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Biomedical subjects

M Ito

Publications and source records attributed to M Ito.

At least 433 records · Page 24Linked to original sources
PubMed2000

Interleukin 18 and interleukin 1beta production is decreased in HIV type 1-seropositive hemophiliacs but not in HIV type 1-seropositive nonhemophiliacs.

In Japan, the proportion of hemophiliacs infected with human immunodeficiency virus type 1 (HIV-1) is 40%, whereas more than 90% are infected with hepatitis C virus (HCV). To evaluate the immunological status of hemophiliacs infected with HIV-1, we investigated the pattern of cytokine production in peripheral blood mononuclear cells (PBMCs) of HIV-1-seropositive and -seronegative hemophiliacs, HIV-1-seropositive non-hemophiliacs, and healthy individuals. The production of IL-18 and IL-1beta from PBMCs stimulated with Staphylococcus aureus Cowan strain 1 (SAC) in the HIV-1-seropositive hemophiliacs was significantly decreased in comparison with the other groups. On the other hand, IL-12 production in both HIV-1-seropositive groups was significantly lower than in HIV-1-seronegative groups. TNF-alpha and IL-6 production was similar among the four groups. In contrast, plasma levels of TGF-beta1 were increased in HIV-1-seropositive hemophiliacs, HIV-1-seropositive nonhemophiliacs, and HIV-1-seronegative hemophiliacs, with the highest levels being in HIV-1-seropositive hemophiliacs, suggesting that coinfection with HIV-1 and HCV increases the level of plasma TGF-beta in HIV-1-seropositive hemophiliacs. Treatment of PBMCs from healthy individuals with TGF-beta1 inhibited IL-18 and IL-1beta production without affecting IL-6, IL-10, or TNF-alpha production. Suppression of the expression of caspase 1 mRNA, which is known to be an IL-1beta-converting enzyme and which also cleaves the precursor of IL-18, was observed in the SAC-stimulated PBMCs from healthy individuals after treatment with TGF-beta1 and in the SAC-stimulated PBMCs from HIV-1-seropositive hemophiliacs, suggesting that the decreased production of IL-18 and IL-1beta in HIV-1-seropositive hemophiliacs may be related to the downregulation of caspase 1 mRNA induced by high levels of TGF-beta1 in plasma.

Adolescent↗
PubMed2000

Performance of bent-crystal monochromators for high-energy synchrotron radiation.

Two monochromators for high-energy synchrotron radiation have been studied at the elliptical multipole wiggler beamline BL08W of SPring-8. Both monochromator crystals are bent and indirectly water-cooled. In the 100 keV monochromator an efficient cooling scheme is employed. A monochromatic beam is successfully focused using new benders for the 100 keV and 300 keV monochromators. Measured radii of curvature of the bent crystal agree qualitatively with calculation.

Journal Article↗
PubMed2000

Isolation and characterization of cDNA clones for the e1beta and E2 subunits of the branched-chain alpha-ketoacid dehydrogenase complex in Arabidopsis.

Branched-chain alpha-ketoacid dehydrogenase (BCKDH) has been known in mammals to be a key enzyme of the catabolic pathway of branched-chain amino acids. We have isolated two cDNA clones encoding the E1beta and E2 subunits of BCKDH, respectively, from Arabidopsis thaliana. Proteins encoded in these cDNA sequences had putative mitochondrial targeting sequences and conserved domains reported for their mammalian counterparts. Northern blot and immunoblot analyses showed that transcripts from the respective genes and E2 protein markedly accumulated in leaves kept in the dark. We found that the activity of BCKDH in the leaf extracts also increased when plants were placed in the dark. Addition of sucrose to detached leaves inhibited the accumulation of transcripts, whereas application of a photosynthesis inhibitor strongly induced the expression of these genes even under light illumination. These observations indicate that the cellular sugar level is likely responsible for the dark-induced expression of these genes. The transcript levels of these genes were also high in senescing leaves, in which photosynthetic activity is low and free amino acids from degraded protein are likely to serve as an alternative energy source.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2000

Expression of coxsackievirus and adenovirus receptor in hearts of rats with experimental autoimmune myocarditis.

The expression of coxsackievirus and adenovirus receptor (CAR) was dominant in the brains and hearts of mice until the newborn phase. There is no detailed information concerning the relation between the expression of CAR and development of hearts. It is also uncertain whether CAR is able to be induced in adult hearts after cardiac injury. We demonstrated that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats. The expression of CAR in rat hearts with experimental autoimmune myocarditis, which was induced by immunization of purified cardiac myosin, was serially investigated. Active myocarditis was observed from day 15 after immunization. By immunohistochemistry, cardiomyocytes were strongly stained for CAR antibody from days 24 to 42. CAR mRNA was also detected from days 18 to 30 by using reverse transcription-polymerase chain reaction. In the next experiment, the induction of CAR on isolated cardiomyocytes was investigated. CAR was barely detectable in cultured cardiomyocytes by Western blot analysis after isolation. This molecule gradually appeared along with the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR in cultured cardiomyocytes increased after supplement with conditioned medium of rat splenocytes activated by concanavalin A. In conclusion, rat CAR is expressed strongly in the hearts of newborn rats and is suppressed in those of adult rats. The expression of CAR is enhanced during the active phase of experimental autoimmune myocarditis and is induced by inflammatory mediators. CAR may play a role in cell-to-cell contact and adhesion of cardiomyocytes.

Aging↗
PubMed2000

CD27-mediated activation of murine NK cells.

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell-dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-gamma production of freshly isolated NK cells and enhanced the proliferation and IFN-gamma production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN-gamma-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70.

Adjuvants, Immunologic↗
PubMed2000

Anabolic response of mouse bone-marrow-derived stromal cell clone ST2 cells to low-intensity pulsed ultrasound.

The effects of 20-min exposure to low-intensity, pulsed ultrasound were investigated in ST2 cells of bone marrow stromal origin. They responded to ultrasound with elevated levels of IGF mRNAs, osteocalcin, and bone sialoprotein mRNAs. The upregulated expression of these messages appeared in a biphasic manner, with the first peak resistant to the protein synthesis inhibitor cycloheximide, and a second peak that was eliminated by NS398, an inhibitor of the inducive prostaglandin G/H synthase (cyclooxygenase-2). A cumulative effect of mechanical loading called the memory effect, which has been observed in vivo, can be explained from such a biphasic anabolic reaction mediated by prostaglandins. The upregulation of IGF or osteocalcin mRNAs can be observed even at 24 h after the initiation of the 20-min exposure to ultrasound. Our results suggest that this low-intensity, pulsed ultrasound, which has been clinically used to accelerate the healing processes of fractured bone, induces a direct anabolic reaction of osteogenic cells, leading to bone matrix formation.

Animals↗
PubMed2000

Purification and characterization of a neutral ceramidase from mouse liver. A single protein catalyzes the reversible reaction in which ceramide is both hydrolyzed and synthesized.

We report here a novel ceramidase that was purified more than 150, 000-fold from the membrane fraction of mouse liver. The enzyme was a monomeric polypeptide having a molecular mass of 94 kDa and was highly glycosylated with N-glycans. The amino acid sequence of a fragment obtained from the purified enzyme was homologous to those deduced from the genes encoding an alkaline ceramidase of Pseudomonas aeruginosa and a hypotheical protein of the slime mold Dictyostelium discoideum. However, no significant sequence similarities were found in other known functional proteins including acid ceramidases of humans and mice. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, sulfatide, GM1a, or sphingomyelin. The enzyme exhibited the highest activity around pH 7.5 and was thus identified as a type of neutral ceramidase. The apparent K(m) and V(max) values for C12-4-nitrobenzo-2-oxa-1, 3-diazole-ceramide and C16-(14)C-ceramide were 22.3 microM and 29.1 micromol/min/mg and 72.4 microM and 3.6 micromol/min/mg, respectively. This study also clearly demonstrated that the purified 94-kDa ceramidase catalyzed the condensation of fatty acid to sphingosine to generate ceramide, but did not catalyze acyl-CoA-dependent acyl-transfer reaction.

Alkaline Ceramidase↗
PubMed2000

Stable transfectants of smooth muscle cell line lacking the expression of myosin light chain kinase and their characterization with respect to the actomyosin system.

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666-6671).

Actomyosin↗
PubMed2000

Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors.

To overcome low efficiency of retroviral infection into immature T cells, we modified reaggregation fetal thymus organ culture by closely packed co-culture with virus-producing cells (VPC). The viral vector was constructed in chimeric vector, pMX, with IRES and tailless-rat CD2 as a surface marker of infected cells. A rearranged TCR beta gene (Vbeta8.2) was further inserted into the construct for investigating effect of the introduced gene in T cell development. Using this system, we succeeded to transfer the viral vector into immature thymocytes at a remarkably higher efficiency compared to conventional methods using medium containing retrovirus. Moreover, the introduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice to induce in the transition of CD4-CD8- double-negative (DN) into CD4+CD8+ double-positive (DP) cells by transducing beta-selection signaling. Thus, our modified reaggregation culture system is useful for studying the molecular mechanism of T cell development due to a highly efficient gene transfer into immature T cells.

Animals↗
PubMed2000

Phrenic nerve conduction in infancy and early childhood.

Diaphragmatic action potentials (DAPs) were mapped on the thorax bilaterally in 16 neurologically normal infants and 8 boys aged 1 to 4 years during artificial ventilation after thoracic surgery. Transcutaneous stimulation was used to activate the phrenic nerve at the supraclavicular fossa at the end of an artificial inspiration. The DAPs were of positive polarity and were recorded on the ipsilateral anterolateral chest wall over the sixth to the eighth intercostal spaces, with a maximal peak at the seventh intercostal space. The DAP latencies gradually decreased from 6 to 8 ms at birth to about 5 ms at the age of 1 year, despite an increase of conduction distance. Statistical analyses revealed that DAP amplitude did not correlate with age. The latencies and amplitudes of the DAPs displayed little interside variation. The results are valuable not only as a reference for the diagnosis of patients with phrenic nerve palsy, but also as an indicator of the normal development of the phrenic nerve.

Action Potentials↗
PubMed2000

Mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency.

Defects in the pyruvate dehydrogenase (PDH) complex are an important cause of primary lactic acidosis, a frequent manifestation of metabolic disease in children. Clinical symptoms can vary considerably in patients with PDH complex deficiencies, and almost equal numbers of affected males and females have been identified, suggesting an autosomal recessive mode of inheritance of the disease. However, the great majority of PDH complex deficiencies result from mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1). The major factors that contribute to the clinical variation in E1alpha deficiency and its resemblance to a recessive disease are developmental lethality in some males with severe mutations and the pattern of X-inactivation in females. To date, 37 different missense/nonsense and 39 different insertion/deletion mutations have been identified in the E1alpha subunit gene of 130 patients (61 females and 69 males) from 123 unrelated families. Insertion/deletion mutations occur preferentially in exons 10 and 11, while missense/nonsense mutations are found in all exons. In males, the majority of missense/nonsense mutations are found in exons 3, 7, 8 and 11, and three recurrent mutations at codons R72, R263 and R378 account for half of these patients with missense/nonsense mutations (25 of 50). A significantly lower number of females is found with missense/nonsense mutations (25). However, 36 females out of 55 affected patients have insertion/deletion mutations. The total number of female and male patients is thus almost the same, although a difference in the distribution of the type of mutations is evident between both sexes. In many families, the parents of the affected patients were studied for the presence of the PDHA1 mutation. The mutation was never present in the somatic cells of the father; in 63 mothers studied, 16 were carriers (25%). In four families, the origin of the new mutation was determined to be twice paternal and twice maternal.

Amino Acid Sequence↗
PubMed2000

Syndecan-4 deficiency impairs the fetal vessels in the placental labyrinth.

Syndecan-4 is a transmembrane protein bearing heparan sulfate chains, involved in anticoagulation and focal adhesion formation. Here, we revealed that syndecan-4 was expressed in the fetal vessels in the placental labyrinth by in situ hybridization and immunohistochemical staining. At 17.5 gestational days, the area of degenerated fetal vessels in the placental labyrinth was more diffuse and larger in Synd4(-/-) embryos than wild-type controls. Calcium and fibrin(ogen) depositions in the degenerated vessels were also more extensive and more severe in the placentas of Synd4(-/-) embryos. These findings suggest that syndecan-4 deficiency impairs the fetal vessels in the placenta, probably due to a deficit in the anticoagulation mechanism. This article is the first report demonstrating that among a large number of core proteins of heparan sulfate proteoglycans, a defect of a single core protein caused impaired anticoagulation in a specific site.

Animals↗
PubMed2000

A rat model for investigation of bladder dysfunction associated with demyelinating disease resembling multiple sclerosis.

Myelin basic protein (MBP) can be used as an antigen for inducing experimental allergic encephalomyelitis (EAE). In various studies, EAE animals have been used as an experimental model of demyelinating diseases. The aim of this study was to determine whether EAE, induced by MBP in rats, can be useful for investigation of bladder dysfunction associated with demyelinating disease. Female Lewis rats were used. In Study 1, the time course of behavioral and cystometric changes were observed consecutively after MBP sensitization. In Study 2, the correlations between behavioral, cystometric, and histologic abnormalities were studied. The degree of paralysis and histologic findings were evaluated. In Study 1, transient hind limb paralysis was observed in all rats. Cystometric findings were characterized by three different patterns: 1) detrusor areflexia (DA), 2) detrusor hyperactivity (DH), and 3) normal. Ten (77%) of the 13 rats given MBP showed bladder dysfunction, including DA (seven), DA/DH (two) and DH (one). Study 2 showed DA in 10 rats, DH in one, and normal findings in nine animals. The difference in degree of paralysis between the DA and the cystometrically normal animals was statistically significant (P<0.01). The mean value of the degree of inflammation in the spinal cord (L6-S1) in the DA group was significantly (P<0.05) higher than that in the cystometrically normal group. The degrees of paralysis and spinal inflammation were weakly correlated (R = 0.47, P = 0.05). The present rat model seems useful for studies of bladder dysfunction associated with spinal myelitis/demyelinating diseases.

Animals↗
PubMed2000

Abnormal down-regulation of PKC is responsible for giant granule formation in fibroblasts from CHS (beige) mice--a thiol proteinase inhibitor, E-64-d, prevents giant granule formation in beige fibroblasts.

We have previously reported that the abnormally rapid down-regulation of protein kinase C (PKC) activity is responsible for the cellular dysfunction in natural killer (NK) cells and polymorphonuclear leukocytes (PMNs) from Chediak-Higashi syndrome (beige) mice. In this report, we examined whether the down-regulation of PKC is associated with giant granule formation in fibroblasts from beige mice. In cultured beige fibroblasts, the membrane-bound PKC activity declined significantly after phorbol ester stimulation. We found that E-64-d, which is a thiol proteinase inhibitor and protects PKC from calpain-mediated proteolysis, reversed the declined PKC activity and prevented giant granule formation in beige fibroblasts. Moreover, E-64-d corrected the reduced lysosomal elastase and cathepsin G activity in beige fibroblasts. In contrast, specific PKC inhibitors, chelerythrin and calphostin C, promoted giant granule formation in normal fibroblasts. We also demonstrate that ceramide production is enhanced in beige fibroblasts and is involved in the rapid down-regulation of PKC. These results suggest that the accelerated breakdown of PKC observed in beige fibroblasts is caused by enhanced ceramide production and is also responsible for giant granule formation.

Animals↗
PubMed2000

Ability of osteoclast formation from peripheral monocytes using anti-fusion regulatory protein-1/CD98/4F2 monoclonal antibodies in patients with osteoporosis.

We investigated the difference in osteoclast formation between patients with osteoporosis and two healthy control groups by inducing it from peripheral blood monocytes with use of anti-fusion regulatory protein- monoclonal antibody. The group of patients with osteoporosis consisted of 35 women and excluded secondary osteoporosis, and the control groups consisted of 12 young healthy volunteers (control I) or 10 individuals age-matched to the patients with osteoporosis (control II). Osteoclast formation declined with age between the two control groups, but this decline was not significant. Fusion rate and the mean number of nuclei in osteoclasts were significantly less in the patients with osteoporosis than in the young or age-matched controls. It was clearly demonstrated that the ability of monocytes to fuse declines significantly in patients with osteoporosis.

Adult↗
PubMed2000

Spindle-shaped cells derived from giant-cell tumor of bone support differentiation of blood monocytes to osteoclast-like cells.

Spindle-shaped cells were established from four giant-cell tumors of bone. When human blood monocytes were co-cultured with these cells, multinucleated giant-cell formation of monocytes was induced. Intriguingly, even when a filter (pore size: 0.45 microm) was interposed between monocytes and the spindle-shaped cells, polykaryocytes also appeared. These multinucleated giant cells were positive for tartrate-resistant acid phosphatase, expressed calcitonin receptor, and showed bone-resorption activity, characteristics of osteoclast-like cells. These findings indicate that soluble factors secreted from these cells play an important role in osteoclast-like cell formation from blood monocytes. These data additionally suggest that these cells support osteoclast-like cell formation in giant-cell tumors of bone. The cells also expressed mannose receptor, fibronectin, receptor activator of nuclear factorkappaB, and several cytokine mRNAs, including interleukin-6, receptor activator of nuclear factorkappaB ligand/osteoclast differentiation factor/osteoprotegerin ligand, and macrophage colony-stimulating factor. However, all of these molecules except receptor activator of nuclear factorkappaB ligand mRNA could also be detected in control HeLa and CV-1 cells. Although the soluble receptor activator of nuclear factorkappaB ligand has not been found under physiological conditions, it is possible that it is cleaved by cellular proteases and the truncated receptor activator of nuclear factorkappaB is released from cells. Identification of the soluble factors capable of inducing osteoclast formation from blood monocytes is a pressing problem to be solved.

Bone Neoplasms↗
PubMed2000

Glycyrrhizin improves the resistance of MAIDS mice to opportunistic infection of Candida albicans through the modulation of MAIDS-associated type 2 T cell responses.

Compared with normal mice, MAIDS mice (mice infected with LP-BM5 murine leukemia virus) exhibited an increase up to 100 times greater in susceptibility to infection with Candida albicans. The impaired resistance of MAIDS mice to the infection was recovered to levels observed in normal mice by the administration of glycyrrhizin (GR), an active component of licorice roots. MAIDS mice inoculated with CD4(+) T cells from GR-treated mice were also resistant to C. albicans infection. Normal mice inoculated with CD4(+) T helper type 2 cells (Th2 cells) from MAIDS mice were susceptible to C. albicans infection at the same levels shown in MAIDS mice. The susceptibility of normal mice inoculated with type 2 T cells was reversible by (i) administration of GR and (ii) inoculation of CD4(+) T cells from GR-treated mice and injection of a mixture of mAbs targeted against type 2 cytokines (IL-4 and IL-10). Type 2 cytokines were not detected in sera of MAIDS mice inoculated with CD4(+) T cells from GR-treated mice, while they were present in sera of MAIDS mice treated with saline. These results suggest that, by inducing CD4(+) T cells which suppress type 2 cytokine production by MAIDS-associated Th2 cells, GR improves the resistance of MAIDS mice to C. albicans infection.

AIDS-Related Opportunistic Infections↗