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Biomedical subjects

C Miyamoto

Publications and source records attributed to C Miyamoto.

At least 19 recordsLinked to original sources
PubMed2001

Toxic shock syndrome toxin-1 accelerated collagen-induced arthritis in mice.

The aim of this study was to explore the roles of toxic shock syndrome toxin-1 (TSST-1) in collagen-induced arthritis (CIA). DBA/1 mice were immunized with type II collagen (CII) and treated with TSST-1. Intraperitoneal and intravenous injections of TSST-1 aggravated CIA, enhancing its incidence and severity. CIA was accompanied by an increase in anti-CII IgG Ab levels. Intraperitoneal administration with TSST-1 enhanced IFN-gamma, TNF-alpha, and IL-4 production in DBA/1 mice. We discovered the mRNA expressions of IFN-gamma, IL-2, TNF-alpha, IL-1beta, and iNOS in spleen cells stimulated with TSST-1 in vitro. However, IL-12 and IL-4 mRNA expression were seen constitutively without stimulation. Only a little increase of IL-12 and IL-4 mRNA expression was seen at 2-3 h after treatment with TSST-1. Our experiments demonstrated that CIA was aggravated by the treatment with TSST-1, which may have induced various proinflammatory cytokines and the production of both Th1 and Th2 cytokines.

Animals↗
PubMed2001

Genetic polymorphism of the MxA gene promoter and interferon responsiveness of hepatitis C patients: revisited by analyzing two SNP sites (-123 and -88) in vivo and in vitro.

We have previously reported a single nucleotide polymorphism (SNP) at nucleotide (nt) position -88 (G or T) within an interferon-stimulated response element-like sequence in the promoter region of the MxA gene, which correlated with responsiveness of hepatitis C patients to interferon. Upstream of it, we then identified another SNP (C or A at nt -123) and investigated whether this SNP also correlates with interferon responsiveness. The two SNPs showed a high linkage to each other: all the individuals having G at -88 had C at -123, and 73% of those having T at -88 had A at -123. As was expected from this observation, the SNP at -123 also exhibited a correlation with interferon responsiveness (C/C homozygotes were more frequent among nonresponders than among responders: 65% of 107 vs. 40% of 52, p = 0.0028). These in vivo data from patients were further supported by results from in vitro experiments. The MxA promoter sequence with A at -123 and T at -88 showed about 4-fold higher activity of upregulating the downstream reporter gene than that with C at -123 and G at -88, in a luciferase reporter assay.

GTP-Binding Proteins↗
PubMed2001

Treatment of malignant astrocytomas with repetitive resections: a longitudinal study.

BACKGROUND: The impact of repeated surgical resection on the survivorship of patients with malignant astrocytomas is an issue of some controversy in the medical literature. OBJECTIVES: To clarify this issue through a retrospective analysis of treatment outcomes in a brain tumor clinic. METHODS: The patient records from the Brain Tumor Clinic at Hahnemann University Hospital for the period 1988 to 2000 were reviewed. From these, 112 cases of glioblastoma multiforme and 50 cases of anaplastic astrocytoma were chosen for analysis. RESULTS: The group of patients with glioblastomas showed a median survival of 415 days. When analyzed as subgroups based on the number of surgical resections, the median survival was 393 days in the group with biopsy only, 380 days in the group with one surgical resection, and 548 days in the group with two or three resections. Using the Kaplan-Meier method to generate survival plots and the log rank test to compare groups, repeat debulking was found to be a significant predictor of survival (P = 0.173). The group of patients with anaplastic astrocytomas showed a median survival of 1,311 days. When analyzed by subgroups, the patients with biopsy only had a median survival of 544 days, those with one debulking 1,589 days and those with two or three debulkings 1,421 days. There was a trend toward increased survival with debulking and the log rank test again showed statistical significance (P = 0.1998). CONCLUSIONS: This study indicates that repeated surgical resections offer increased survival for both glioblastomas and anaplastic astrocytomas.

Adult↗
PubMed2001

[Ocular complications in patients infected with human immunodeficiency virus seen at the Acquired Immunodeficiency Syndrome Clinical Center].

PURPOSE: To examine the ocular complications in patients infected with human immunodeficiency virus(HIV) in Japan. METHODS: The medical records of 322 patients seen at the acquired immunodeficiency syndrome(AIDS) Clinical Center from July 1, 1997 through December 31, 1998 were reviewed, and the HIV-associated ocular complications were correlated with serum CD 4+ T-lymphocyte counts. RESULTS: Ocular complications were found in 51 patients: 35 cases with retinal microvasculopathy, 17 cases with cytomegalovirus retinitis(9 quiescent, 6 active, and 2 recurrent), and 1 case each with tuberculous uveitis, phthisis bulbi after necrotizing herpetic retinopathy, conjunctival Kaposi's sarcoma, papilledema, divergence palsy, hemianopia, and abducens palsy. Retinal microvasculopathy was present in patients with CD 4+ T-lymphocyte counts above 500/mm3, but was more common in patients with cell counts below 200/mm3. Among 6 patients with active cytomegalovirus retinitis, 5 patients had a CD 4+ T-lymphocyte count below 50/mm3 at the onset of retinitis, while one patient developed retinitis after the cell count increased to over 200/mm3 with highly active antiretroviral therapy. CONCLUSION: Cytomegalovirus retinopathy may occur in patients with a CD 4+ T-lymphocyte count of more than 200/mm3.

AIDS-Related Opportunistic Infections↗
PubMed2000

Cloning and functional studies of a luxO regulator LuxT from Vibrio harveyi.

LuxO is the central regulator integrating the quorum sensing signals controlling autoinduction of luminescence in Vibrio harveyi. We have previously purified to homogeneity a new lux regulator, LuxT, that binds to the luxO promoter. Based on the sequence of the tryptic peptides of LuxT, degenerate oligonucleotides were designed for PCR of the genomic DNA. A 273 bp PCR DNA fragment containing sequences encoding the tryptic peptides was extended by inverse PCR to obtain the complete gene (luxT) encoding a protein of 153 amino acids which shares homology with the AcrR/TetR family of transcriptional regulators. The recombinant and native LuxT gave the same footprint binding between 117 and 149 bp upstream from the luxO initiation codon. Gene disruption of luxT in V. harveyi increased luxO expression and affected the cell density dependent induction of luminescence showing that LuxT was a repressor of luxO. As LuxT also affected the survival of the V. harveyi cells at high salt concentration and homologous proteins are present in other bacterial species, including the pathogen, Vibrio cholerae, the LuxT regulatory protein appears to be a general rather than a lux-specific regulator.

Amino Acid Sequence↗
PubMed2000

Plaque radiotherapy for uveal melanoma: long-term visual outcome in 1106 consecutive patients.

OBJECTIVE: To identify clinical predictive factors for visual outcome in a large series of patients who underwent plaque radiotherapy for uveal melanoma. DESIGN: Clinical factors, including patient data, tumor features, and radiation variables, were analyzed for their impact on visual acuity using Cox proportional hazards regression models. PARTICIPANTS: Patients with uveal melanoma and initial visual acuity of 20/100 or better in the affected eye who were treated with plaque radiotherapy between July 1976 and June 1992. MAIN OUTCOME MEASURES: Two end points were used to evaluate posttreatment visual acuity: (1) final visual acuity (good [20/20-20/100] vs poor [20/200 to no light perception]) and (2) loss of visual acuity (minimal [<5 lines Snellen visual acuity] vs moderate [> or = 5 lines Snellen visual acuity]). RESULTS: Of 1300 consecutive patients with uveal melanoma treated by plaque radiotherapy, 1106 had a visual acuity of 20/100 or better at the time of treatment. In this group, poor visual acuity was found in 34% at 5 years and 68% at 10 years of follow-up. From multivariable analysis, clinical factors that best predicted poor visual acuity were increasing tumor thickness, proximity to foveola of less than 5 mm, notched plaque shape, tumor recurrence, patient age 60 years or older, subretinal fluid, cobalt isotope, anterior tumor margin posterior to equator, and worse initial visual acuity. Moderate loss of visual acuity of 5 Snellen lines or more was found in 33% at 5 years and 69% at 10 years of follow-up. From multivariable analysis, clinical factors that best predicted moderate visual acuity loss included increasing tumor thickness, worse initial visual acuity, notched plaque shape, tumor recurrence, proximity to foveola of less than 5 mm, patient age of 60 years or older, subretinal fluid, and diabetes mellitus or hypertension. When analyzing visual outcome with regard to tumor thickness, ultimate poor visual acuity of 20/200 or worse at 5 years was found in 24% with a small melanoma (< or = 3.0 mm), 30% with a medium melanoma (3.1-8.0 mm), and 64% with a large melanoma (>8.0 mm). When analyzing visual outcome with regard to tumor proximity to visually important structures, tumors less than 5 mm from the optic disc or foveola demonstrated poor visual acuity in 35% at 5 years, whereas those 5 mm or more from the optic disc and foveola showed poor visual acuity in 25% at 5 years. CONCLUSIONS: Ultimate visual acuity after plaque radiotherapy for uveal melanoma depends on many factors, including patient age and general health, initial visual acuity, tumor location and size, subretinal fluid, radioactive isotope, and final tumor control. At 10 years' follow-up, 68% of patients demonstrate poor visual acuity. Visual acuity is most effectively preserved in eyes with small tumors outside a radius of 5 mm from the optic disc and foveola. Arch Ophthalmol. 2000;118:1219-1228

Adolescent↗
PubMed2000

Purification and characterization of a luxO promoter binding protein LuxT from Vibrio harveyi.

Bioluminescence in the marine bacterium Vibrio harveyi is cell density dependent and is regulated by small molecules (autoinducers) excreted by the bacteria. The autoinducer signals are relayed to a central regulator, LuxO, which acts in its phosphorylated form as a repressor of the lux operon at the early stages of cell growth. We report in these studies the purification to homogeneity of a luxO DNA binding protein (LuxT) from V. harveyi after five major chromatography steps, including a highly effective DNA affinity chromatography step and reverse-phase HPLC. Regeneration of binding activity was accomplished after HPLC and SDS-PAGE by renaturation of LuxT from guanidine hydrochloride. It was also demonstrated that the functional LuxT was a dimer of 17 kDa that bound tightly (K(d) = 2 nM) to the luxO promoter. The sequences of three tryptic peptides obtained on digestion of the purified protein did not match any sequences in the Protein Data Bank, indicating that LuxT is a new V. harveyi lux regulatory protein.

Bacterial Proteins↗
PubMed2000

Copper(II)-catalyzed oxidation of d-alpha-tocopherol by oxygen in aqueous solution.

alpha-Tocopherol (alpha-Toc) was solubilized in aqueous solutions using 13 solubilizing agents and the products of oxidation by oxygen in the presence and the absence of Cu(II) were analyzed by HPLC. In the presence of Cu(II), the oxidation was accelerated and 5-formyl-7,8-dimethyltocol and alpha-tocoquinone were the major oxidation products. Their yields greatly increased in the presence of Cu(II). The yields and the rates of formation of the products were dependent on the properties of solubilizing agents and other conditions as well as the presence of Cu(II) or other metal ions. It is suggested that slight changes in the structure of the solubilizing agents affect the course of the reaction.

Catalysis↗
PubMed2000

Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate.

Phosphotransacetylase (Pta) catalyzes the reversible conversion of acetyl-coenzyme A (CoA) to acetyl phosphate. Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha. During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type. PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio). The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation. These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta. A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation.

Acyltransferases↗
PubMed1999

A novel human gene encoding HECT domain and RCC1-like repeats interacts with cyclins and is potentially regulated by the tumor suppressor proteins.

Cyclin E-Cdk2 is an evolutionary conserved cyclin-dependent kinase (CDK) complex that drives the G1 to S phase transition of the cell cycle. A novel cDNA encoding a HECT family protein also containing RCC1-like repeats was isolated by a yeast two-hybrid screening using both cyclin E and its inhibitor p21. The protein product of this cDNA, Ceb1, interacts with various cyclin subunits of CDKs in mammalian cells. Expression of Ceb1 is specifically detected in testis and ovary and is highly elevated when the functions of the tumor suppressor proteins, p53 and RB, are compromised by mutations or viral oncoproteins. The present results suggest that Ceb1 may play a critical role when its expression and the CDK activity are upregulated by inactivation of p53 and RB.

Amino Acid Motifs↗
PubMed1999

Covalent modification of all members of human cullin family proteins by NEDD8.

Recently we found that NEDD8, a ubiquitin-like protein, was linked covalently to human cullin-4A (abbreviated Cul-4A) by a new ubiquitin-related pathway that is analogous to but distinct from the ligating system for SUMO1, another ubiquitin-like protein. However, it remained unknown whether the other five members of the family of human cullin/Cdc53 proteins are modified by NEDD8. Here we report that all Hs-Cul family proteins, such as Cul-1, Cul-2, Cul-3, Cul-4B, and Cul-5, in addition to Cul-4A, were modified by covalent attachment of NEDD8 in rabbit reticulocyte lysates. Moreover, by comprehensive Northern-blot analyses, we examined multiple tissue distributions of the messages for all Cul-family proteins, NEDD8, and the NEDD8-ligating system consisting of APP-BP1/hUba3, and hUbc12, which function as E1- and E2-like enzymes, respectively. The expressions of Cul-1, Cul-2, and Cul-3 resembled each other and were apparently correlated to those of NEDD8 and the NEDD8-ligating system in various human cells and tissues. However, the mRNA levels of Cul-4A, Cul-4B, and Cul-5 differed considerably from each other as well as from other Cul-family proteins. The enhanced expression of all Cul-family proteins except Cul-5 was observed in a variety of tumor cell lines.

Cell Cycle Proteins↗
PubMed1999

Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution.

Ubiquitin-conjugating enzymes (Ubc) are involved in ubiquitination of proteins in the protein degradation pathway of eukaryotic cells. Ubc transfers the ubiquitin (Ub) molecules to target proteins by forming a thioester bond between their active site cysteine residue and the C-terminal glycine residue of ubiquitin. Here, we report on the NMR assignment and secondary structure of class I human ubiquitin-conjugating enzyme 2b (HsUbc2b). Chemical shift perturbation studies allowed us to map the contact area and binding interface between ubiquitin and HsUbc2b by1H-15N HSQC NMR spectroscopy. The serine mutant of the active site Cys88 of HsUbc2b was employed to obtain a relatively stable covalent ubiquitin complex of HsUbc2b(C88S). Changes in chemical shifts of amide protons and nitrogen atoms induced by the formation of the covalent complex were measured by preparing two segmentally labeled complexes with either ubiquitin or HsUbc2b(C88S)15N-labeled. In ubiquitin, the interaction is primarily sensed by the C-terminal segment Val70 - Gly76, and residues Lys48 and Gln49. The surface area on ubiquitin, as defined by these residues, overlaps partially with the presumed binding site with ubiquitin-activating enzyme (E1). In HsUbc2b, most of the affected residues cluster in the vicinity of the active site, namely, around the active site Cys88 itself, the second alpha-helix, and the flexible loop which connects helices alpha2 and alpha3 and which is adjacent to the active site. An additional site on HsUbc2b for a weak interaction with ubiquitin could be detected in a titration study where the two proteins were not covalently linked. This site is located on the backside of HsUbc2b opposite to the active site and is part of the beta-sheet. The covalent and non-covalent interaction sites are clearly separated on the HsUbc2b surface, while no such clear-cut segregation of the interaction area was observed on ubiquitin.

Amino Acid Sequence↗
PubMed1999

Down-regulation of p27(Kip1) by two mechanisms, ubiquitin-mediated degradation and proteolytic processing.

The intracellular level of p27(Kip1), a cyclin-dependent kinase (CDK) inhibitory protein, is rapidly reduced at the G1/S transition phase when the cell cycle pause ceases. In this study, we demonstrated that two posttranslational mechanisms were involved in p27(Kip1) breakdown: degradation via the ubiquitin (Ub)-proteasome pathway and proteolytic processing that rapidly eliminates the cyclin-binding domain. We confirmed that p27(Kip1) was ubiquitinated in vitro as well as in vivo. The p27(Kip1) -ubiquitination activity was higher at the G1/S boundary than during the G0/G1 phase, and p27(Kip1) ubiquitination was reduced significantly when the lysine residues at positions 134, 153, and 165 were replaced by arginine, suggesting that these lysine residues are the targets for Ub conjugation. In parallel with its Ub-dependent degradation, p27(Kip1) was processed rapidly at its N terminus, reducing its molecular mass from 27 to 22 kDa, by a ubiquitination-independent but adenosine triphosphate (ATP)-dependent mechanism with higher activity during the S than the G0/G1 phase. This 22-kDa intermediate had no cyclin-binding domain at its N terminus and virtually no CDK2 kinase inhibitory activity. These results suggest that p27(Kip1) is eliminated by two independent mechanisms, ubiquitin-mediated degradation and ubiquitin-independent processing, during progression from the G1 to S phase.

3T3 Cells↗
PubMed1998

Radiotherapy for unilesional mycosis fungoides.

PURPOSE: To evaluate the treatment outcome and natural history of patients with the diagnosis of unilesional mycosis fungoides, treated according to a prospective radiotherapy protocol in our institution since July 1975. METHODS AND MATERIALS: A total of 325 patients with the diagnosis of mycosis fungoides have been referred to the Department of Radiation Oncology at Allegheny University of Health Sciences from July 1975 through September 1996. Of these, 18 patients (5%) were classified as having unilesional mycosis fungoides and were irradiated with a curative intent using local electron fields. One patient received 22 Gy; 1 patient received 40 Gy, and the rest of the patients 30.6 Gy. Daily fractions ranged from 1.8 to 2.0 Gy. Treatments prior to radiation consisted of topical steroids and/or antifungal creams in the majority of patients, with temporary partial responses. One patient had received 2 years of topical mechlorethamine (HN2) and another patient had received topical carmustine solution (BCNU) without response prior to irradiation. RESULTS: The responses were measured clinically; posttreatment skin biopsy was not performed routinely unless there was clinical evidence of disease persistence. Complete response rate was 100%; all treated lesions cleared completely within 4 to 8 weeks after the completion of radiation. With a median follow-up of 43 months (range 12 to 240 months), 2 relapses have occurred, 2 and 71 months after the completion of radiation. Both relapses were confined to the skin and were remote from the original site. Both relapses responded to topical application of HN2. There have been no recurrences in the irradiated field nor systemic dissemination. No long-term side effects were found related to treatment, and all the patients are currently alive and without evidence of disease. Actuarial relapse-free and overall survival at 10 years are, respectively, 86.2% and 100%. CONCLUSION: Unilesional mycosis fungoides has a long natural history, is possibly the earliest manifestation of a malignant process, and local treatments, including local radiotherapy, result in long-term disease-free intervals and, possibly, cure. Total skin electron beam radiotherapy is not indicated for this disease entity.

Adult↗
PubMed1998

Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes.

BACKGROUND: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. RESULTS: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. CONCLUSION: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Cell Differentiation↗
PubMed1998

Principles of treatment of malignant gliomas in adults: an overview.

The cornerstone of conventional treatments of malignant gliomas in adults has been surgical debulking, radiation therapy and chemotherapy. Almost always a combination of these treatments is used. With these conventional treatments the outcome, as measured by survival and quality of life, has remained universally dismal. Novel treatments, which are at different stages of laboratory and clinical trials, may offer a ray of hope for treatment of malignant gliomas. Development of these methods are directly related to the discoveries, over the past two decades, of cellular and molecular mechanisms involved in the genesis of brain tumors. Understanding of the mechanisms of tumor genesis may open new avenues of effective treatments for this devastating cancer.

Adult↗
PubMed1997

Human endothelin receptors ET(A) and ET(B) expressed in baculovirus-infected insect cells--direct application for signal transduction analysis.

We expressed human endothelin receptors, ET(A) and ET(B), in insect Sf9 cells infected by recombinant baculoviruses that contained the respective cDNAs. Ligand-binding experiments showed that the two expressed receptors have the same affinities as observed for the receptors in mammalian cells, i.e. the ET(A) receptor showed an affinity order of ET-1 > or = ET-2 >> ET-3, and the ET(B) receptor remained nonselective for three isopeptide ligands. The ET(B) receptor was purified by affinity chromatography with K9-biotinyl-ET-1 without losing the ligand-binding activity from the membrane of infected Sf9 cells. Protein chemical analysis of the purified ET(B) receptor showed that it is glycosylated, and that the N-terminal 38-amino-acid peptide is susceptible to proteolytic digestion, resulting in a small 35-kDa receptor like that found in the human placenta. Surprisingly, the infected and unlysed cells showed a strong intracellular Ca2+ concentration increase ([Ca2+]i), which was generated by a unique signal-transduction pathway consisting of the insect GTP-binding protein and human endothelin receptors expressed in the late phase of virus infection. Due mainly to an efficient expression (over 200,000 receptors/cell), to a low background owing to no endogenous homolog receptor in insect Sf9 cells, and to a sensitive fluorescent reagent Fura-2, this insect Sf9 cell system can detect the [Ca2+]i induced by picomolar levels of endothelin-receptor. We propose that this highly sensitive system be used to screen for potential antagonists/agonists of endothelin receptors.

Amino Acid Sequence↗