PubMed Health⌕ Search

Biomedical subjects

M Kotaka

Publications and source records attributed to M Kotaka.

At least 19 recordsLinked to original sources
PubMed2006

pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells.

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.

Animals↗
PubMed2001

Translocation of a human focal adhesion LIM-only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization.

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our results, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagenesis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle.

Actins↗
PubMed2001

Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET).

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.

Base Sequence↗
PubMed2000

Interaction of hCLIM1, an enigma family protein, with alpha-actinin 2.

Enigma proteins are proteins that possess a PDZ domain at the amino terminal and one to three LIM domains at the carboxyl terminal. They are cytoplasmic proteins that are involved with the cytoskeleton and signal transduction pathway. By virtue of the two protein interacting domains, they are capable of protein-protein interactions. Here we report a study on a human Enigma protein hCLIM1, in particular. Our study describes the interaction of the human 36 kDa carboxyl terminal LIM domain protein (hCLIM1), the human homologue of CLP36 in rat, with alpha-actinin 2, the skeletal muscle isoform of alpha-actinin. hCLIM1 protein was shown to interact with alpha-actinin 2 by yeast two-hybrid screening and immunochemical analyses. Yeast two-hybrid analyses also demonstrated that the LIM domain of hCLIM1 binds to the EF-hand region of alpha-actinin 2, defining a new mode of LIM domain interactions. Immunofluorescent study demonstrates that hCLIM1 colocalizes with alpha-actinin at the Z-disks in human myocardium. Taken together, our experimental results suggest that hCLIM1is a novel cytoskeletal protein and may act as an adapter that brings other proteins to the cytoskeleton.

Actinin↗
PubMed2000

Protein-protein interaction of FHL2, a LIM domain protein preferentially expressed in human heart, with hCDC47.

In the yeast two-hybrid library screening, the heart-specific FHL2 protein was found to interact with hCDC47. In vitro interaction study between FHL2 protein and hCDC47 was demonstrated. From the results of domain studies by the yeast two-hybrid assay, the second and third LIM domains in conjunction with the first half LIM domain of FHL2 were identified to be important in binding with hCDC47. Besides, in Northern blot hybridization of human cancer cell lines, the highest FHL2 mRNA expression was detected in colorectal adenocarcinoma SW480 and HeLa cell S3. Our results imply that FHL2 protein may associate with cancer development and may act as a molecular adapter to form a multicomplex with hCDC47 in the nucleus, thus it plays an important role in the specification or maintenance of the terminal differentiated phenotype of heart muscle cells.

Base Sequence↗
PubMed1999

Characterization of a brain-specific nuclear LIM domain protein (FHL1B) which is an alternatively spliced variant of FHL1.

We have amplified and sequenced a novel, alternatively spliced variant of a human gene coding for the four-and-a-half LIM domain protein 1 (FHL1). This gene is located at chromosome Xq27 and the spliced variant is named FHL1B. The ORF of FHL1B cDNA codes for a LIM-only protein that possesses a zinc finger and three tandem repeats of LIM domains at the N-terminus with an active bipartite nuclear localization signal (NLS) motif and a possible RBP-J binding region at the C-terminus. FHL1B and FHL1 have the same N-terminal three-and-a-half LIM domains but different C-terminal protein sequences, due to the presence of an additional alternative exon 4b in FHL1B causing a frame-shift in the 3'coding region. RT-PCR results revealed that the expression of FHL1 is not restricted in skeletal muscle and heart, but is widely distributed in other tissues, including brain, placenta, lung, liver, kidney and pancreas, albeit as a low abundance transcript. In contrast, FHL1B is specifically expressed in brain. The C-terminal alternative region in FHL1B is sufficient to localize FHL1B in the nucleus of mammalian cell. FHL1B is probably related to neural differentiation and certain fragile X syndrome.

Alternative Splicing↗
PubMed1999

Characterization of the human 36-kDa carboxyl terminal LIM domain protein (hCLIM1).

We characterized a human cDNA clone encoding a 36-kDa carboxyl terminal LIM domain protein with a PDZ domain at the amino terminal. This full-length cDNA clone has a predicted open reading frame (ORF) of 329 amino-acid residues. The ORF of this cDNA encodes the human homolog of rat CLP36, and the putative protein is named human 36-kDa carboxyl terminal LIM domain protein (hCLIM1, nomenclature approved by the HUGO/GDB Nomenclature Committee). The hCLIM1 probe was used to hybridize with poly(A)+ RNA of various human tissues. Strong signals were detected in heart and skeletal muscle; moderate signals were detected in spleen, small intestine, colon, placenta, and lung; weaker levels were detected in liver, thymus, kidney, prostate, and pancreas; and no observable signals were detected in brain, testis, ovary, and peripheral blood leukocytes. The hCLIM1 gene was studied by fluorescence in situ hybridization (FISH), somatic cell hybrid analysis, and radiation hybrid mapping, and it is located at the human chromosome 10q26.

Amino Acid Sequence↗
PubMed1998

Chromosomal mapping of a skeletal muscle specific LIM-only protein FHL3 to the distal end of the short arm of human chromosome 1.

Four-and-a-half LIM domain proteins (FHL) possess four tandem repeats of LIM domain and an extra zinc finger. FHL family LIM proteins are unique when compared with other LIM-only proteins because they possess an odd number of zinc fingers. In this study, the tissue distribution and chromosomal mapping of skeletal muscle LIM protein FHL3 were reported. When the FHL3 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, and virtually no signal could be detected in heart, brain, placenta, lung, liver, kidney and pancreas. Using radiation hybrid technique, FHL3 gene was mapped to the distal end of the short arm of chromosome 1 (123.26 cR from the top of the Chr1 linkage group) and this region (near 1p34) is related to several human malignancies.

Animals↗
PubMed1994

[Factorial structure of mutural cognition between parents and child].

Tsujioka's Children's Report of Parental Behavior Inventory (EICA) and its modification adapted to the parents were administered to the high school students (75 boys and 63 girls) and their parents. The results were analyzed by means of the Cliff's Procrustian factor analysis. In the first-order factor analysis, very congruent eight primary factors were obtained in two kinds of samples (boy-parents sample and girl-parents sample). These factors were 1. Acceptance in mother, 2. Autonomy in parents, 3. Identification in child, 4. Acceptance in father, 5. Autonomy in child, 6. Control in father, 7. Control in mother, and 8. Emotional support in child. Sex differences in children were examined in terms of the means and the standard deviations.

Adolescent↗
PubMed1992

[Clinical evaluation of the laryngeal mask airway using capnogram].

The laryngeal mask airway (LMA) was clinically evaluated using capnogram in patients who breathed spontaneously under the combination of general and spinal anesthesia. Using the LMA, the airway was maintained without jaw lift and no remarkable hemodynamic change was observed during its insertion and removal. Capnogram was useful to confirm the intact airway and to monitor the respiration. Respiratory depression was observed after thiopental administration and enflurane inhalation. The respiration recovered promptly following the increase of respiratory rate (RR) and tidal volume at first, and it made a further recovery following increase in RR. The use of the LMA under light inhalation anesthesia is hence a useful combination with regional anesthesia.

Adult↗
PubMed1990

Experimental influenza A virus myocarditis in mice. Light and electron microscopic, virologic, and hemodynamic study.

To elucidate the heart involvement associated with influenza virus infection, the authors studied the hearts of influenza A/PR/8/34 virus-inoculated ICR mice by light and electron microscopy, cardiac catheterization, virus assay, and indirect immunofluorescence. Light microscopy showed small necrotic foci with inflammatory cell infiltration spreading in the myocardium on days 3 to 7 and evidence of healing by day 9 after inoculation. Electron microscopy demonstrated that necrotic cell debris was phagocytosed by macrophages, and that degenerating cardiocytes, macrophages, and lymphocytes were often in close contact, suggesting immunologic interactions, and that platelet thrombi were present in some capillaries on days 3 to 5. Both systolic and diastolic functions of the left ventricle (LV) were impaired on days 3 to 9 and recovered almost to normal by day 14. The virus could be isolated from the heart on days 3 to 7. Immunofluorescent preparations showed virus antigens in the vascular walls and cardiocytes until day 7. These results suggest that the acute cardiac injury was related to cytotoxic immunologic interactions, virus-induced cytolysis and, at least in part, to ischemia due to intracapillary thrombosis. Compared with coxsackie B3 myocarditis in mice, the influenza myocarditis was mild in degree and short in duration, but the influenza infection is a most common and repetitive disease in humans. The clinical implications of this animal model with myocarditis are discussed.

Animals↗
PubMed1989

Cell-mediated immune cardiocyte injury in viral myocarditis of mice and patients.

The cellular immune mechanism of cardiocyte injury in viral myocarditis was investigated by observing and analyzing the interactions among cardiocytes, T cells, B cells, natural killer (NK) cells and macrophages in situ in the myocardium of our murine model (C3H/He mice) and of human patients. In murine coxsackie B3 virus myocarditis, lymphocyte subsets were identified by light and electron microscopic immunohistochemical techniques with antibodies against specific antigens of pan T (Thy 1.2), helper/inducer T (Th/i) (Lyt 1+), cytotoxic/suppressor T (Tc/s) (Lyt 2+), B (Ig+) and asialo GM1+ cells in the myocardium. In the acute phase of myocarditis, asialo GM1+ (mostly NK) cells predominated over pan T cells and peaked on day 9. Pan T cells then reached a peak on day 14. The T4/T8 (Lyt 1+/Lyt 2+) ratio was 1.3 +/- 0.5 on day 5 and reached a peak of 9.1 +/- 3.6 with an increase of Lyt 1+ cells on day 14. Thereafter, NK cells and T cells gradually decreased and could still be seen in fibrotic foci even 3 and 12 months later. B cells were so scarce that no quantitative evaluation could be made. Electron microscopy revealed that macrophages were in close contact with Th/i cells, target cardiocytes and less commonly, B cells; Tc/s and NK cells also occasionally conjugated with apparently viable or degenerated cardiocytes. Some lymphocytes were located in widened intercellular spaces of dissociated intercalated discs, and in intracytoplasmic widened confines of some cardiocytes (emperipolesis). These results suggest that in the acute phase of myocarditis, NK cells initiate the reaction, and then sensitized cytotoxic T cells and activated macrophages aggravate cell-mediated injury by their close contacts with target cardiocytes; close contacts among macrophages; Th/i cells and a few B cells, and the increased T4/T8 ratio may facilitate regulation of the complex immune network; in the chronic phase, residual but active NK and cytotoxic T cells may sustain cytotoxicity. In the endomyocardial biopsies obtained from 8 patients with viral or idiopathic myocarditis from 3 to 48 days after the clinical onset, conventional electron microscopy revealed actual contacts among cardiocytes, macrophages and lymphocytes. As in our murine model, some lymphocytes had emperipolesed in cardiocytes or were located in widened spaces of dissociated intercalated discs. In 4 of these 8 patients infiltration of Leu 2a+ Tc/s, Leu 3+ Th/i and Leu 7+ cells was identified immunohistochemically, and T4/T8 ratios varied widely from 0.1 to 3.8 in the endomyocardial biopsides.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1987

In situ analysis with monoclonal antibodies of lymphocyte subsets in myocardial biopsies from patients with dilated cardiomyopathy and idiopathic (viral) myocarditis.

This light- and electron-microscopic immunohistochemical study using monoclonal antibodies analyzes of the in situ lymphocyte subsets in endomyocardial biopsies from 11 patients with dilated cardiomyopathy (DCM) and three patients with idiopathic (viral) myocarditis (MYO). In the DCM patients both Leu 2a+ cytotoxic/suppressor T cells (Tc/s) and Leu 3+ helper/inducer T cells (Th/i) were identifiable in the myocardial lesions, and mean Th/i/Tc/s (T4/T8) ratio was 0.7 +/- 0.6 (mean +/- SD). In 9 of the 11 DCM patients, Tc/s were more numerous than Th/i cells, so the T4/T8 ratio was less than 1.0. On the other hand, the T4/T8 ratios varied widely in the three MYO patients; one of them had marked mononuclear cell infiltrates with many Th/i in the inflammatory foci and a T4/T8 ratio of 2.6. Immunoelectron microscopy revealed some Th/i in close contact with macrophages. These T cells in the myocardium of DCM and MYO patients appeared to be in vivo effector cells playing an important role in cell-mediated immune responses.

Adult↗
PubMed1986

Immunohistochemical study of the myocardium in murine Coxsackie B3 virus myocarditis using monoclonal antibodies--significance of Lyt 1 antigen-bearing lymphocytes in cell-mediated immunity.

This light- and electron-microscopic study using monoclonal antibodies provides an immunohistochemical demonstration of lymphocytes in situ in the myocardium in murine coxsackie B3 virus myocarditis. On the 7th and 9th days after virus inoculation, we observed many necrotic cardiocyte foci infiltrated with numerous inflammatory cells including macrophages and T lymphocytes. There were only a few Lyt 1-bearing lymphocytes in this phase of inoculation. On the 14th day there were many Lyt 1-bearing cells among the T cells in the foci. By immuno-electron microscopy, some Lyt 1-bearing T lymphocytes were seen in close contact with macrophages and/or other lymphocytes. These Lyt 1-bearing lymphocytes appeared to represent helper T cells, although Lyt 2.3+ lymphocytes were not examined. It is well known that activation of helper T cells leads to stimulation of B cells and cytotoxic T cells. It is assumed, therefore, that Lyt 1-bearing T lymphocytes that infiltrated into the myocardial foci play an important role in immune responsiveness at this stage of viral myocarditis.

Animals↗
PubMed1985

Viral and idiopathic myocarditis in Japan: a questionnaire survey.

In order to study the current clinical status of viral and idiopathic myocarditis in Japan, we conducted a questionnaire survey and collected data for 218 cases from 62 institutions. The diagnosis was based on clinical and laboratory findings alone in 45% of the cases, and it included endomyocardial biopsy in 24% and autopsy in 9% of the patients. Endomyocardial biopsies were available in 40% of the patients; definite cellular infiltrations were identified in half the cases. Regardless of the biopsy findings or availability of biopsy, males predominated in the patient population; the mean age range was 30-39 years for both sexes. Cardiac symptoms and signs were common in addition to "common cold" symptoms; ECG abnormalities, leukocytosis, accelerated erythrocyte sedimentation rate, positive CRP, and increased cardiac enzyme levels were also very common in the acute phase of the disease. Serologic tests for virus titers, performed in 80% of the cases, were positive in 21%. There was no apparent correlation between serologic results and endomyocardial biopsy findings. In this survey, complete recovery occurred in 43%, cure with sequelae in 40%, recurrences in 3%, and death in 13% of the total patient population.

Adolescent↗
PubMed1985

Cell-mediated immunity in Coxsackie B3 virus myocarditis in mice--in situ characterization by monoclonal antibody of mononuclear cell infiltrates.

This light- and electron-microscopic study using monoclonal antibody and anti-immunoglobulin antibodies in murine Coxsackie B3 virus myocarditis provides an immunohistochemical demonstration of surface antigens of lymphocytes. On the 7th and 9th days after inoculation, many necrotic cardiocytes were surrounded by numerous cellular infiltrates, in which macrophages and T lymphocytes predominated, whereas immunoglobulin-bearing B lymphocytes represented a minority. Immuno-electron microscopy showed some T lymphocytes in close contact with other lymphocytes, macrophages, and the sarcolemma of cardiocytes. After the 30th day, significant numbers of T lymphocytes and macrophages were still identifiable in and around the fibrotic foci. Our study suggests that cell-mediated immunity plays a protective role by lysing and scavenging virus-infected cardiocytes and cell debris at least in the early stage of myocarditis. The residual T lymphocytes in the chronic stage suggest their involvement in sustained cardiocyte injury.

Animals↗
PubMed1984

Experimental coxsackie B3 virus myocarditis in golden hamsters. II. Evaluation of left ventricular function in intact in situ heart 14 months after inoculation.

The hemodynamic changes of the left ventricle (LV) of golden hamsters surviving for 14 months after acute coxsackie B3 virus myocarditis were assessed with the use of a high fidelity micromanometer pressure system. Of 25 infected hamsters, 10 survived to the 14th month, and 4 of these had cardiomegaly. Body weight (BW) was 150.0 +/- 20.7 g (mean +/- SD) (controls, 164.5 +/- 20.1 g, NS); heart weight (HW), 0.499 +/- 0.084 g (controls, 0.448 +/- 0.035 g, NS); and HW/BW, 3.39 +/- 0.79 X 10(-3) (controls, 2.74 +/- 0.23 X 10(-3), p less than 0.05). The hemodynamic data under anesthesia were: HR, 378 +/- 42 (controls, 414 +/- 43, NS); LVSP, 108 +/- 16 mmHg (controls, 126 +/- 16, NS); LVDP, 4.0 +/- 4.8 mmHg (controls, 0.6 +/- 0.7, NS); LVEDP, 9.7 +/- 7.5 mmHg (controls, 3.4 +/- 1.4, NS); peak positive dp/dt, 4960 +/- 1431 mmHg/sec (controls, 6714 +/- 1326, p less than 0.05); (dp/dt)/DP40, 56.8 +/- 9.8 sec-1 (controls, 73.1 +/- 7.0, p less than 0.01); peak negative dp/dt, 3876 +/- 1072 mmHg/sec (controls, 4971 +/- 599, p less than 0.05); and time constant T of LV pressure fall, 7.7 +/- 1.3 msec (controls, 5.9 +/- 0.7, p less than 0.01). Five hamsters had congestion of the lungs and liver with or without an elevation of LVEDP. One of them had an organizing thrombus in the left atrium, and one had an aneurysm in the LV free wall. Though markedly varied in extent, residual myocardial fibrosis was always evident in the hearts in which isovolumic contractility and early diastolic relaxation of the LV were significantly impaired. In a clinical extension of these findings, it may be that some cases of dilated cardiomyopathy in man develop in a way similar to the pathological processes noted in this experiment.

Animals↗