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Biomedical subjects

M Hayashi

Publications and source records attributed to M Hayashi.

At least 145 records · Page 8Linked to original sources
PubMed2001

Relationship between tumor necrosis factor-alpha production and oxidative stress in the failing hearts of patients with dilated cardiomyopathy.

OBJECTIVES: This study evaluated oxidative stress in the failing ventricle in patients with dilated cardiomyopathy (DCM). BACKGROUND: Oxidative stress appears to increase in the failing myocardium and may contribute to ventricular dysfunction in patients with DCM. Tumor necrosis factor-alpha (TNF-alpha), which is expressed in the failing heart, may stimulate oxidative stress. METHODS: We measured plasma oxidized low density lipoprotein (oxLDL) by sandwich enzyme-linked immunosorbent assay using specific antibodies against oxLDL in the aortic root (AO) and the coronary sinus (CS) in control subjects (n = 8) and in 22 patients with DCM and mild congestive heart failure. We also measured the plasma levels of TNF-alpha and angiotensin II. RESULTS: There was no difference in oxLDL between the AO and CS in control subjects. In contrast, plasma oxLDL was significantly higher in the CS than the AO in patients with DCM, suggesting that the transcardiac gradient ofoxLDL reflects oxidative stress in the failing heart in these patients. Plasma TNF-alpha levels were significantly higher in the CS than the AO with a significant positive correlation of the transcardiac gradient of TNF-alpha and the transcardiac gradient of oxLDL. Moreover, a significant negative correlation existed between the transcardiac gradient of oxLDL and left ventricular ejection fraction. The transcardiac gradient of plasma oxLDL was significantly lower in 6 patients who received carvedilol than in 16 patients who did not receive carvedilol. CONCLUSIONS: These findings indicate that the transcardiac gradient of oxLDL may be a marker of oxidative stress in the heart and that left ventricular dysfunction may be partly due to the oxidative stress in patients with DCM. In addition, TNF-alpha may stimulate oxidative stress in the failing heart in patients with DCM.

Adolescent↗
PubMed2001

Intravenous atrial natriuretic peptide prevents left ventricular remodeling in patients with first anterior acute myocardial infarction.

OBJECTIVES: The study evaluates the effect of atrial natriuretic peptide (ANP) compared with nitroglycerin (GTN) on left ventricular (LV) remodeling after first anterior acute myocardial infarction (AMI). BACKGROUND: Compared with GTN, ANP suppresses the renin-angiotensin-aldosterone system and endothelin-1 (ET-1), which stimulate LV remodeling. METHODS: Sixty patients with a first anterior AMI were randomly divided into the ANP (n = 30) or GTN (n = 30) groups after direct percutaneous transluminal coronary angioplasty. We evaluated LV ejection fraction (LVEF), end-diastolic volume index (LVEDVI) and end-systolic volume index (LVESVI) at the acute phase and after one month. We also measured neurohumoral factors during study drug infusion. RESULTS: There was no difference in the baseline characteristics or LVEF (46.9+/-1.0 vs. 46.8+/-1.3%) between the two groups. Although there was no difference in hemodynamics during the infusion periods, the LVEF was significantly improved after one month compared with the baseline value in both groups, but it was improved more in the ANP group than in the GTN group (54.6+/-1.1%, 50.8+/-1.3%, p < 0.05). Left ventricular enlargement was prevented in the ANP group (LVEDVI, 85.8+/-3.1 ml/m2 to 87.3+/-2.7 ml/m2; p = ns, LVESVI, 45.6+/-1.8 ml/m2 to 41.0+/-2.1 ml/m2, p < 0.05) but not in the GTN group (LVEDVI, 86.2+/-4.1 to 100.2+/-3.7, p < 0.01; LVESVI, 46.3+/-2.8 ml/m2 to 51.1+/-3.0 ml/m2, p = ns). During the infusion, ANP suppressed plasma levels of aldosterone, angiotensin II and ET-1 compared with GTN. CONCLUSIONS: These findings indicate that in patients with a first anterior AMI, an ANP infusion can prevent LV remodeling better than can GTN, and effectively suppresses aldosterone, angiotensin II and ET-1.

Atrial Natriuretic Factor↗
PubMed2001

Recent progress in the Na(+)-translocating NADH-quinone reductase from the marine Vibrio alginolyticus.

The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump. The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.

Amino Acid Sequence↗
PubMed2001

D-Aspartate is stored in secretory granules and released through a Ca(2+)-dependent pathway in a subset of rat pheochromocytoma PC12 cells.

D-Aspartate in mammalian neuronal and neuroendocrine cells is suggested to play a regulatory role(s) in the neuroendocrine function. Although D-aspartate is known to be released from neuroendocrine cells, the mechanism underlying the release is less understood. Rat pheochromocytoma PC12 cells contain an appreciable amount of D-aspartate (257 +/- 31 pmol/10(7) cells). Indirect immunofluorescence microscopy with specific antibodies against d-aspartate indicated that the amino acid is present within a particulate structure, which is co-localized with dopamine and chromogranin A, markers for secretory granules, but not with synaptophysin, a marker for synaptic-like microvesicles. After sucrose density gradient centrifugation of the postnuclear particulate fraction, about 80% of the d-aspartate was recovered in the secretory granule fraction. Upon the addition of KCl, an appreciable amount of D-aspartate (about 40 pmol/10(7) cells at 10 min) was released from cultured cells on incubation in the presence of Ca(2+) in the medium. The addition of also triggered d-aspartate release. Botulinum neurotoxin type E inhibited about 40% of KCl- and Ca(2+)-dependent d-aspartate release followed by specific cleavage of 25-kDa synaptosomal-associated protein. alpha-Latrotoxin increased the intracellular [Ca(2+)] and caused the Ca(2+)-dependent d-aspartate release. Bafilomycin A1 dissipated the intracellular acidic regions and inhibited 40% of the Ca(2+)-dependent D-aspartate release. These properties are similar to those of the exocytosis of dopamine. Furthermore, digitonin-permeabilized cells took up radiolabeled d-aspartate depending on MgATP, which is sensitive to bafilomycin A1 or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile. Taken together, these results strongly suggest that d-aspartate is stored in secretory granules and then secreted through a Ca(2+)-dependent exocytotic mechanism. Exocytosis of D-aspartate further supports the role(s) of D-aspartate as a chemical transmitter in neuroendocrine cells.

Adrenal Gland Neoplasms↗
PubMed2001

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts).

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCtheta) of perfringolysin O (theta-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCtheta on a sucrose gradient. BCtheta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCtheta binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCtheta binding to FLDF. The staining patterns of BCtheta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCtheta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCtheta binding does not cause any damage to cell membranes, indicating that BCtheta is a useful probe for the detection of membrane rafts in living cells.

Bacterial Toxins↗
PubMed2001

Hyperosmolarity reduces GLUT4 endocytosis and increases its exocytosis from a VAMP2-independent pool in l6 muscle cells.

The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored. Here we make use of L6 myoblasts stably expressing GLUT4 with an exofacially directed Myc-tag (GLUT4myc) to determine the exocytic and endocytic rates of the transporter. Insulin caused a rapid (t(12) = 4 min) gain, whereas hyperosmolarity (0.45 m sucrose) caused a slow (t(12) = 20 min) gain in surface GLUT4myc molecules. With prior insulin stimulation followed by addition of hypertonic sucrose, the increase in surface GLUT4myc was partly additive. Unlike the effect of insulin, the GLUT4myc gain caused by hyperosmolarity was insensitive to wortmannin or to tetanus toxin cleavage of VAMP2 and VAMP3. Disappearance of GLUT4myc from the cell surface was rapid (t(12) = 1.5 min). Insulin had no effect on the initial rate of GLUT4myc internalization. In contrast, hyperosmolarity almost completely abolished GLUT4myc internalization. Surface GLUT4myc accumulation in response to hyperosmolarity was only partially blocked by inhibition of tyrosine kinases with erbstatin analog (erbstatin A) and genistein. However, neither inhibitor interfered with the ability of hyperosmolarity to block GLUT4myc internalization. We propose that hyperosmolarity increases surface GLUT4myc by preventing GLUT4 endocytosis and stimulating its exocytosis via a pathway independent of phosphatidylinositol 3-kinase activity and of VAMP2 or VAMP3. A tetanus toxin-insensitive v-SNARE such as TI-VAMP detected in these cells, might mediate membrane fusion of the hyperosmolarity-sensitive pool.

Androstadienes↗
PubMed2001

Mutation spectrum of o-aminoazotoluene in the cII gene of lambda/lacZ transgenic mice (MutaMouse).

The o-aminoazotoluene (AAT) has been evaluated as a possible human carcinogen by the International Agency for Research on Cancer. In rodents, it is carcinogenic mainly in the liver, and also in lung following long term administration. We previously examined in lambda/lacZ transgenic mice for the induction of lacZ mutations in liver, lung, urinary bladder, colon, kidney, bone marrow, and testis. AAT induced gene mutations strongly in the liver and colon. In the present report, we reveal the molecular nature of mutations induced by AAT in the lambda cII gene (the cII gene, a phenotypically selectable marker in the lambda transgene, has 294bp, which makes it easier to sequence than the original target, the 3kb lacZ gene). The cII mutant frequency in liver and colon was five and nine times higher, respectively, in AAT-treated mice than in control mice. Sequence analysis revealed that AAT induced G:C to T:A transversions, whereas spontaneous mutations consisted primarily of G:C to A:T transitions at CpG sites.

Animals↗
PubMed2001

Design of nanoparticles composed of graft copolymers for oral peptide delivery.

The development of a dosage form that improves the absorption of peptide and protein drugs via the gastrointestinal tract is one of the greatest challenges in the pharmaceutical field. Many researchers have taken up the challenge, using approaches including mucoadhesive drug delivery, colon delivery, particulate drug delivery such as nanoparticles, microcapsules, liposomes, emulsions, micelles, and so on. The objective of this article is to provide the reader with outlines of novel nanoparticle technologies for oral peptide delivery based on polymer chemistry. The physicochemical properties of nanoparticles and their behavior on exposure to physiological media are greatly dominated by their chemical structures and surface characteristics. We will especially focus on the design of nanoparticles composed of novel graft copolymers having a hydrophobic backbone and hydrophilic branches as drug carriers.

Administration, Oral↗
PubMed2001

A homologue of N-ethylmaleimide-sensitive factor in the malaria parasite Plasmodium falciparum is exported and localized in vesicular structures in the cytoplasm of infected erythrocytes in the brefeldin A-sensitive pathway.

N-Ethylmaleimide-sensitive factor (NSF) and its homologues play a central role in vesicular trafficking in eukaryotic cells. We have identified a NSF homologue in Plasmodium falciparum (PfNSF). The reported PfNSF gene sequence (GenBank accession number CAB10575) indicated that PfNSF comprises 783 amino acids with a calculated molecular weight of 89,133. The overall identities of its gene and amino acid sequences with those of rat NSF are 50.9 and 48.8%, respectively. Reverse transcription-polymerase chain reaction analysis and Northern blotting with total P. falciparum RNA indicated expression of the PfNSF gene. Polyclonal antibodies against a conserved region of NSF specifically recognized an 89-kDa polypeptide in the parasite cells. After homogenization of the parasite cells, approximately 90% of an 89-kDa polypeptide is associated with particulate fraction, suggesting membrane-bound nature of PfNSF. PfNSF was present within both the parasite cells and the vesicular structure outside of the parasite cells. The export of PfNSF outside of the parasite cells appears to occur at the early trophozoite stage and to terminate at the merozoite stage. The export of PfNSF is inhibited by brefeldin A, with 9 microM causing 50% inhibition. Immunoelectromicroscopy indicated that intracellular PfNSF was associated with organelles such as food vacuoles and that extracellular PfNSF was associated with vesicular structures in the erythrocyte cytoplasm. These results indicate that PfNSF expressed in the malaria parasite is exported to the extracellular space and then localized in intraerythrocytic vesicles in a brefeldin A-sensitive manner. It is suggested that a vesicular transport mechanism is involved in protein export targeted to erythrocyte membranes during intraerythrocytic development of the malaria parasite.

Amino Acid Sequence↗
PubMed2001

BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase.

Activation of the mammalian mitogen-activated protein kinase known as BMK1 is required for growth factor-induced cell proliferation. To understand the mechanism by which BMK1 mediates this cellular response, this kinase was used as bait in a yeast two-hybrid-based library screening. Here, we report the identification of serum and glucocorticoid-inducible kinase (SGK) as a cellular protein that physically interacts with BMK1. During growth factor-induced cell stimulation, BMK1 activates SGK by phosphorylation at serine 78. This BMK1-mediated phosphorylation event is necessary for the activation of SGK and, more importantly, for cell proliferation induced by growth factors.

Cell Division↗
PubMed2001

Lethal form of chondrodysplasia punctata with normal plasmalogen and cholesterol biosynthesis.

We present a male autopsied case of chondrodysplasia punctata with abnormal face, symmetrical proximal limb shortness, severe psychomotor developmental delay, respiratory muscle weakness, and death at the age of 2 years. Although his clinical manifestations were similar to those of rhizomelic chondrodysplasia punctata (RCDP), biochemical studies using skin fibroblasts did not document the peroxisomal dysfunction described in RCDP. In addition, the sterol profile, for which abnormalities have recently been reported in cases of X-linked dominant form chondrodysplasia punctata (CDPX2), was normal both in the liver and in the fibroblasts. This patient may represent a new lethal form of chondrodysplasia punctata.

Calcinosis↗
PubMed2001

Antithrombotic effects of a synthetic inhibitor of activated factor X, JTV-803, in animals.

JTV-803, 4-[(2-amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy)methyl]-1-(4-pyridinyl)piperidine-4-carboxylic acid monomethanesulfonate trihydrate, at > or = 0.1 mg/kg/h inhibited the increase in plasma thrombin-antithrombin III complex in response to continuous infusion of thromboplastin in rats. JTV-803 inhibited thrombus formation in an arteriovenous shunt model by intravenous infusion at > or = 0.3 mg/kg/h and prolonged the occlusion time of photochemically induced arterial thrombus in the middle cerebral artery at >1.5 mg/kg/0.5 h. Activated partial thromboplastin time was prolonged at 10 mg/kg/h. Intravenous administration of JTV-803 prolonged bleeding time at 30 mg/kg/h, a dose 10-100 times higher than the dose that inhibited thrombus formation. Compared with thrombin inhibitor, JTV-803 had less of an effect on the bleeding time. In the arteriovenous shunt model in cynomolgus monkey, JTV-803 prolonged the occlusion time when administered by continuous infusion at 0.3 mg/kg/h or orally at 10 mg/kg. These results suggest that the human factor Xa inhibitor JTV-803 is an orally active anticoagulant that does not affect bleeding time and is useful for the prevention of thrombus.

Animals↗
PubMed2001

FMN is covalently attached to a threonine residue in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus.

The Na(+)-translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits. From post-source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr-235 in the NqrB subunit and to Thr-223 in the NqrC subunit. The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide.

Amino Acid Sequence↗
PubMed2001

Diphenylpyraline-responsive parkinsonism in cerebrotendinous xanthomatosis: long-term follow up of three patients.

A long-term follow-up study was made of three patients with cerebrotendinous xanthomatosis (CTX) associated with parkinsonism, two of whom were siblings. Besides typical CTX symptoms, all three patients showed severe parkinsonism. This observation has been rarely reported in CTX. The fact that the two siblings showed parkinsonism strongly suggests the genetic propensity to parkinsonism in these CTX patients. Positron emission tomography studies of the two patients revealed presynaptic dysfunction of the nigro-striatal dopaminergic system. Treatment with the reductase inhibitor hydroxymethyl glutaryl coenzyme successfully corrected the serum cholestanol level in the early stage of the disease, which, however, did not arrest the progression of clinical symptoms, particularly their parkinsonism. Clinically, levodopa had a little effect on parkinsonism, whereas an antihistamine drug, diphenylpyraline hydrochloride (DPP) had excellent effects on all three patients throughout the long-term follow up. The mechanism of the action of DPP on parkinsonism is unclear, however, the drug seems to be a therapeutic choice for treating parkinsonism in CTX.

Adult↗
PubMed2001

Recovery of flagellar dynein function in a Chlamydomonas actin/dynein-deficient mutant upon introduction of muscle actin by electroporation.

Flagellar and ciliary inner-arm dyneins contain actin as a subunit; however, the function of this actin subunit remains unknown. As a first step toward experimental manipulation of actin in dynein, we developed a method for introducing exogenous actin into Chlamydomonas cells by electroporation. A non-motile mutant, ida5oda1, lacking inner-arm dyneins due to the absence of conventional actin, was electroporated in the presence of rabbit skeletal muscle actin. About 20% of the electroporated cells recovered motility under optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recovered with fluorescently labeled actin; in this case, axonemes became fluorescent after electroporation, suggesting that actin was in fact incorporated as a dynein subunit. The feasibility of incorporating a substantial amount of macromolecules by electroporation will be useful not only for studying actin function, but also for a variety of studies using Chlamydomonas in which no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules.

Actins↗
PubMed2001

Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

Animals↗