Myocyte-specific enhancer factor 2 and thyroid hormone receptor associate and synergistically activate the alpha-cardiac myosin heavy-chain gene.

The muscle-specific regulatory region of the alpha-cardiac myosin heavy-chain (MHC) gene contains the thyroid hormone response element (TRE) and two A/T-rich DNA sequences, designated A/T1 and A/T2, the putative myocyte-specific enhancer factor 2 (MEF2) binding sites. We investigated the roles of the TRE and MEF2 binding sites and the potential interaction between thyroid hormone receptor (TR) and MEF2 proteins regulating the alpha-MHC promoter. Deletion mutation analysis indicated that both the A/T2 motif and TRE were required for muscle-specific expression of the alpha-MHC gene.

Neuropathology of HTLV-1-associated myelopathy (HAM/TSP).

In order to clarify pathogenesis of HAM/TSP, we performed a detailed neuropathologic analysis of seven autopsy patients with HAM/TSP. Inflammatory infiltrates of mononuclear cells and degeneration of myelin and axons were noted in the middle to lower thoracic spinal cords and were continuously extended to the entire spinal cord. Horizontal distribution of inflammatory lesions was symmetric at any spinal levels.

Angiotensin II and serum differentially regulate expression of cyclins, activity of cyclin-dependent kinases, and phosphorylation of retinoblastoma gene product in neonatal cardiac myocytes.

The hypertrophic response in cardiac myocytes and the mitogenic response in other cell types share various early cellular responses. However, how the subsequent cell growth response, such as cell cycle machinery, is regulated in cardiac hypertrophy is not understood. Using cultured neonatal rat cardiac myocytes, we examined the effect of angiotensin II (Ang II), a hypertrophic stimulus, on mRNA and protein expression of cyclins and cyclin-dependent protein kinases (cdks), activity of cdks, and phosphorylation of retinoblastoma gene product (pRb).

Tyrosine kinases mediation of c-fos expression by cell swelling in cardiac myocytes.

We investigated the signal transduction mechanism initiated by hypotonic cell swelling in cardiac myocytes using c-fos expression as a nuclear marker. Treatment of myocytes with hypotonic culture media rapidly induced c-fos expression, whereas hypertonic stress had no effect on it. Extensive pharmacological studies indicated that only tyrosine kinase inhibitors suppressed the hypotonic swelling-induced c-fos expression; down-regulation of protein kinase C or a Ca2+ chelator (EGTA) had no effect.

Overview of the molecular mechanisms of cardiac development.

Over the past 4 years remarkable progress has been made in the field of heart development. Several genes that regulate heart development in various model organisms have been cloned, and their functions have been analyzed in vitro and in vivo using molecular biological and genetic engineering techniques. We expect that understanding the molecular mechanisms of cardiac development will lead to better understanding of the pathogenesis of congenital heart disease and hopefully to new therapeutic approaches not only to congenital heart disease but also acquired heart disease such as heart failure.

The cellular and molecular response of cardiac myocytes to mechanical stress.

External load plays a critical role in determining muscle mass and its phenotype in cardiac myocytes. Cardiac myocytes have the ability to sense mechanical stretch and convert it into intracellular growth signals, which lead to hypertrophy. Mechanical stretch of cardiac myocytes in vitro causes activation of multiple second messenger systems that are very similar to growth factor-induced cell signaling systems.

Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling-induced ERK activation and c-fos gene expression in cardiac myocytes.

Hypotonic stress causes rapid cell swelling and initiates various cellular adaptive processes. However, it is unknown how cells initially sense low osmolarity and convert it into intracellular signals. We investigated the signal transduction mechanism initiated by hypotonic cell swelling in cardiac myocytes using c-fos expression as a nuclear marker.

Intrathecal humoral immune response in HAM/TSP in relation to HLA haplotype analysis.

Introduction: In HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), we correlated human leukocyte antigen (HLA) haplotypes to the fine specificities of intrathecally synthesized IgG antibodies against HTLV-1.

Patients And Methods: HLA haplotypes of HAM/TSP patients were determined by the standard NIH microcytotoxicity test and family HLA studies. IgG antibodies against HTLV-1 synthetic peptides in paired CSF and serum were measured by enzyme immunoassay, and intrathecal synthesis of antibodies was evaluated.

Therapeutic trials in 200 patients with HTLV-I-associated myelopathy/ tropical spastic paraparesis.

We report here the results of therapeutic trials in 200 patients with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) conducted in our department between 1986 and 1993. Motor disability grades were improved by more than one grade in 69.5% (91/131) of patients by oral administration of prednisolone, 50% (3/6) by eperisone hydrochloride only, 43.

Mutation rates in LTR of HTLV-1 in HAM/TSP patients and the carriers are similarly high to Tax/Rex-coding sequence.

The genomic sequence of human T-cell leukemia virus type 1 (HTLV-1) is highly conserved, although minor sequence variations enable classification of the isolates into several subgroups. We previously reported, however, that the Tax-coding sequence of HTLV-1 genome is highly variable in a random fashion within individuals with HAM/TSP and asymptomatic carriers. Here, we describe frequent base substitutions in the LTR sequence similarly to those in Tax-coding sequence.

The main HTLV-I-harboring cells in the muscles of viral carriers with polymyositis are not macrophages but CD4+ lymphocytes.

We have analyzed muscle biopsy specimens from polymyositis patients who are also positive for human T cell lymphotropic virus type I (HTLV-I) using both immunohistochemistry for surface antigens of lymphocytes and macrophages and in situ polymerase chain reaction for HTLV-I proviral DNA on the same sections. We found HTLV-I in CD4+ cells but not in macrophages. This finding suggests that most of the HTLV-I-containing CD4+ cells are not macrophages but lymphocytes.

Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes.

Infection by human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia and a slowly progressive disease of the central nervous system (CNS), HTLV-I-associated myelopathy/tropical spastic paraparesis, characterized pathologically by inflammation and white matter degeneration in the spinal cord. One of the explanations for the tissue destruction is that HTLV-I infects cells in the CNS, or HTLV-I-infected CD4+ T lymphocytes enter the CNS, and this drives local expansion of virus-specific CD8+ cytotoxic T lymphocytes, which along with cytokines cause the pathological changes. Because both in the circulation and in the cerebrospinal fluid, CD8+ cytotoxic T lymphocytes are primarily reactive to the product of the HTLV-I tax gene, we sought evidence of expression of this gene within cells in the inflammatory lesions.

Detection of human T lymphotrophic virus type I (HTLV-I) DNA and mRNA in individual cells by polymerase chain reaction (PCR) in situ hybridization (ISH) and reverse transcription (RT)-PCR ISH.

Human T lymphotrophic virus type I (HTLV-I) proviral DNA and mRNA in the blood obtained directly from HTLV-I infected adult T cell leukemia (ATL) patients were amplified by the polymerase chain reaction (PCR), and reverse transcription (RT)-PCR, and then were hybridized to fluorescein-labelled probes by means of in situ hybridization (ISH). Before the cytospin samples were prepared, heterogenous cell populations were reproducibly resolved into HTLV-I-positive and -negative distributions. Immunohistochemical staining was performed, using anti-fluorescein monoclonal antibody.

Diversity of intrathecal antibody synthesis against HTLV-I and its relation to HTLV-I associated myelopathy.

The humoral immune response against human T-cell lymphotropic virus type I (HTLV-I) in the central nervous system (CNS) compartment and in the blood was investigated by enzyme immunoassay using 16 synthetic peptides corresponding to HTLV-I core and envelope sequences. We evaluated paired samples of cerebrospinal fluid and serum from HTLV-I seropositive Japanese patients, classified as follows: HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP; n = 39), patients with spinal cord disease ascribed to either HAM/TSP or to some concomitant, HTLV-I-unrelated disease (possible HAM/TSP; n = 6) or carriers without any clinical signs of HAM/TSP (n = 15). HTLV-I-peptide-specific intrathecal antibody synthesis was found in 79% of HAM/TSP patients, but only in 20% of carriers without HAM/TSP.

Characterization of a unique T-cell clone established from a patient with HAM/TSP which recognized HTLV-I-infected T-cell antigens as well as spinal cord tissue antigens.

Five T-cell clones reactive to autologous HTLV-I-infected T-cells (KODA-TV) were established from peripheral blood lymphocytes of a HAM/TSP patient (KODA) by the limiting dilution method. All the clones showed CD3+, CD4+ and CD25+ surface markers and expressed alpha beta+ T-cell receptors to recognize KODA-TV antigens. One of the five T-cell clones (KODA-408) was infected with HTLV-I but the remaining four clones (KODA-400, 404, 405 and 409) were free of HTLV-I infection.

Interferon-alpha is effective in HTLV-I-associated myelopathy: a multicenter, randomized, double-blind, controlled trial.

A double-blind, multi-center study was performed on patients with HTLV-I-associated myelopathy (HAM) to evaluate the therapeutic effect of treatment with natural interferon-alpha (HLBI). Forty-eight HAM patients were enrolled and treated with either 0.3 MU (n = 15), 1.

Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress.

Endothelium exposed to fluid shear stress (FSS) undergoes cell shape change, alignment and microfilament network remodeling in the direction of flow by an unknown mechanism. In this study we explore the role of tyrosine kinase (TK) activity, intracellular calcium ([Ca2+]i), mechanosensitive channels and cytoskeleton in the mechanism of cell shape change and actin stress fiber induction in bovine aortic endothelium (BAE). We report that FSS induces beta-actin mRNA in a time- and magnitude-dependent fashion.

Detection of human T-lymphotropic virus type-I DNA and mRNA in the lymph nodes; using polymerase chain reaction in situ hybridization (PCR/ISH) and reverse transcription (RT-PCR/ISH).

To examine the relationship between human T-lymphotrophic virus type I (HTLV-I) proviral DNA and its expression in the lymph nodes, HTLV-I DNA and tax/rex mRDA were directly amplified by polymerase chain reaction in situ hybridization (PCR/ISH), and reverse transcription (RT)-PCR/ISH [RT-PCR/ISH]. We studied 24 lymph nodes from patients with adult T-cell leukemia/lymphoma (ATLL), incipient ATLL (I-ATLL), and HTLV-I associated lymphadenitis dermatopathic type (HAL-D) and enlarged paracortical type (HAL-EP). In ATLL, 40-60% of the nucleated cells were positive for for HTLV-I proviral DNA by PCR/ISH, while in I-ATLL and HAL, respectively 5-20% and less than 1-5% of cells were positive.

A novel AvaI polymorphism within exon 5 of the rhodopsin gene.

We identified a novel AvaI polymorphism within 3' non-coding region within exon 5 of the human rhodopsin gene and determined the allele frequency in a Japanese population. The polymorphism was found to be due to A/G transversion at nucleotide 5510 of the gene.

Study of hereditary cerebellar degeneration in cats.

Objective: To elucidate the nature of ataxia observed in 3 cats spanning 2 generations.

Design: Experimental breeding was attempted to confirm heritability of the disease and establish the mode of inheritance; the original 3 cats and their offspring were studied.

Animals: Seven diseased cats spanning 3 generations and 11 neurologically normal cats.

cDNA cloning and characterization of murine transcriptional enhancer factor-1-related protein 1, a transcription factor that binds to the M-CAT motif.

The M-CAT motif is a cis-regulatory DNA sequence that is essential for muscle-specific transcription of several genes. Previously, we had shown that both muscle-specific (A1) and ubiquitous (A2) factors bind to an essential M-CAT motif in the myosin heavy chain beta gene and that the ubiquitous factor is transcriptional enhancer factor (TEF)-1. Here we report the isolation of mouse cDNAs encoding two forms (a and b) of a TEF-1-related protein, TEFR1.

The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.

p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein-coupled receptors activate p21 ras in metazoans.