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Biomedical subjects

A Miyazaki

Publications and source records attributed to A Miyazaki.

At least 73 records · Page 4Linked to original sources
PubMed1999

[Glibenclamide inhibits cholesterol metabolism in macrophage].

Sulfonylureas are generally used in the therapeutic treatment of non-insulin-dependent diabetes mellitus. Little is known, however, whether they also affect the lipid metabolism. Using glibenclamide (GB), a typical sulfonylurea, we have investigated its effects on the lipid metabolism in macrophages, J774 and phorbol ester-treated THP-1 cells. In the whole-cell assay system for cholesteryl ester (CE) accumulation that is induced by addition of chemically modified low-density lipoprotein (LDL), such as Ac-LDL and ox-LDL, GB effectively inhibited the CE accumulation of J774 cells in dose-dependent manners. The inhibition was resulted from increase in free cholesterol but not from change in total cholesterol amount. The results suggest that GB acts on acyl-CoA: cholesterol acyltransferase (ACAT) and inhibits its activity. To confirm the possibility, we then tested GB by another assay system using ACAT that was solubilized from the cells and reconstituted into the liposome composed of phosphatidyl choline- cholesterol. GB inhibition was not so much effective as those by CI-976 and NTE-122, known ACAT inhibitors, but the inhibition was complete in the presence of 100 microM GB. Using cell homogenates of PMA-stimulated THP-1 cells, GB also inhibited the ACAT activity to the level of undifferentiated THP-1 cells. These results indicate that GB acts as ACAT inhibitor but the chemical structure is quite different from the conventional ACAT inhibitors, suggesting it can be a seed to generate potential ACAT inhibitors which do not exhibit toxicity in adrenal gland.

Animals↗
PubMed1999

Insulin resistance and classic risk factors in type 2 diabetic patients with different subtypes of ischemic stroke.

OBJECTIVE: In addition to classic risk factors (e.g., hypertension), insulin resistance is an important risk factor for the development of atherosclerosis. To investigate the risk factors for ischemic stroke in type 2 diabetes, we measured insulin sensitivity and several risk factors in 94 Japanese type 2 diabetic patients with different types of stroke. RESEARCH DESIGN AND METHODS: Stroke was classified by magnetic resonance imaging (MRI) and magnetic resonance (MR) angiography into the following subtypes: 1) patients with normal MRI and MR angiography (NOR; n = 30), 2) patients with lacunar infarction (LAC; n = 28), 3) patients with atherothrombotic infarction (ATI; n = 22), and 4) patients with large-artery atherosclerosis (LAA; n = 14). Insulin sensitivity was assessed by the K index of the insulin tolerance test (KITT). RESULTS: Patients with LAC, ATI, and LAA were significantly older and were more likely to be hypertensive than patients with NOR. Significantly higher insulin resistance was observed in patients with LAC, ATI, and LAA than in patients with NOR (KITT 2.21 +/- 0.17, 2.10 +/- 0.17, 2.19 +/- 0.25, and 3.25 +/- 0.21% per min, respectively, P < 0.001). Adjustment for age, sex, BMI, and duration of diabetes did not influence this result. Multiple logistical regression analysis showed that insulin resistance was an independent risk factor for all subtypes of ischemic stroke in type 2 diabetic patients. The same analysis showed that a high pulse pressure was a risk factor for LAC, postprandial C-peptide (hyperinsulinemia) was a risk factor for ATI, and longstanding hyperglycemia was a risk factor for LAA.

Age Factors↗
PubMed1999

A simple and reliable method of detecting variant transthyretins by multidimensional liquid chromatography coupled to electrospray ionization mass spectrometry.

Transthyretin (TTR) variants cause amyloidosis. A method, originally reported by us, of detecting the variants by high performance liquid chromatography/electrospray ionization mass spectrometry (ESIMS) using materials precipitated with anti-TTR antiserum, has been successfully applied by several institutions. The method is simple and reliable, but some variants may not precipitate with the antiserum or may precipitate in different yields compared to normal TTR. Moreover, unidentified minor peaks were observed, which may have been derived from cross reactive materials. We have now devised a new procedure to overcome these problems. An anion exchange and reversed phase liquid chromatography system and ESI mass spectrometer were connected in a tandem fashion using a 6 port valve and a protein trap cartridge. The profile of ion peaks by the method was the same as that by MS with immunoprecipitates. The minor peaks were proved not to be derived from cross reactive materials, and the molecular species of these peaks were characterized. This method is faster than immunoprecipitation method and using no antibody is a great benefit. The method can be applied widely to the study various proteins, when antibodies are not available.

Amyloid Neuropathies↗
PubMed1999

Enhanced amyloidogenicity of sulfonated transthyretin in vitro, a hypothetical etiology of senile amyloidosis.

Genetic variants of transthyretin (TTR) cause systemic amyloidosis and wild-type TTR may also in some situations produce amyloid fibrils. We have analyzed wild-type and variant TTRs by mass spectrometry and found that TTR preparations from all individuals demonstrated free TTR, TTR conjugated with thiol compounds and several minor components. We previously described a component which had a molecular mass 80 Da larger than free TTR and was proved to be TTR conjugated with sulfite. Here, the amyloid fibril formation of the TTR isoforms was monitored by the turbidity at 330 nm, and by a Congo red-binding assay as a function of pH, according to the method of Lai et al. The S-sulfonated TTR showed the highest level of amyloid fibril formation. In contrast, TTR reduced by dithiothreitol, which was free of the S-sulfonated component, showed neither elevation of the turbidity nor the Congo red binding. Commercially purchased TTR without further treatment containing free, S-sulfonated and other species of TTR molecules showed an intermediate elevation. These results suggested that the S-sulfonated wild-type TTR is highly amyloidogenic. Although further experiments are needed to apply the observation to in vivo phenomenon, exogenous sulfite may be a cause of senile systemic amyloidosis.

Amyloid↗
PubMed1999

A new amyloidogenic transthyretin variant, [D38A], detected by electrospray ionization/mass spectrometry.

A new variant of transthyretin (TTR) was detected by mass spectrometry (MS) in a 63-year-old Japanese female patient suffering from amyloidosis. TTR was analyzed by 2-dimensional liquid chromatography coupled with electrospray ionization MS. Variant TTR showed extra peaks in addition to normal TTR peaks. The extra peaks were about 44 Da smaller than normal TTR peaks, and the abundance of variant peaks showed about 80% of the corresponding normal free and adduct peaks. Direct genomic DNA sequencing of TTR exon 2 showed both adenine and cytosine in the position corresponding to the second base of codon 38. This codes for a variant alanine (GCT) as well as the normal aspartic acid (GAT), indicating that the case is heterozygous for the substitution, [D38A].

Amyloid↗
PubMed1999

[Development of new antiatherosclerotic agents--ACAT inhibitors and CETP inhibitors].

Development of new antiatherosclerotic agents were reviewed focusing on ACAT inhibitors and CETP inhibitors. ACAT inhibitors enhance intracellular degradation of VLDL in hepatocytes. Cholesterol absorption in small intestine is inhibited by ACAT inhibitors. Thus, ACAT inhibitors reduce plasma cholesterol levels. In atherosclerotic lesions, ACAT inhibitors suppress foam cell formation (cholesteryl ester accumulation) in macrophages. Since ACAT inhibitors have multiple anti-atherogenic effects, they are considered future drugs controlling hypercholesterolemia and atherosclerosis. CETP inhibitors are expected to increase HDL and decrease LDL. Although the patients with CETP deficiency show high level of HDL, recent studies showed that they are not necessarily resistant to atherosclerosis. The strategy to inhibit CETP for suppressing atherosclerosis has not been established.

Animals↗
PubMed1999

[A case of relapsing polychondritis with oculobulbar symptoms and successful treatment of respiratory failure with BiPAP].

A 66-year-old man developed diplopia, ptosis, dysphagia, and acute respiratory failure. The initial diagnosis was myasthenia gravis and prednisolone had been administrated for three years. Because of recurrent upper respiratory infections, prednisolone was tapered off. Two months later, auricular chondritis, arthritis, and conjunctivitis appeared. He was diagnosed as having relapsing polychondritis on the basis of histological findings of the ear lobe biopsy. Reinstituted prednisolone had the effect on the auricular chondritis, arthritis, and conjunctivitis, but no effect on dysphagia, hoarseness, and respiratory failure caused by the deformity of the pharynx and airway. Tracheal collapse usually causes rapid death, so early tracheostomy and the use of endotracheal prostheses have been recommended in patients with airway obstruction from relapsing polychondritis, but such surgical management can only partially open up the large airways and has no effect on smaller airways. In this case tracheostomy and endoluminal stent placement have helped improve the patient's respiratory failure, but have had little effect on its aggravation at night in the supine position. The use of BiPAP after surgical management can be an effective treatment for airway involvement in relapsing polychondritis probably because it keeps the narrowed airways from collapsing, especially at night.

Acute Disease↗
PubMed1998

Induction of murine macrophage growth by oxidized low density lipoprotein is mediated by granulocyte macrophage colony-stimulating factor.

We have examined whether certain secreted factor(s) is involved in oxidized low density lipoprotein (Ox-LDL)-induced murine macrophage growth. An antibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) effectively inhibited Ox-LDL-induced macrophage growth by >80%. Ox-LDL as well as phospholipase A2-treated acetylated LDL enhanced mRNA levels and protein release of GM-CSF from macrophages, while neither acetylated LDL nor lysophosphatidylcholine (lyso-PC) showed such effects. The maximal induction of GM-CSF by Ox-LDL was noted at 4 h, followed by a time-dependent decrease to a basal level within 24 h. Ox-LDL-induced macrophage growth was inhibited by 75% by replacement of the culture medium at 24 h by a fresh medium containing the same concentration of Ox-LDL, when GM-CSF had already returned to the basal level. Thus, a cytokine(s) other than GM-CSF is also expected to participate in Ox-LDL-induced macrophage growth in a later phase. The Ox-LDL-induced GM-CSF release was inhibited by calphostin C, a protein kinase C inhibitor, and was significantly reduced in macrophages from the knockout mice lacking class A, type I and type II macrophage scavenger receptors (MSR-AI/AII). These results taken together indicate that effective endocytosis of lyso-PC of Ox-LDL by macrophages through MSR-AI/AII and subsequent protein kinase C activation have led to GM-CSF release into the medium which may play a priming role in conjunction with other cytokines in Ox-LDL-induced macrophage growth.

Animals↗
PubMed1998

The very low- and intermediate-density lipoprotein fraction isolated from apolipoprotein E-knockout mice transforms macrophages to foam cells through an apolipoprotein E-independent pathway.

Apolipoprotein E (apoE)-knockout mice develop severe atherosclerosis associated with high levels of very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) in plasma. To investigate the atherogenic role of VLDL and IDL, the lipoprotein fraction containing both VLDL and IDL (apoEko-VLDL/IDL) was isolated from plasma of apoE-knockout mice by ultracentrifugation, and its interaction with macrophages was studied. When peritoneal macrophages obtained from apoE-knockout mice were incubated with apoEko-VLDL/IDL, the level of cellular cholesteryl esters (CE) increased with the concentration of apoEko-VLDL/IDL. The level of cellular cholesteryl [3H]oleate formed reached 15.1 nmol/mg of cell protein upon incubation with 50 microg/mL apoEko-VLDL/IDL for 18 h, which was an 8.4-fold increase over the corresponding level induced by low-density lipoprotein (LDL). The cellular CE mass was also significantly increased by apoEko-VLDL/IDL. Morphologically, after exposure to apoEko-VLDL/IDL, macrophages became strongly stained with Sudan black B. The total binding of [125I]apoEko-VLDL/IDL to macrophages was effectively replaced by more than 80% by an excess of the unlabeled ligand. Specific binding, calculated by subtracting the nonspecific binding from the total binding, exhibited a saturation pattern. Similar results were obtained with cell association and degradation experiments. In addition, the endocytic degradation of [125I]apoEko-VLDL/IDL was partially inhibited by LDL, whereas acetyl-LDL did not show any effect. These results indicated that apoEko-VLDL/IDL in its unmodified form produced significant CE accumulation in macrophages through a specific and apoE-independent pathway. This pathway may explain, in part, the mechanisms of foam cell formation in arterial walls and the subsequent development of atherosclerosis in apoE-knockout mice.

Animals↗
PubMed1998

A novel hepatic stellate (Ito) cell-derived protein, epimorphin, plays a key role in the late stages of liver regeneration.

Limited data exist regarding morphogenesis and differentiation during liver regeneration. We examined the role of epimorphin on liver regeneration. After 70% partial hepatectomy, mouse liver was collected on days 1, 3, 7, and 14 for immunohistochemistry and the detection of epimorphin mRNA and connexin 32. Using primary cultured rat hepatocytes, morphogenesis and differentiation of cells were tested with or without epimorphin. Seven days after cell inoculation, the expression of connexin 32 and the cell-cell communication was tested as a marker of differentiation. Epimorphin was detected exclusively in hepatic stellate cells. Connexin 32 was detected only in hepatocytes. After partial hepatectomy, epimorphin mRNA was detected on day 3 and peaked at day 7, followed by protein expression. Connexin 32 expression showed a similar time course. Cultured hepatocytes formed multicellular spheroids in an active epimorphin-coated culture dish and showed positive dye coupling, whereas the cell-cell communication was lost without active epimorphin. Because epimorphin was expressed late in liver regeneration, it might play a role in morphogenesis and differentiation.

Animals↗
PubMed1998

Simple and defined method to detect the SOD-1 mutants from patients with familial amyotrophic lateral sclerosis by mass spectrometry.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis (FALS). We established a simple and defined method to detect the mutant SODI in erythrocytes by electrospray ionization mass spectrometry (ESIMS) using materials precipitated with specific antiserum. Hemolysate was mixed with anti-SOD1 antiserum and the generated precipitate, which was soluble in the solvent for MS analysis, was injected on to an LC column connected to an ESI-mass spectrometer. MS spectra of the reduced SOD1 prepared from normal individuals showed ion peaks corresponding to free monomer SOD1. The spectra from FALS patients revealed doublet ion peaks corresponding to normal and mutant components. The ratios of mutant to normal SOD1 were about 1/2 in cases of (G37R) and (A4S), and about 0.15 in a case of (H46R). This method provides for the rapid diagnosis using small amount of specimens, and will contribute to elucidate the pathomechanism of FALS through the quantification of SOD1 mutants in erythrocytes and in tissues of nervous systems.

Amino Acid Substitution↗
PubMed1998

Molecular cloning, functional expression and tissue distribution of rat acyl-coenzyme A:cholesterol acyltransferase.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an enzyme catalyzing the intracellular formation of cholesteryl esters from free cholesterol and fatty acyl-CoA. In the present study, we cloned rat ACAT cDNA and determined its tissue distribution. Rat ACAT cDNA, having a coding region of 1635 bp with its deduced protein sequence of 545 amino acids and two typical motifs such as signature sequences and leucine heptad motif, showed 83, 92 and 90% identity with human, mouse, and hamster ACAT, respectively. Expression of rat ACAT cDNA in A293 cells and CHO cells resulted in a 3.0 to 3.5-fold increase in the enzyme activity. Among twelve tissues examined, ACAT activity was highest in adrenal followed by liver and intestine while that of aorta was extremely low. The mRNA level was also the highest in adrenal among four tissues examined. However, in contrast to its high ACAT activity, the liver mRNA level was extremely low (adrenal >> intestine > aorta >> liver). Consistent with mRNA levels, immunohistochemical analyses with a specific ACAT antibody detected significant ACAT signals in adrenal and intestine but a negligible signal in liver. These results indicate that adrenal most abundantly expresses ACAT in rat. Furthermore, rat liver showed a high ACAT activity but an extremely low ACAT mRNA and negligible immunohistochemical reactivity, suggesting the presence of a structurally different ACAT protein(s) in rat liver.

Amino Acid Sequence↗
PubMed1998

Biochemical evidence for oligomerization of rat adrenal acyl-coenzyme A:cholesterol acyltransferase.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) in rat adrenal was compared with that in rat liver. Immunoblot analyses of the microsomal fractions from adrenal with an anti-human ACAT antibody detected a 45 kDa protein. Upon pretreatment of these microsomal fractions with chemical cross-linkers such as BS3 and Sulfo-EGS, the 45 kDa band decreased with a concomitant increase in high molecular weight proteins (55, approximately 100, and approximately 230 kDa), suggesting that ACAT constitutes oligomers of 45 kDa monomers associated with a 10 kDa protein. In sharp contrast, the same immunoblot analysis of rat liver microsomal fractions identified a 50 kDa protein which was not cross-linked by these cross-linkers. Moreover, when four ACAT inhibitors were tested for their effects on adrenal and liver enzymes, NTE-122, CI-976, and E5324 were more effective for the liver enzyme, whereas 58-035 was much more effective for adrenal ACAT. These biochemical and pharmacological observations support the notion that the rat liver ACAT protein is distinct from the adrenal counterpart.

Adrenal Glands↗