prevention of the development of AIG in these mice in which 3d-Tx was performed. The prevention induced was parietal cell specific, since the development of AIO was not inhibited in female mice. Moreover, the injection of BALB/c liver cells, Mls-matched (BALB/c) and -disparate (DBA/2) B blasts which resulted in V beta 6 T cell deletion, as well as the injection of staphylococcal enterotoxin B failed to prevent the diseases. These findings suggested that recognition of an autoantigen in the thymus is necessary for the induction of tolerance, and that involvement of Mls-1 antigens in the pathogenesis of AIG, as has been suggested previously (Schwartz, R. H., Cell 1989. 57: 1073), was unlikely. T cells that suppress the development of organ-specific autoimmune diseases in 3d-Tx mice seem to maintain the unresponsiveness of autoreactive T cells at the periphery in normal mice. In agreement with our previous observations, we found that intraperitoneal (i.p.) injection of spleen cells from 3-month-old normal mice into 3d-Tx mice on day 10 after birth prevented the development of AIG, whereas spleen cells from age-matched AIG+ (mice with AIG) or AIG- (mice without AIG) 3d-Tx mice failed to do this. This implies that the suppressor cells probably affect the differentiation of effector-precursor to effector. In fact, these suppressor cells did not inhibit the adoptive transfer of AIG to nu/nu BALB/c mice by spleen cells from 3d-Tx mice manifesting AIG. By negative selection using monoclonal antibody and complement, it was confirmed that the phenotype of the suppressor cell was CD4. In contrast to 3d-Tx, 10d-Tx did not induce AIG, indicating the peripheralization of the suppressor cell by that time. On the other hand, intrathymic injection of parietal cells immediately after birth did not affect suppressor cell generation, implying that some T cells, including suppressor cells, escape thymus selection. We postulate that these cells correspond to the precursors of the autoreactive effector T cells and suppressor T cells that are present in normal mice.

Animals↗
PubMed1993

Identification of the hydrophobic ligand-binding region in recombinant glutathione S-transferase P and its binding effect on the conformational state of the enzyme.

Recombinant glutathione S-transferase P (GST-P) was purified in a homogeneous state. Fatty acid analysis of the enzyme revealed that the final enzyme preparation endogenously bound fatty acids, mostly palmitic acid or stearic acid, which were difficult to dissociate from the complex. Temperature-dependent analysis by 1H NMR indicated that the molecular motion of fatty acids was strongly restrained under physiological conditions, which was significantly different from that of serum albumin. On the other hand, there existed another hydrophobic ligand-binding region in GST-P, to which 1-amino-8-naphthalenesulfonic acid and bilirubin would bind with relatively lower affinity than the endogenously bound fatty acid. The hydrophobic ligand-binding region was determined to be around 141-156 residues from the N-terminus by procedures including association of the enzyme to fatty acid-linked Sepharose and affinity labeling with fluorescent fatty acid. Furthermore, circular dichroism analysis showed that the binding of hydrophobic ligand to GST-P produced a remarkable conformational change of the enzyme, which led to states devoid of transferase activity. In addition, the hydrophobic ligand binding caused a significant fluorescence quenching of tryptophan 38, which was assumed to be located at the active center of GST-P. It could be the result of a conformational change of the active center of the enzyme.

Affinity Labels↗
PubMed1993

Cytosolic protein phosphatase may turn off activated NADPH oxidase in guinea pig neutrophils.

Protein phosphatase inhibitors, okadaic acid and calyculin A, potentiated and elongated N-formyl-methionyl-leucyl-phenylalanine-induced superoxide anion (O2-) production in guinea pig neutrophils. The activity of NADPH oxidase in the membrane fraction prepared from phorbol 12-myristate 13-acetate-stimulated neutrophils was inactivated by the addition of the cytosol from resting neutrophils, such inactivation of NADPH oxidase was also suppressed by the protein phosphatase inhibitors. We previously reported that phosphorylation of the 46-kDa protein by protein kinase C is one of the activation mechanisms of NADPH oxidase-dependent superoxide anion production. In the cytosol fraction, we found protein phosphatase activity that catalyzed dephosphorylation of 32P-labeled phosphoproteins including the 46-kDa protein. Dephosphorylation of the 46-kDa protein was inhibited by the addition of okadaic acid and calyculin A. These results indicate that dephosphorylation of the 46-kDa protein by protein phosphatase is involved in the inactivation of NADPH oxidase. NADPH oxidase activity in guinea pig neutrophil may be regulated by the phosphorylation/dephosphorylation state of the 46-kDa protein by protein kinase C and protein phosphatase.

Animals↗
PubMed1993

Thyrotropin-releasing hormone (TRH) is the major prolactin-releasing factor in the bullfrog hypothalamus.

A substance exhibiting potent activity in stimulating the release of prolactin from bullfrog (Rana catesbeiana) pituitary in vitro was isolated from an acid extract of bullfrog hypothalami by gel-filtration chromatography (Sephadex G-15), ion-exchange chromatography (Mono-S HR 5/5), and reverse-phase high-performance liquid chromatography (TSK-gel ODS-120T). Its amino acid composition was similar to that of synthetic thyrotropin-releasing hormone (TRH). Radioimmunoassay confirmed that the substance had TRH immunoreactivity. Moreover, it exhibited the same chromatographic behavior as that of synthetic TRH. These results clearly indicate that the isolated hypothalamic substance is TRH, and that it is the major prolactin-releasing factor present in the bullfrog hypothalamus.

Amino Acids↗
PubMed1993

Heterogeneous expression of ornithine decarboxylase gene in the proximal tubule of the mouse kidney following testosterone treatment.

The expression of the ornithine decarboxylase (ODC) gene in the mouse kidney following testosterone treatment was examined using in situ hybridization histochemistry. Testosterone (n = 5) or vehicle (n = 5) was subcutaneously injected (1 mg/animal) into male BALB/c mice (8 weeks in age) 14 h before sacrifice. Animals were sacrificed under ether anesthesia, their kidneys were removed and immediately frozen in liquid nitrogen. Frozen sections (10-microns-thick) were cut on a cryostat. Sections were hybridized with 35S-labeled sense or antisense RNA probe. The hybridization continued for 24 h at 50 degrees C and emulsion autoradiography was subsequently performed. A marked increase in ODC mRNA was exclusively detected in the proximal tubule of the renal cortex in the testosterone-treated animals. The hybridization signal was greater in the outer portion of the proximal tubule than in the inner portion. No significant hybridization signal was detected either in the distal tubule, renal corpuscle or peritubular tissues. These results indicate that testosterone induces the expression of the ODC gene in the proximal tubule of the renal cortex, leading to the increase in ODC activity in the same region.

Animals↗
PubMed1993

Pharmacokinetics of flosequinan in elderly patients with chronic congestive heart failure.

We have investigated the pharmacokinetics of the direct vasodilator flosequinan in elderly patients with congestive heart failure. Eight patients received a single dose of 50 mg, and 8 patients received once-daily treatment with 25 mg for two weeks. In the single dose study, the tmax of flosequinan was 2.5 h, Cmax was 1.17 microgram.ml-1 and t1/2 was 5.63 h. The tmax of the metabolite BTS 53554 was 20.3 h, Cmax was 1.44 microgram.ml-1 and t1/2 was 62.0 h. BTS 53554 accumulated gradually in the 14-day repeated dose study and steady-state was reached after approximately 2 weeks. Flosequinan was not found to accumulate. Adverse reactions were not observed in either the single or repeated dose study. It is advisable to consider renal function and body weight when flosequinan is to be administered to elderly patients with congestive heart failure. The initial dose should be 25 mg.

Aged↗
PubMed1993

Acetyl-low density lipoprotein receptors on rat mesangial cells.

To elucidate whether mesangial cells have any scavenger functions for modified lipoproteins, surface binding and cholesteryl ester (CE) formation by acetyl-low density lipoproteins (acetyl-LDL) have been studied in cultured rat renal mesangial cells. Specific binding kinetics for acetyl-LDL were observed with Kd = 28.3 micrograms/ml and Bmax = 1.1 ng/micrograms cell protein at 0 degrees C. The fluorescence microscopic finding demonstrated the enhanced uptake of DiI-acetyl-LDL in mesangial cells. Incorporation of [14C]oleate into CE was enhanced to 6-fold by loading 30 micrograms/ml of acetyl-LDL on 10 micrograms/ml of [14C]oleate-bovine serum albumin conjugate as compared with the control without lipoproteins (P < 0.05). The CE formation was completely inhibited by chloroquine. The light microscopic finding demonstrated the increased CE deposition by acetyl-LDL, resulting in foam cell formation. These results indicate biochemically and morphologically that the mesangial cells take up acetyl-LDL by receptor-mediated endocytosis, and that cholesterols in acetyl-LDL are converted to CE, resulting in an increased cellular cholesterol content. In conclusion, mesangial cells may have a scavenger function similar to macrophages.

Animals↗
PubMed1993

Immunocytochemical localization of copper-zinc superoxide dismutase in mouse olfactory mucosa.

Copper-zinc superoxide dismutase (Cu-Zn SOD) has been localized in mouse nasal mucosa. Immunocytochemical staining using polyclonal antibody against Cu-Zn SOD revealed endogenous Cu-Zn SOD in sustentacular cells in the olfactory area, and in ciliated epithelial cells in the respiratory area of the olfactory mucosa. Since these cells are located in the superficial portion of the olfactory mucosa, Cu-Zn SOD may serve as an intracellular antioxidant.

Animals↗
PubMed1993

Localization of a gene for Fukuyama type congenital muscular dystrophy to chromosome 9q31-33.

Fukuyama type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy associated with an anomaly of the brain. Twenty-one FCMD families, 13 of them with consanguineous marriages, were analysed by genetic linkage analyses with polymorphic microsatellite markers to map the FCMD gene. Significant lod scores were obtained with the markers D9S58 (Zmax = 5.81 at theta = 0.06), D9S59 (Zmax = 4.33 at theta = 0.02), and HXB (Zmax = 3.28 at theta = 0.09) on chromosome 9q31-33. Multipoint analysis placed FCMD between D9S58 and D9S59, with a maximum lod score of 16.93. These markers will be useful for presymptomatic, prenatal and carrier diagnosis of family members carrying FCMD, and they represent important resources for the identification of a gene responsible for FCMD.

Chromosome Mapping↗
PubMed1993

Concomitant enhancement of the response to Mls-1a antigens and the induction of post-thymectomy autoimmune gastritis in BALB/c mice.

We examined the role of Mls antigens in the induction of autoimmune gastritis (AIG) in BALB/c and DBA/2 mice subjected to thymectomy. The prevalence of AIG in Mls-1b mice which underwent thymectomy on day 3 after birth (3d-Tx) was 78% (mean), while in Mls-1a DBA/2 mice it was < 6%. Whereas AIG-negative 3d-Tx DBA/2 mice produced 2-mercaptoethanol (2-ME)-sensitive antiparietal cell autoantibody, AIG-positive BALB/c mice made 2-ME-resistant anti-parietal cell autoantibody. In addition, the prevalence of AIG in 3d-Tx BALB/c mice which were rendered tolerant to MIs-1a antigens by injection of bone marrow cells from (BALB/c x DBA/2)F1 mice within 24 h after birth was decreased compared with the non-tolerant control mice; the prevalence being 80% in the controls and 30% in the tolerant animals. Thus, the activation of helper T cells, including T cells responding to Mls-1a antigens and including immunoglobulin class switch, appeared to be closely associated with the induction of AIG. Flowcytometric analysis confirmed that CD4- V beta 6 T cells had increased in the regional lymph nodes of the stomach in AIG mice. However, an increase in the number of V beta 11 T cells, which are known to increase in 3d-Tx mice, occurred in the CD8, but not in the CD4 T cell population. Injection of MoAb to L3T4, but not Lyt2, V beta 6- or V beta 8-TCR, into 3d-Tx BALB/c and syngeneic nude mice which had received spleen cells of 3d-Tx BALB/c mice bearing AIG completely abrogated the development of AIG, despite there being remarkable decreases in T cells expressing relevant markers to the injected antibodies in all the mice. These findings suggest that the increase of V beta 6+ L3T4+ T cells in AIG mice was concomitant with the activation of AIG-inducing V beta 6- L3T4+ T cells.

Animals↗
PubMed1993

A case of antirabies inoculation encephalitis with a long clinical course.

In 1950, a 24-year-old man developed gait disturbance, incontinence of urine and increasing lethargy a week after a course of antirabies vaccination. He was diagnosed as having post-rabies vaccination encephalitis and had been in hospital for 37 years before he died in 1987. This is one of the rare cases in which the course of this disease was traced for an exceptionally long period. His personality began to deteriorate at an early stage of the illness, but his intellectual faculties seemed to be maintained rather well until a few years prior to his death. A neuropathological study revealed disseminated, patchy and somewhere perivascularly located demyelinated lesions in the cerebral white matter. Inflammatory lymphocytic infiltration was also remarkable in the CNS regions. But while the superimposed lesions due to convulsive attacks, traumatic contusion and terminal anoxia were remarkable, the whole aspect of neuropathological changes in rabies inoculation encephalitis cannot be observed.

Adult↗