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Biomedical subjects

R Kubota

Publications and source records attributed to R Kubota.

At least 55 records · Page 3Linked to original sources
PubMed1997

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. Immunohistochemical analysis demonstrated T-cell dominance. The numbers of CD4+ T cells and CD8+ T cells were equally present in patients with shorter clinical course. Apoptosis of helper/inducer T cells were observed in the presence of TIA1+ cytotoxic T cells in these active inflammatory lesions. Inflammatory infiltrates were markedly decreased and CD8+/TIA1- T cells were predominated over CD4+ cells in patients with prolonged clinical course. HTLV-1 proviral DNA amounts in the freshly frozen spinal cord measured by quantitative PCR were well correlated with the numbers of infiltrated CD4+ cells. In situ PCR of HTLV-1 proviral DNA using multi-primary pairs demonstrated the presence of HTLV-1 infected cells exclusively in the mononuclear infiltrates of perivascular areas. From these findings, it is suggested that the target of the inflammatory process seen in HAM/TSP lesions may be HTLV-1 infected CD4+ T cells infiltrating the spinal cord.

Apoptosis↗
PubMed1996

Chromosomal mapping of a family of human glutamine synthetase genes: functional gene (GLUL) on 1q25, pseudogene (GLULP) on 9p13, and three related genes (GLULL1, GLULL2, GLULL3) on 5q33, 11p15, and 11q24.

Glutamine synthetase (GS) is a ubiquitous enzyme that catalyzes the ATP-dependent conversion of glutamate to glutamine using ammonia as the nitrogen source. Using human GS cDNA as a probe, a bacterial artificial chromosome (BAC) library consisting of two-fold coverage of the human genome was screened, and 18 clones were obtained. The restriction analysis of the human insert DNAs provided a basis to divide these 18 BAC clones into five groups, suggesting the existence of a GS gene family in the human genome. PCR analysis using three sets of primers designed from the reported sequences for GS cDNA and a processed pseudogene identified the corresponding BAC clones. Fluorescence in situ hybridization analysis revealed the chromosomal localization of these five genes: the GS gene to 1q25 (GLUL), the processed pseudogene to 9p13 (GLULP), and three related gene to 5q33 (GLULL1), 11p15 (GLULL2), and 11q24 (GLULL3), respectively.

Chromosome Mapping↗
PubMed1996

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. These observations indicate that frequent mutations are not unique to the sequence encoding the most effective antigen for cytotoxic T lymphocytes, but also seen in the LTR, a non-coding sequence. Thus, frequent mutations seem to occur during the viral replication process rather than the selection of rare mutants by immune surveillance.

Base Sequence↗
PubMed1996

Feasibility of fluorine-18-fluorophenylalanine for tumor imaging compared with carbon-11-L-methionine.

UNLABELLED: L-[methyl-11C]methionine (11C-Met) is a useful tracer for tumor imaging with PET. The drawbacks include a short half-life and high physiological accumulation in abdominal organs. To overcome these shortfalls, the feasible use of [18F]fluorophenylalanine (18F-Phe), which shares the same amino acid transport system with Met, for tumor imaging was examined. METHODS: The time course of tissue distribution of 18F-Phe and the tumor uptake response to radiotherapy were compared with 14C-Met and [3H] thymidine (3H-Thd) in the rat AH109A tumor model. Intratumoral distribution of 18F-Phe was compared with 14C-Met and 14C-Thd using double-tracer macroautoradiography (ARG). We also evaluated whole-body ARG. RESULTS: Tumor uptake of 18F-Phe peaked at 60 min postinjection and was higher than that of the liver, intestine and kidney but lower than the pancreas. Tumor uptake of 18F-Phe was similar to that of 14C-Met. Tumor-to-blood and tumor-to-muscle ratios were higher in 14C-Met compared with that of 18F-Phe because of the rapid blood clearance of 14C-Met. With whole-body ARG, the tumor was clearly visualized with high contrast. Radiotherapeutic response of tumor uptake of 18F-Phe was as rapid as that with 14C-Met and with 3H-Thd. Intratumoral distribution of 18F-Phe and 14C-Met were identical, and 18F-Phe and 14C-Thd were similar. CONCLUSION: Fluorine-18-Phe seems to be a potentially useful amino acid tracer for tumor imaging with a longer half-life than 11C, with higher tumor contrast in the abdomen than Met and a similar sensitive response to radiotherapy.

Animals↗
PubMed1996

Re-evaluation of myocardial FDG uptake in hyperglycemia.

UNLABELLED: Myocardial [18F] fluorodeoxyglucose (FDG) uptake depends on several metabolic variables in vivo. The effect of different levels of experimentally induced hyperglycemia on myocardial FDG uptake was examined. METHODS: FDG uptake was studied in young Donryu rats 1 hr after intravenous injection under various pretreatments that increased serum glucose levels. Serum samples were analyzed for glucose, insulin and free fatty acids. Myocardial distribution of FDG was examined with autoradiography. RESULTS: Administration of glucose (n = 42), triiodothyronine (n = 7), epinephrine (n = 7), dehydroascorbic acid (n = 5) and 4 mg streptozotocin (Szt, n = 10) increased glucose levels to 120-200 mg/dl. Dexamethasone (Dex, n = 34) and 6 mg Szt (n = 6) increased glucose levels to 200-450 mg/dl. Myocardial FDG uptake increased proportionately with increases in serum glucose level up to 200 mg/dl. In severe hyperglycemia (serum glucose: 200-450 mg/dl), however, the FDG uptake decreased and did not correlate with blood glucose level. A study of fractional FDG uptake calibrated by the arterial FDG curve confirmed the same results. Heterogeneous distribution of FDG was observed in the myocardium, both in fasting and in severe hyperglycemic conditions. The pattern of FDG uptake by skeletal muscles was similar to that of the myocardium, although the uptake was lower than that in the myocardium. Changes in insulin and free fatty acids levels could not explain the FDG uptake pattern in severe hyperglycemia. Blood FDG uptake level remained constant regardless of glucose level. CONCLUSION: Hyperglycemia induced a biphasic pattern of myocardial FDG uptake, common with skeletal muscles. The understanding of myocardial FDG uptake characteristics and their dependence on blood glucose is helpful in interpreting myocardial FDG-PET images.

Animals↗
PubMed1995

Human T-cell leukemia virus type-1 (HTLV-1) Tax is expressed at the same level in infected cells of HTLV-1-associated myelopathy or tropical spastic paraparesis patients as in asymptomatic carriers but at a lower level in adult T-cell leukemia cells.

Patients with human T-cell leukemia virus type-1 (HTLV-1)-associated myelopathy (HAM/TSP) generally harbor a much greater population of HTLV-1-infected T cells in peripheral blood mononuclear cells (PBMCs) than asymptomatic carriers. These cells are not malignant but frequently proliferate clonally. To investigate the possibility that higher expression of the viral activator Tax induces preferential proliferation of infected nonmalignant T cells in HAM/TSP patients, the expression of Tax mRNA in fresh PBMCs was analyzed by reverse transcriptase-mediated polymerase chain reaction. Total amount of Tax mRNA was higher in HAM/TSP patients than in carriers, but the expression level was almost the same as that in asymptomatic carriers when compared per infected cell. The expression levels in adult T-cell leukemia patients were significantly lower than those in HAM/TSP patients and asymptomatic carriers. These results indicate that tax gene is not expressed at a continuously high level in HAM/TSP patients who carry a high population of infected T cells, even in those with clonally proliferated infected T cells.

Base Sequence↗
PubMed1995

Effects of radiotherapy on the cellular uptake of carbon-14 labeled L-methionine in tumor tissue.

In order to examine in vivo effects of irradiation on tumor uptake of L-[methyl-11C]methionine at a cellular level, the distribution of L-[methyl-14C]methionine (Met) in a rat AH109A tumor model was investigated using microautoradiography. Silver grain density of tumor cell layer decreased rapidly within the first day after 20 Gy of irradiation, and continued to fall during day 2. Grain density of granulation tissue was 25% of tumor cells and was unchanged after irradiation. Macrophages and necrotic tissue showed low grain density and a small post-irradiation decrease. One day after irradiation, tumor cell showed giant cell formation and decreased cell density per unit area. The number of grains was greater in giant tumor cells than in non-irradiated tumor cell. Rapid response of Met uptake by tumor cells without a significant uptake by granulation tissue and macrophages suggest that 11C-Met is a suitable tracer for monitoring tumor radiotherapy with positron emission tomography.

Animals↗
PubMed1995

Sequence analysis of human T cell lymphotropic virus type I (HTLV-I) Env genes amplified from central nervous system tissues of patients with HTLV-I-associated myelopathy or leukemia.

Human T cell lymphotropic virus type I (HTLV-I) is a retrovirus that has been linked to HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), a chronic or inflammatory neurological disease with some resemblance to multiple sclerosis. We used the polymerase chain reaction to amplify viral env genes in foci of inflammation and demyelination in the nervous system to adduce additional evidence of the association of HTLV-I with the neuropathological changes in HAM/TSP, and document in this report such an association. We also sought evidence of a distinct viral species in the lesions by amplifying, cloning and sequencing the env genes from tissues sections in which there were pathological changes. We did not find changes in the env gene that correlated with HTLV-I-associated neurological disease vs adult T cell leukemia or with the nervous system vs peripheral blood and lymphoid organs. We did, however, find evidence of extensive mutation and possibly deletions in the env gene in HTLV-I-associated neurological disease. We interpret these findings of increased genetic diversity as a reflection of higher rates of viral replication in HTLV-I-associated myelopathy that support a model of pathogenesis in which increased viral replication activates immune cells that subsequently enter the nervous system and cause injury by immunopathological mechanisms.

Adult↗
PubMed1995

Frequent mutation in pX region of HTLV-1 is observed in HAM/TSP patients, but is not specifically associated with the central nervous system lesions.

Human T-cell leukemia virus type 1 (HTLV-1) is an etiologic agent of adult T-cell leukemia (ATL) and of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Recently it has been reported that defective HTLV-1 provirus was detected frequently in the central nervous system (CNS) lesions of HAM/TSP patients. Here we investigated sequence variations of the pX region of HTLV-1 in the CNS and peripheral blood lymphocytes (PBL) of the same patient. The results analyzing 9-13 clones isolated from each specimen indicated that the pX region is highly variable within a patient with HAM/TSP, and the mutations were found at almost random positions within the sequences analyzed. The frequency and pattern of those mutations did not appear to differ significantly between the CNS and PBL of the same patient, although they differed among patients. Similarly, frequent mutations were observed in an asymptomatic carrier of HTLV-1, although the variability was moderate, suggesting that the high variability of the pX sequence is not a specific event in HAM/TSP. However, one asymptomatic carrier showed much less frequent variations very similarly to an ATL patient; both of them harbored clonally expanded infected cells. Thus the apparent low variability was explained by clonal selection of a single species of the provirus by the clonal proliferation of infected cells. These results clearly indicate that mutations including defectives are not specifically associated with the CNS lesions in HAM/TSP patients, but suggest that the random mutations simply reflect the rate of viral replication in individuals and the variants were not inherited frequently.

Adult↗
PubMed1995

High accumulation of fluorine-18-fluorodeoxyglucose in turpentine-induced inflammatory tissue.

UNLABELLED: Fluorine-18-2-deoxy-2-fluoro-D-glucose ([18F]FDG) uptake and distribution in an experimentally induced inflammatory tissue were investigated. METHODS: Rats were subcutaneously inoculated with turpentine oil to induce inflammation and used for tissue distribution studies and autoradiography. RESULTS: Time course study of [18F]FDG tissue distribution showed that the uptake in inflammatory tissue increased gradually until 60 min and then decreased. A longitudinal study of [18F]FDG tissue distribution showed that the uptake increased progressively to a peak 4 days after inoculation and then decreased. On the fourth day postinoculation, a section of inflammatory tissue showed characteristic changes of chronic inflammation. Macro- and micro-autoradiography showed a high density of silver grains in the abscess wall consisting of an inflammatory cell layer and granulation tissue. Grain counting on micro-autoradiography of the abscess wall showed that the highest grain density was found in the marginal zone of young fibroblasts, endothelial cells of vessels and phagocytes of neutrophils and macrophages, followed by that in the neutrophil layer and granulation tissue. CONCLUSION: Our results indicate that [18F]FDG PET may be useful in detecting and monitoring chronic inflammatory processes.

Animals↗
PubMed1995

Methionine uptake by tumor tissue: a microautoradiographic comparison with FDG.

UNLABELLED: L-methyl-11C-methionine (11C-Met) and 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) are used for tumor diagnosis and treatment evaluation by PET. In order to examine the role of these tracers in cancer imaging, intratumoral properties of 14C-Met were studied and compared to those of 18F-FDG. METHODS: The distribution of 14C-Met in various cellular elements of two different mouse malignant tumor tissues, MH134 and FM3A, was analyzed serially using microautoradiography within a period of 120 min after injection of the tracer. RESULTS: Carbon-14-Met and 18F-FDG showed different distributions in tumor tissue. Carbon-14-Met uptake by the tumor was mostly by viable cancer cells. The uptake by macrophages and other cellular components was low. The uptake was higher in the highly proliferative tumor but did not reflect protein synthesis. The rapid and slow growing tumors demonstrated that 14C-Met uptake ratio was lower than that of 18F-FDG, reflecting de novo DNA synthesis ratio. CONCLUSION: Carbon-14-Met uptake represents the presence of viable cancer cells. Carbon-11-Met may be suitable for treatment evaluation of individual tumors but not growth rates of different tumors. Fluorine-18-FDG reflects tumor-host immune system reaction and is an excellent tool for pretreatment evaluation of tumors and determination of tumor proliferative activity.

Animals↗
PubMed1994

HTLV-I proviral DNA amount correlates with infiltrating CD4+ lymphocytes in the spinal cord from patients with HTLV-I-associated myelopathy.

A quantitative method utilizing polymerase chain reaction was employed to evaluate the amount of human T-cell leukemia virus type I (HTLV-I) proviral DNA in the affected spinal cords from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Central nervous system (CNS) tissues were obtained at post-mortem from five patients with HAM/TSP, who vary in the duration of illness from 2.5-10 years, and one patient with adult T-cell leukemia (ATL), who had leukemic cell infiltration in the CNS. The presence of HTLV-I pX and pol sequences in the CNS tissues were demonstrated in all patients examined. In HAM/TSP, the proviral DNA quantified in the thoracic cord was 0.002-2 copies per 100 tissue cells, and that in the peripheral blood mononuclear cells (PBMC) was 2-8 copies per 100 PBMC. The proviral DNA amount in the thoracic cord of the patient with ATL was 0.4 copies per 100 tissue cells. An apparent propensity for the amount of integrated HTLV-I in the thoracic cord to decrease with the disease duration in patients with HAM/TSP was observed. The decline in HTLV-I proviral DNA amount in the thoracic cord lesions was paralleled with the alteration of proportion of CD4+ T lymphocytes in patients with HAM/TSP. These findings suggest that preferential virus reservoir may be infiltrating CD4+ T lymphocytes in the spinal cord lesions of patients with HAM/TSP, and HTLV-I infection in the CNS of patients is declining with the disease duration in spite of the chronic course of neurological manifestations at least in some patients with HAM/TSP.

Aged↗
PubMed1994

Investigation of tumor invasion with [111In]antimyosin.

Accumulation of [111In]antimyosin in various types of tumors has been reported. In order to test the possibility that the destruction of muscle tissue by tumor invasion could induce high uptake of [111In]antimyosin, we investigated the distribution of [111In]antimyosin in tumor and muscle using autoradiography (ARG). High uptake of [111In]antimyosin was observed both in rabbit papilloma VX2 and rat hepatoma AH109A by scintigraphy and tissue distribution studies, and its concentration was found to be highest at the periphery and outer surface of the tumors using whole-body ARG. However, in micro- and macro-ARG, high grain density was observed in granulation tissue with macrophage infiltration between the tumor and muscle, not in the muscle tissue invaded by tumor cell. Significant non-specific accumulation of [111In]antimyosin in inflammatory tissue surrounding the tumor was suggested.

Animals↗
PubMed1994

A spontaneous point mutation in the human T-cell leukemia virus type 1 pX gene leads to expression of a novel doubly spliced pX-mRNA that encodes a 25-kD, amino-terminal deleted rex protein.

Primary RNA transcripts of the human T-cell leukemia virus type 1 (HTLV-1) are processed into mature mRNA by a complex series of splicing events. In this paper, we report the finding of a novel doubly spliced pX mRNA in two out of eight HTLV-1-infected cell lines and in one out of 13 peripheral blood mononuclear cells from HTLV-1-infected individuals. The second splicing for this novel pX mRNA is different from that for the known doubly spliced pX mRNA. A novel acceptor site in this splicing was generated by a single point mutation (G to A) at nucleotide 7,337 of the pX gene. This mRNA contained a complete open reading frame that encodes an amino-terminal truncated p27rex protein with 189 amino acids. A new 25-kD protein was detected in the cell lines expressing the novel pX mRNA by an antibody against the carboxy-terminal peptide of p27rex and was termed p25rex. Although the function of p25rex is not clear, we clarified that p25rex is a cytoplasmic phosphoprotein and its function is different from the transcriptional regulator function of p27rex. The possibility that the mutated virus is replicable only in cells coinfected with the wild type HTLV-1 may explain why the incidence of the mutants observed here is low.

Amino Acid Sequence↗
PubMed1994

Apoptosis of T lymphocytes in the spinal cord lesions in HTLV-I-associated myelopathy: a possible mechanism to control viral infection in the central nervous system.

Immunocytochemical staining of spinal cords from five autopsied patients with HAM/TSP was performed using the monoclonal antibody TIA-1, a marker of cytotoxic T lymphocytes (CTL). Many TIA-1+, CD8+ cells are distributed in active inflammatory lesions. The number of TIA-1+ cells is related to the amount of HTLV-I proviral DNA in situ. The protein TIA-1 has been associated with the induction of apoptosis in target cells. In active inflammatory lesions, we found cells undergoing apoptosis, most of them identified as helper-inducer CD45RO T lymphocytes, which were consistent with in vivo cellular tropism of HTLV-I in patients with HAM/TSP. These findings suggest that CTL-induced apoptosis of T lymphocytes may be one of the possible mechanisms which eliminate HTLV-I-infected cells from the central nervous system. In addition, many T lymphocytes in the inflammatory lesions expressed bcl-2 oncoprotein, suggesting that infiltrated T lymphocytes may be resistant to apoptosis. Expression of bcl-2 oncoprotein may explain the longstanding inflammatory process in the central nervous system of HAM/TSP.

Antibodies, Monoclonal↗