Biomedical subjects

Nobuyuki Tanaka

Publications and source records attributed to Nobuyuki Tanaka.

At least 19 recordsLinked to original sources

PubMed2006

AMSH, an ESCRT-III associated enzyme, deubiquitinates cargo on MVB/late endosomes.

The appropriate sorting of vesicular cargo, including cell-surface proteins, is critical for many cellular functions. Ubiquitinated cargo is targeted to endosomes and digested by lysosomal enzymes. We previously identified AMSH, a deubiquitination enzyme (DUB), to be involved in vesicular transport. Here, we purified an AMSH-binding protein, CHMP3, which is an ESCRT-III subunit. ESCRT-III functions on maturing endosomes, indicating AMSH might also play a role in MVB/late endosomes. Expression of an AMSH mutant lacking CHMP3-binding ability resulted in aberrant endosomes with accumulations of ubiquitinated cargo. Nevertheless, CHMP3-binding capability was not essential for AMSH's in vitro DUB activity or its endosomal localization, suggesting that, in vivo, the deubiquitination of endosomal cargo is CHMP3-dependent. Ubiquitinated cargo also accumulated on endosomes when catalytically inactive AMSH was expressed or AMSH was depleted. These results suggest that both the DUB activity of AMSH and its CHMP3-binding ability are required to clear ubiquitinated cargo from endosomes.

Catalysis↗

PubMed2006

Murine leukemia virus vector integration favors promoter regions and regional hot spots in a human T-cell line.

Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We mapped 340 MLV vector integration sites in the infected human T-cell clones we established. The data showed that MLV preferred integration near the transcription start sites (+/-5kb), near CpG islands (+/-1kb), and within the first intron of RefSeq genes. We also identified MLV integration hot spots that contained three or more integrations within a 100kb region. RT-PCR revealed that mRNA-levels of T-cell clones that contained MLV integrations near transcription start sites or introns were dysregulated compared to the uninfected cells. These studies help define the profile of MLV integration in T-cells and the risks associated with MLV-based gene therapy.

Cell Line↗

PubMed2006

Survival of parvovirus B19-infected cells by cellular autophagy.

Human parvovirus B19 (B19) is a well-known pathogenic agent which causes apoptosis in erythrocyte lineage cells. Here, we provide the first evidence that mitochondrial autophagy is specifically found in the B19-infected cells. The protein expression ratio for LC3-II/LC3-I increased significantly in infected cells, indicating possible involvement of cellular autophagy in the infection process. Immunofluorescence confocal microscopy analyses revealed that B19 infection induced an intracellular autophagosome as judged by endogenous LC3 staining. Moreover, inhibition of autophagy by 3-MA significantly facilitated B19-infection-mediated cell death. These results suggest a novel mechanism by which B19-infected cells survive by cellular autophagy.

Adenine↗

PubMed2006

The SK channel blocker apamin inhibits slow afterhyperpolarization currents in rat gonadotropin-releasing hormone neurones.

Gonadotropin-releasing hormone (GnRH) neurones play an essential role in the hypothalamo-pituitary-gonadal axis. As for other neurones, the discharge pattern of action potentials is important for GnRH neurones to properly function. In the case of a luteinizing hormone (LH) surge, for example, GnRH neurones are likely to continuously fire for more than an hour. For this type of firing, GnRH neurones must have a certain intrinsic property. To address this issue, we investigated the voltage-gated Ca(2+) currents and Ca(2+)-activated voltage-independent K(+) currents underlying afterhyperpolarization, because they affect cell excitability. Dispersed GnRH neurones from adult GnRH-EGFP (enhanced green fluorescent protein) transgenic rats were cultured overnight and then used for an electrophysiological experiment involving the perforated patch-clamp configuration. The GnRH neurones showed five subtypes of voltage-gated Ca(2+) currents, i.e. the T-, L-, N-, P/Q- and R-types. The GnRH neurones also showed a slow afterhyperpolarization current (I(sAHP)), but not a medium one. It is reported that the SK channel blocker apamin (10 pm-100 nm) blocks a medium afterhyperpolarization current but not an I(sAHP). In contrast to previous reports, the I(sAHP) observed in rat GnRH neurones was potently blocked by apamin. In addition, the GnRH neurones expressed transcripts for SK1-3 channels. The results indicate that rat GnRH neurones express all five subtypes of voltage-gated Ca(2+) channels and exhibit an apamin-sensitive I(sAHP), which may allow continuous firing in response to a relatively strong depolarizing input.

Action Potentials↗

PubMed2006

Development of novel sustained-release system, disintegration-controlled matrix tablet (DCMT) with solid dispersion granules of nilvadipine (II): in vivo evaluation.

A novel sustained-release (SR) system, disintegration-controlled matrix tablet (DCMT), was developed for poorly water-soluble drugs. DCMT, consisting of wax and solid dispersion (SD) granules containing a disintegrant, could control the release of nilvadipine (NiD), a model compound, by its disintegration. In the present study, two DCMTs (DCMT-1 and DCMT-2) with different release rates of NiD were orally administered to beagle dogs, and in vivo absorption of NiD from DCMTs was compared with that from immediate-release (IR) tablets. DCMTs successfully sustained the absorption of NiD longer than IR tablets, while they did not decrease the bioavailability of NiD. DCMT-2, providing the slower release of NiD than DCMT-1, prolonged the absorption longer than DCMT-1. In vivo absorption profiles of NiD from DCMTs were significantly correlated with in vitro release profiles, suggesting that the release property from DCMTs would maintain regardless of the change in physiological condition through the gastrointestinal tract. Furthermore, the food intake did not affect the absorption of NiD after oral administration of DCMT-2. The present results strongly indicate that the DCMT system would be a promising SR system, which could improve the solubility and sustain the absorption of poorly water-soluble drugs.

Administration, Oral↗

PubMed2006

Muscle fatigue decreases short-interval intracortical inhibition after exhaustive intermittent tasks.

OBJECTIVE: Central fatigue is the inability of central commands to recruit maximum evocable muscle force during voluntary contraction. Here, we investigate how fatigue affects the inhibitory circuits of the motor cortex. METHODS: MEPs, short interval intracortical inhibition (SICI) and intracortical facilitation (ICF) were evaluated using a paired pulse transcranial magnetic stimulation (TMS) paradigm before, during and after a series of 5 isometric contractions of the FDI muscle to 50% maximal voluntary contraction (MVC). Each contraction lasted 2 min and was separated from the next by a pause of 2 min 40 s. Twelve male healthy subjects (range from 22 to 51 years) participated in experiment 1, in which the intensity of test stimulus was constant throughout the experiment. Eight of the same subjects (range from 26 to 51 years) participated in experiment 2, in which the intensity of test stimulus was adjusted so that the amplitude of the test MEP was kept constant throughout the measurement. RESULTS: As expected, test MEPs gradually decreased with progressive fatigue and recovered to control values with 5-10 min of rest. Because of the change in MEP amplitude, changes in percent SICI (reduced inhibition) and percent ICF (increased facilitation) in experiment 1 are difficult to interpret. When the test MEP was maintained at a constant size in experiment 2 there was no change in percent ICF, but the reduction in SICI was still present although it recovered to control values within the first 5-10 min of rest. CONCLUSIONS: SICI in FDI decreases transiently after a series of fatiguing isometric contractions. This decrease may compensate to some extent for reduced cortical excitability after muscle fatigue.

Adult↗

PubMed2006

Application of HSVtk suicide gene to X-SCID gene therapy: ganciclovir treatment offsets gene corrected X-SCID B cells.

Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human gamma c chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the gamma c chain, the cells were treated with ganciclovir (GCV). The gamma c chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the gamma c chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial.

Antiviral Agents↗

PubMed2006

Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines.

BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-). METHODS: A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone. RESULTS: Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation. CONCLUSIONS: This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.

Bacteriophages↗

PubMed2006

Clathrin anchors deubiquitinating enzymes, AMSH and AMSH-like protein, on early endosomes.

Endosomal sorting of ubiquitinated membrane proteins for trafficking to lysosomes is executed by a complex of two ubiquitin-binding proteins, Hrs and STAM, that localizes on a microdomain of early endosomes with a flat clathrin coat. AMSH is a deubiquitinating enzyme that interacts with STAM and is implicated in the down-regulation of epidermal growth factor receptor. AMSH has a close homolog, AMSH-like protein (AMSH-LP). Here we show that AMSH-LP is also a deubiquitinating enzyme that acts on early endosomes. We further show that AMSH and AMSH-LP bind to the terminal domain of clathrin heavy chain via a novel clathrin-binding site conserved between these proteins. Exogenously expressed AMSH and AMSH-LP co-localized with clathrin on early endosomes. However, deletion of the clathrin-binding site from the proteins, as well as RNA interference-mediated depletion of clathrin heavy chain, resulted in a failure of AMSH and AMSH-LP to localize on endosomes. In contrast, a mutant of AMSH that lacks the ability to bind STAM localized normally on endosomes. We suggest that AMSH and AMSH-LP are anchored on the early endosomal membrane via interaction with the clathrin coat.

Amino Acid Sequence↗

PubMed2006

Arthroscopic and open synovectomy of the elbow in rheumatoid arthritis.

BACKGROUND: Synovectomy has been advocated for early treatment of the rheumatoid elbow. It has not been determined whether arthroscopic or open synovectomy is better and whether a preoperative arc of flexion of >90 degrees is an important prognostic factor. METHODS: Arthroscopic or open synovectomy was performed in fifty-eight elbows in fifty-three patients with rheumatoid arthritis and radiographic changes in the joint of Larsen grade 2 or less. Clinical symptoms, recurrent synovitis, postoperative complications, and radiographic changes were assessed ten to eighteen years (average, thirteen years) postoperatively. RESULTS: Eleven (48%) of twenty-three elbows in which arthroscopic synovectomy had been performed and sixteen (70%) of twenty-three elbows in which open synovectomy had been performed were mildly or not painful at the latest follow-up evaluation. However, no significant difference was detected between the overall clinical results of arthroscopic synovectomy and those of open synovectomy. In elbows with a preoperative arc of flexion of <90 degrees , the clinical results of the two procedures were comparable. In elbows with a preoperative arc of flexion of <90 degrees , arthroscopic synovectomy provided significantly (p < 0.05) better function than open surgery after mid-term follow-up, and motion and function continued to be better in those patients at the most recent follow-up evaluation. Recurrent synovitis was observed in six elbows that had arthroscopic synovectomy and in three that had open synovectomy, and the Larsen grade increased in both groups. Three elbows with a preoperative arc of flexion of <90 degrees underwent a total elbow arthroplasty to treat ankylosis after open synovectomy. Surgical complications were uncommon and not severe. CONCLUSIONS: Arthroscopic synovectomy of the elbow is a reliable procedure. One of the most favorable indications for either arthroscopic or open synovectomy is a preoperative arc of elbow flexion of >/=90 degrees in patients with early rheumatoid arthritis.

Adult↗

PubMed2006

On site experiments of the slanted soil treatment systems for domestic gray water.

In order to make a breakthrough for the acute problem of water shortage in the world, the key words "decentralization and re-use" are very important for new sustainable sanitation systems that will be developed. Therefore, we focused on a new treatments system called "a slanted soil treatment system" which combines a biotoilet system with a domestic grey water treatment system. Because this system is a low cost and compact system, the system can be easily introduced to homes in urban areas or in the suburbs of cities in many developing countries. In this study, we performed on site experiments carried out on Shikoku Island, Japan, for several years. We obtained the following results. The slanted soil treatment system could remove organic pollutants and total nitrogen and total phosphorus in grey water effectively. Furthermore, the system performance became high in the case of the high concentration of the influent water. The nitrification reaction and denitrification reaction were speculated to exist due to aerobic zones and anaerobic zones present in the slanted soil treatment system. The slanted soil treatment system could perform for approximately 3 years with zero maintenance. The plug flow model of 1st order reaction kinetics could describe the reaction in the slanted soil treatment system. However, it is necessary to improve the system to maintain the performance in all seasons.

Biodegradation, Environmental↗

PubMed2006

Reduction of eosinophils in small airways by inhaled steroids is insufficient in patients with adult asthma.

BACKGROUND: Recent reports suggest that small airway as well as large airway involvement in asthma is important. We investigate the therapeutic effects of a meter-dose inhaler of chrolofluorocarbon-beclomethasone dipropionate (CFC-BDP) and dry-powder fluticasone (DP-FP). METHODS: Lung specimens obtained at operation due for small size lung cancer in 16 asthmatic patients and 16 controls were evaluated immunohistochemically using antibodies of EG2 (eosinophil), AA1 (mast cell), CD68 (macrophage), and CD34 (pluripotent hematopoietic stem cell). We calculated the number of each cell type in 5 fields in the inner and outer areas of large airways (luminal diameter; > or =2 mm) and small airways (<2 mm) using computer software. RESULTS: In asthmatic patients eosinophils were significantly increased in both inner and outer areas of small airways and the number of CD34+ cells was significantly elevated in inner areas as compared with controls. Although the density of eosinophils in the inner area of large airways was significantly suppressed (p < 0.02), there was no such suppression in the inner areas of small airways in asthmatic patients treated with CFC-BDP or DP-FP. CONCLUSIONS: It was speculated that inhaled CFC-BDP and DP-FP might deposit mainly in large airways and fail to fully reach small airways, consequently allowing eosinophilic inflammation to continue in small airways.

Administration, Inhalation↗

PubMed2005

Short-term effects of thermotherapy for spasticity on tibial nerve F-waves in post-stroke patients.

Thermotherapy is generally considered appropriate for post-stroke patients with spasticity, yet its acute antispastic effects have not been comprehensively investigated. F-wave parameters have been used to demonstrate changes in motor neuron excitability in spasticity and pharmacological antispastic therapy. The present study aimed to confirm the efficacy of thermotherapy for spasticity by evaluating alterations in F-wave parameters in ten male post-stroke patients with spastic hemiparesis (mean age: 49.0+/-15.0 years) and ten healthy male controls (mean age: 48.7+/-4.4 years). The subjects were immersed in water at 41 degrees C for 10 min. Recordings were made over the abductor hallucis muscle, and antidromic stimulation was performed on the tibial nerve at the ankle. Twenty F-waves were recorded before, immediately after, and 30 min following thermotherapy for each subject. F-wave amplitude and F-wave/M-response ratio were determined. Changes in body temperature and surface-skin temperature were monitored simultaneously. The mean and maximum values of both F-wave parameters were higher on the affected side before thermotherapy. In the post-stroke patients, the mean and maximum values of both parameters were significantly reduced after thermotherapy (P<0.01). Hence, the antispastic effects of thermotherapy were indicated by decreased F-wave parameters. Body temperature was significantly increased both immediately after and 30 min after thermotherapy in all subjects. This appeared to play an important role in decreased spasticity. Surface-skin temperature increased immediately after thermotherapy in both groups and returned to baseline 30 min later. These findings demonstrate that thermotherapy is an effective nonpharmacological antispastic treatment that might facilitate stroke rehabilitation.

Adult↗

PubMed2005

Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase.

Abdominal aortic aneurysm (AAA) is a common disease among elderly people that, when surgical treatment is inapplicable, results in progressive expansion and rupture of the aorta with high mortality. Although nonsurgical treatment for AAA is much awaited, few options are available because its molecular pathogenesis remains elusive. Here, we identify JNK as a proximal signaling molecule in the pathogenesis of AAA. Human AAA tissue showed a high level of phosphorylated JNK. We show that JNK programs a gene expression pattern in different cell types that cooperatively enhances the degradation of the extracellular matrix while suppressing biosynthetic enzymes of the extracellular matrix. Selective inhibition of JNK in vivo not only prevented the development of AAA but also caused regression of established AAA in two mouse models. Thus, JNK promotes abnormal extracellular matrix metabolism in the tissue of AAA and may represent a therapeutic target.

Adenoviridae↗

PubMed2005

TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo.

TGF-beta-activated kinase 1 (TAK1), a member of the MAPKKK family, is thought to be a key modulator of the inducible transcription factors NF-kappaB and AP-1 and, therefore, plays a crucial role in regulating the genes that mediate inflammation. Although in vitro biochemical studies have revealed the existence of a TAK1 complex, which includes TAK1 and the adapter proteins TAB1 and TAB2, it remains unclear which members of this complex are essential for signaling. To analyze the function of TAK1 in vivo, we have deleted the Tak1 gene in mice, with the resulting phenotype being early embryonic lethality. Using embryonic fibroblasts lacking TAK1, TAB1, or TAB2, we have found that TNFR1, IL-1R, TLR3, and TLR4-mediated NF-kappaB and AP-1 activation are severely impaired in Tak1(m/m) cells, but they are normal in Tab1(-/-) and Tab2(-/-) cells. In addition, Tak1(m/m) cells are highly sensitive to TNF-induced apoptosis. TAK1 mediates IKK activation in TNF-alpha and IL-1 signaling pathways, where it functions downstream of RIP1-TRAF2 and MyD88-IRAK1-TRAF6, respectively. However, TAK1 is not required for NF-kappaB activation through the alternative pathway following LT-beta signaling. In the TGF-beta signaling pathway, TAK1 deletion leads to impaired NF-kappaB and c-Jun N-terminal kinase (JNK) activation without impacting Smad2 activation or TGF-beta-induced gene expression. Therefore, our studies suggests that TAK1 acts as an upstream activating kinase for IKKbeta and JNK, but not IKKalpha, revealing an unexpectedly specific role of TAK1 in inflammatory signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Development of novel sustained-release system, disintegration-controlled matrix tablet (DCMT) with solid dispersion granules of nilvadipine.

The goal of this study is to develop a novel sustained-release (SR) system for poorly water-soluble drugs by applying solid dispersion (SD) technique for improving the solubility. The developed SR system, disintegration-controlled matrix tablet (DCMT), consists of hydrogenated soybean oil (HSO) as wax and SD granules containing low-substituted hydroxypropylcellulose (L-HPC) as a disintegrant. In this study, nilvadipine (NiD) was chosen as a model compound. Sustained-release profiles of NiD from DCMT were identically controlled in several dissolution mediums in spite of varying pH and agitation speed. The release of NiD from DCMT was sustained more effectively by increasing the amount of wax or by decreasing the amount of disintegrant, and supersaturation of NiD was achieved without any re-crystallization in dissolution medium. The release rate of NiD from DCMT was controlled by the disintegration rate of tablet. The release profile of NiD was described by the Hixson-Crowell's model better than zero-order kinetics, first-order kinetics and Higuchi's model, which supports that the release of NiD from DCMT is regulated by the disintegration of the tablet. From this study, it was clarified that DCMT was one of the promising SR systems applying SD for the poorly water-soluble drugs.

Calcium Channel Blockers↗

PubMed2005

Volume of a wash and the other conditions for maximum therapeutic effect of arthroscopic lavage in rheumatoid knees.

The objective of this study was to determine the appropriate volume of saline to obtain a beneficial effect of arthroscopic lavage and the prognostic factors related to the clinical effect in a refractory rheumatoid knee. Arthroscopic lavage or arthrocentesis was performed in a random manner in 142 patients with relapsing rheumatoid arthritis. All patients were followed up for 24 months. Intra-articular lavage with 5 l (p < 0.01) or 3 l (p < 0.05) of saline gave better clinical results than did arthrocentesis. There was no significant difference between the cumulative incidence of recurrence of knee synovitis in patients who underwent arthrocentesis and that in patients who underwent lavage with 1 l of saline. Cox regression analysis showed that patients with knee arthritis of more than 6 months in duration and with Larsen grade II or less were more responsive to lavage with 5 or 3 l of saline. The duration of the effect of lavage with 3 l of saline was increased 2.9-fold (p = 0.04) by injection of both steroids and high molecular weight hyaluronan. No bleeding or infectious complications occurred after those procedures. Arthroscopic lavage with 5 l of saline is safe and has good therapeutic effects for rheumatoid knees with mild destruction in which arthritis has persisted for more than 6 months. Lavage with 3 l of saline is recommended when intra-articular injection of corticosteroid and high molecular weight hyaluronan is performed after lavage.

Arthritis, Rheumatoid↗

PubMed2005

Actions of brain-derived neurotrophic factor on spinal nociceptive transmission during inflammation in the rat.

The aim of the current study was to investigate whether, and if so how, brain-derived neurotrophic factor (BDNF) acts to develop the spinal sensitization underlying inflammation-induced hyperalgesia. In spinal cord slice preparations from rats with inflammation induced by complete Freund's adjuvant (CFA), BDNF, but not nerve growth factor (NGF) or neurotrophin-3 (NT-3), acted presynaptically to increase the frequency of excitatory miniature EPSCs in substantia gelatinosa (SG) neurones of the CFA-treated, but not untreated rats, through activation of lidocaine (lignocaine)-sensitive, TTX-resistant Na+ channels. This effect was observed in the spinal cord slices of the CFA-treated rat only 2-4 days after the CFA injection. On the other hand, the number of monosynaptic Abeta afferent inputs to the SG significantly increased 1 week after the onset of the inflammation, and this increase was significantly suppressed by treatment with anti-BDNF antiserum administered 1 day before and just after the CFA injection. In addition, the treatment with anti-BDNF antiserum significantly attenuated the CFA-induced hyperalgesia and/or allodynia. These findings, taken together, suggest that BDNF, which is considered to be released from the sensitized primary afferents, increases the excitability of SG neurones through its action on the presynaptic terminals. BDNF may thereafter induce monosynaptic Abeta afferents to the SG, thereby developing hyperalgesia and/or allodynia during inflammation.

Animals↗