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R Kubota

Publications and source records attributed to R Kubota.

At least 37 records · Page 2Linked to original sources
PubMed2000

The myocilin (MYOC) gene expression in the human trabecular meshwork.

PURPOSE: We previously reported a novel cytoskeletal protein with a myosin-like domain which is localized in the ciliary rootlet and basal body of connecting cilium of photoreceptor and hence we named it 'myocilin'. It was soon realized that myocilin is identical to a protein called TIGR (trabecular meshwork inducible glucocorticoid response protein) which was found to be responsible for the pathogenesis of juvenile open angle glaucoma. In this study, we employed in situ RNA hybridization to examine the myocilin (MYOC)/ TIGR gene expression in the trabecular meshworks of glaucomatous and nonglaucomatous eyes. METHODS: The glaucomatous specimens were obtained by trabeculectomy from the patients with primary open angle glaucoma (POAG), chronic angle closure glaucoma (CACG) and steroid glaucoma, respectively, and the nonglaucomatous specimens were obtained from a victim of traffic accident at autopsy and from a patient with maxillary sinus carcinoma at enucleation for the operation. The in situ RNA hybridization was carried out with digoxigenin-labeled sense and antisense RNA probes. RESULTS: In all cases, hybridization signals were detected primarily in the trabecular meshwork cells and secondarily in the fibroblast-like cells of corneoscleral wall. CONCLUSIONS: Myocilin gene is expressed clearly in the trabecular meshwork cells of both glaucomatous and nonglaucomatous eyes.

Aged↗
PubMed1999

Activation of macrophages/microglia with the calcium-binding proteins MRP14 and MRP8 is related to the lesional activities in the spinal cord of HTLV-I associated myelopathy.

Macrophages and microglia may play an important role in the pathogenesis of chronic inflammatory process in HTLV-I associated myelopathy (HAM) and tropical spastic paraparesis (TSP). However, the etiology and cellular mechanism of chronic inflammation are poorly understood in HAM/TSP. To help to define the roles of macrophages and microglia we analyzed the various patterns of macrophage and microglia activation in the central nervous system (CNS) of HAM/TSP using several monoclonal antibodies recognizing the different states of activation. The results indicate that a large number of macrophages and microglia express both MRP14 and MRP8 in active-chronic inflammatory lesions of the patients with a short duration of illness (2.5 years). In the patient whose duration of illness was 4.5 years, perivascular and parenchymal macrophages and microglia were reactive for MRP8 but not for MRP14. In contrast, MRP14 and MRP8 were negative on the perivascular and parenchymal macrophages and microglia in inactive-chronic lesions and in controls. This study suggests that (a) activated macrophages and microglia as well as CD4+ T lymphocytes and CD8+ cytotoxic T lymphocytes are main components of the inflammatory process in the CNS in HAM/TSP, (b) activation of macrophages and microglia is related to the amount of HTLV-I proviral DNA in situ.

Aged↗
PubMed1999

Rapid detection and identification of two lineages of influenza B strains with monoclonal antibodies.

Monoclonal antibodies (Mabs) against influenza B virus were obtained by immunizing mice with B/Nagasaki/1/87, one of the strains of the B/Victoria group. Immunoprecipitation analysis revealed that individual Mabs precipitated the nucleoprotein (NP), the matrix protein (M) or the hemagglutinin protein (HA). By using these Mabs by the peroxidase-antiperoxidase (PAP) staining method, a rapid detection and identification method for influenza B virus was established. Monolayers of Madin-Darby canine kidney cells in microplates were infected with each-strain and incubated for about 24 h, and then were subjected to the PAP staining method using the Mabs as the first antibody. Influenza B virus strains are classified into two major phylogenetic trees, the B/Victoria group and the B/Yamagata group. When anti-NP and anti-M antibodies were used in the PAP staining method, all 13 influenza B virus strains isolated from clinical specimens between 1940 and 1994 were detected regardless of the antigenic drift of the influenza virus. On the other hand, several anti-HA Mabs which reacted specifically with the strains of the B/Victoria group, did not react with any strain of the B/Yamagata group. In the 1996/97 influenza season in Osaka Prefecture in Japan, two antigenically distinct groups of influenza B virus strains were isolated. They belonged to different phylogenetic trees and were clearly distinguishable by the PAP staining method with anti-HA Mabs.

Animals↗
PubMed1998

The effect of human beta2-microglobulin on major histocompatibility complex I peptide loading and the engineering of a high affinity variant. Implications for peptide-based vaccines.

The ability to directly load cell surface major histocompatibility complex (MHC) class I molecules with peptides provides a potentially powerful approach toward the development of vaccines to generate cell-mediated immunity. We demonstrate that exogenous beta2-microglobulin (beta2m) stabilizes human cell surface MHC I molecules and facilitates their loading with exogenous peptides. Additionally, using three-dimensional crystal structures and known interaction sites between MHC I heavy chains and beta2m, we engineered variants of human beta2m (hbeta2m) with a single serine substitution at residue 55. This alteration was predicted to promote hydrophobic interactions at the MHC I heavy chain/beta2m interface and displace an ordered water molecule. Compared with hbeta2m, the serine to valine substitution at residue 55 had improved ability to bind to cell surface HLA-A1, HLA-A2, and HLA-A3 molecules, facilitate exogenous peptide loading, and promote recognition by peptide-specific T cells. The inclusion of hbeta2m or higher affinity variants when pulsing cells with MHC-restricted peptides increases the efficiency of peptide loading 50-80-fold. Therefore, the inclusion of hbeta2m in peptide-based vaccines may increase cell surface antigen densities above thresholds that allow recognition of peptide antigens by the immune system, particularly for cryptic, subdominant, or marginally antigenic peptides.

Antigen Presentation↗
PubMed1998

Mouse myocilin (Myoc) gene expression in ocular tissues.

Human myocilin is identical to TIGR (trabecular meshwork inducible glucocorticoid response) which is responsible for the pathogenesis of juvenile-onset primary open angle glaucoma (GLCIA). We have isolated cDNA for mouse myocilin (Myoc) and investigated mouse myocilin gene expression in ocular tissues with in situ RNA hybridization. Hybridization signals were observed in the iris, ciliary body, trabecular meshwork, sclera, and retina in the mouse eye. The marked signals were seen in trabecular meshwork cells and the anterior portion of sclera. These findings suggest that myocilin mutation could affect the capacity of aqueous outflow and cause elevation of the intraocular pressure which is involved in the pathogenesis of glaucoma.

Amino Acid Sequence↗
PubMed1998

Demonstration of human T lymphotropic virus type I (HTLV-I) tax-specific CD8+ lymphocytes directly in peripheral blood of HTLV-I-associated myelopathy/tropical spastic paraparesis patients by intracellular cytokine detection.

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease caused by HTLV-I infection and has been associated with elevated levels of several proinflammatory cytokines in both serum and cerebrospinal fluid. It is unknown what kind of cells secrete these cytokines and if HTLV-I Ags are associated with this phenomenon. Here, we investigated the expression of cytokines in PBL from eight HAM/TSP patients, nine HTLV-I-infected asymptomatic carriers, and seven healthy controls by flow cytometry combined with intracellular cytokine staining. PBL were cultured with brefeldin A without mitogen and IL-2 for 14 h. Under these conditions, CD8+ cells produced proinflammatory cytokines including IFN-gamma, TNF-alpha, and IL-2, which were significantly elevated in HAM/TSP patients. The proportion of CD8+ cells producing IFN-gamma in HAM/TSP patients, asymptomatic carriers, and healthy controls were, on average, 4.9, 0.4, and 0.3%, respectively. IFN-gamma production by these CD8+ cells was suppressed by anti-HLA-class I Ab. Purified CD8+ cells from an HLA-A2 HAM/TSP patient produced IFN-gamma by cocultivation with autologous CD4 cells, the main reservoir of HTLV-I in vivo, or allogenic HLA-A2+ B cells pulsed with a known immunodominant HTLV-I tax peptide. These data suggest that high levels of circulating HTLV-I-specific CD8+ T lymphocytes have the potential to produce proinflammatory cytokines and may promote inflammatory responses to HTLV-I in HAM/TSP patients.

CD8-Positive T-Lymphocytes↗
PubMed1998

Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8(+) lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8(+) T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

Flow Cytometry↗
PubMed1998

Genomic organization of the human myocilin gene (MYOC) responsible for primary open angle glaucoma (GLC1A).

Myocilin is a newly found cytoskeletal protein involved in the morphogenesis of the basal body, a major microtubule organizing center, of the ciliated epithelium. It was recently realized that myocilin is virtually identical to the independently reported protein TIGR (trabecular meshwork-induced glucocorticoid response), which is responsible for the pathogenesis of chromosome 1q-linked primary open angle glaucoma (GLC1A). In this paper, we determined the genomic organization of the myocilin (MYOC/TIGR) gene by analyzing the nucleotide sequence of the BAC clones containing the MYOC/TIGR gene. The MYOC/TIGR gene consists of three exons. Each of the two splice donor and acceptor sites agrees well with the GT/AG rule. Primer sets to amplify each of the three exons are designed. The 5'-flanking region of MYOC gene contains the TGTTCT sequence overlapped with a palindromic sequence TTCTTTTTAAAAAGAA, which appears to be a glucocorticoid responsive element. There is also a unique sequence of dinucleotide repeat [(GT)2AA(GT)4AC(GT)13] which may also serve as a regulatory element. These results should aid in further detection of the MYOC/TIGR gene mutation and in depth understanding of the tissue-specific MYOC gene regulation.

Amino Acid Sequence↗
PubMed1998

Reduction in HTLV-I proviral load and spontaneous lymphoproliferation in HTLV-I-associated myelopathy/tropical spastic paraparesis patients treated with humanized anti-Tac.

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease that results from an interaction of retroviral infection and immune activation. In this study, five doses (1 mg/kg) of humanized anti-Tac antibody were administered to 9 HAM/TSP patients at weeks 0, 2, 6, 10, and 14. Preliminary immunological studies on HAM/TSP patients treated with humanized anti-Tac indicate that there is a selective down-regulation of activated T cells and a decrease in the HTLV-I viral load in peripheral blood lymphocytes, most likely through the selective removal of HTLV-I-infected, activated CD4+ lymphocytes.

Antibodies↗
PubMed1998

Application of subtype-specific monoclonal antibodies for rapid detection and identification of influenza A and B viruses.

We established a rapid method for the identification of influenza A and B virus strains: the peroxidase-antiperoxidase (PAP) staining method with two subtype-specific murine monoclonal antibodies, C179 (H1 and H2 specific) and F49 (H3 specific), and an anti-influenza B virus rabbit polyclonal serum. The types and subtypes of 160 strains were examined, and 158 strains were identified to be the same by the hemagglutination-inhibition (HI) test and the PAP method. In contrast to the results by the HI test, two strains were revealed to be a mixture of two subtypes (H1 and H3) by the PAP method, which was confirmed by plaque cloning. We further analyzed clinical specimens by the PAP method by directly inoculating specimens into Madin-Darby canine kidney cells in microplates. After 40 h of incubation, the types and subtypes of viruses in 52 of 152 specimens were clearly identified. Since the reactivities of the two monoclonal antibodies are not influenced by the antigenic drift of influenza virus, the newly developed method should be applicable not only for rapid diagnosis but also for the epidemiological study of influenza.

Animals↗
PubMed1997

Cloning of the cDNA for a new member of the immunoglobulin superfamily (ISLR) containing leucine-rich repeat (LRR).

We isolated cDNAs for a novel protein with a calculated molecular mass of 46 kDa, containing a leucine-rich repeat (LRR) with conserved flanking sequences and a C2-type immunoglobulin (Ig)-like domain. This novel protein was considered to be a new member of the Ig superfamily and was named ISLR (immunoglobulin superfamily containing LRR). These domains are known to be important for protein-protein interaction or cell adhesion, and therefore it is possible that the novel protein ISLR may also interact with other proteins or cells. Northern blot analysis showed the presence of a 2.4-kb transcript in various human tissues including retina, heart, skeletal muscle, prostate, ovary, small intestine, thyroid, adrenal cortex, testis, stomach, and spinal cord as well as fetal lung and fetal kidney. The ISLR gene was mapped on human chromosome 15q23-q24 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1997

Human T cell leukemia virus type I (HTLV-I)-specific CD8+ CTL clones from patients with HTLV-I-associated neurologic disease secrete proinflammatory cytokines, chemokines, and matrix metalloproteinase.

Human T cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurologic disease characterized by marked degeneration of the spinal cord and the presence of infiltrating CD8+ T cells and macrophages. HAM/TSP patients have very high frequencies of HTLV-I-specific CD8+ CTL in peripheral blood and in cerebrospinal fluid. In this study, we show that HAM/TSP patients also have elevated levels of peripheral blood CD8+ T cells that produce intracellular IFN-gamma. To address the potential role of soluble mediators secreted by CD8+ T cells in the pathogenesis of HAM/TSP, we have analyzed the capacity of a panel of nine HTLV-I-specific CD8+ CTL clones derived from three HAM/TSP patients to secrete cytokines, chemokines, and matrix metalloproteinases. The results demonstrate that the majority of these CTL clones secrete IFN-gamma, TNF-alpha, macrophage-inflammatory protein-1alpha and -1beta, IL-16, and matrix metalloproteinase-9. These findings indicate that HTLV-I-specific CD8+ CTL are an important source of proinflammatory soluble mediators that may contribute significantly to the pathogenesis of HAM/TSP.

Chemokines↗
PubMed1997

Human BAC library: construction and rapid screening.

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.

B-Lymphocytes↗
PubMed1997

A novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression, and chromosomal mapping.

We have isolated a human cDNA clone encoding a novel acidic protein of MW 55,000 that we designated "myocilin" since it has homology to myosin and is localized preferentially in the ciliary rootlet and basal body of the connecting cilium of photoreceptor cells. The deduced amino acid sequence of human myocilin showed significant homologies with nonmuscle myosin of Dictyostelium discoideum in the N-terminal region and also with olfactomedin of bullfrog in the C-terminal region. Myocilin contained a leucine zipper-like motif similar to that seen in kinectin and other cytoskeletal proteins. These findings suggest that myocilin is a novel cytoskeletal protein involved in the morphogenesis of ciliated neuroepithelium such as photoreceptor cells. The myocilin gene (MYOC) was mapped to human chromosome 1q23-q24 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1997

Human retina-specific amine oxidase (RAO): cDNA cloning, tissue expression, and chromosomal mapping.

In search of candidate genes for hereditary retinal disease, we have employed a subtractive and differential cDNA cloning strategy and isolated a novel retina-specific cDNA. Nucleotide sequence analysis revealed an open reading frame of 2187 bp, which encodes a 729-amino-acid protein with a calculated molecular mass of 80,644 Da. The putative protein contained a conserved domain of copper amine oxidase, which is found in various species from bacteria to mammals. It showed the highest homology to bovine serum amine oxidase, which is believed to control the level of serum biogenic amines. Northern blot analysis of human adult and fetal tissues revealed that the protein is expressed abundantly and specifically in retina as a 2.7-kb transcript. Thus, we considered this protein a human retina-specific amine oxidase (RAO). The RAO gene (AOC2) was mapped by fluorescence in situ hybridization to human chromosome 17q21. We propose that AOC2 may be a candidate gene for hereditary ocular diseases.

Adult↗
PubMed1997

cis-Acting inhibitory elements within the pol-env region of human T-cell leukemia virus type 1 possibly involved in viral persistence.

Human T-cell leukemia virus type 1 (HTLV-1) remains latent throughout the life of the carrier, with cells containing the provirus and viral gene expression efficiently down-regulated. On a molecular level, exactly how viruses are down-regulated in vivo remains unresolved. We described here the possibility that down-regulation results from the presence of inhibitory elements within the gag-env region of the provirus in fresh peripheral blood mononuclear cells from carriers. In vitro experiments then revealed that potent cis-acting inhibitory elements (CIEs) are indeed contained in two discrete fragments from the pol region and weaker ones in the env region. The effect of CIEs is relieved by the HTLV-1 posttranscriptional regulator Rex through binding to the Rex-responsive element (RxRE), suggesting that Rex might interfere with pre-mRNA degradation and/or activate the export of mRNA molecules harboring both of the inhibitory elements and RxRE on the same RNA molecule. Thus, we propose the hypothesis that such functions of CIEs may be involved in HTLV-1 persistence.

Cell Line↗