Biomedical subjects

Toshikazu Kubo

Publications and source records attributed to Toshikazu Kubo.

15 recordsLinked to original sources

PubMed2003

A novel marker for terminal Schwann cells, homocysteine-responsive ER-resident protein, as isolated by a single cell PCR-differential display.

Terminal Schwann cells (TSCs) that cover motor neuron terminals are known to play important roles in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. We have established a method of selectively and efficiently collecting TSCs so that cDNA analysis can be done properly. The expression of 1-2% of whole mRNAs was compared between myelinating Schwann cells (MSCs) and TSCs, and it turned out that approximately one-third of the bands could be categorized as cell-type-specific bands. TSCs thus constitute a distinct entity from the viewpoint of gene expression. As one of the cDNA clones belonging to TSC-specific bands was identified homocysteine-responsive ER-resident protein (Herp), and in situ hybridization confirmed that Herp mRNA is expressed in TSCs on motor nerve terminals but not in MSCs, both in developing and adult rats. In conclusion, we have been able to identify Herp as a novel molecular marker for TSCs.

Animals↗

PubMed2003

Hsp70 prevents nitric oxide-induced apoptosis in articular chondrocytes.

OBJECTIVE: To deliver and overexpress the hsp70 gene in cultured chondrocytes to investigate its effect on nitric oxide (NO)-induced apoptosis of chondrocytes. METHODS: Primary chondrocyte cultures were established from rabbit joints. The cells were transduced with an empty adenovirus vector (Ax1w) or an adenovirus vector harboring the hsp70 E-tag fusion gene (AxSHEwt). Apoptosis was induced by sodium nitroprusside (SNP) dihydrate, which generates NO, or by staurosporine, which is a proapoptotic agent dependent upon Bax or Bak protein. Cell viability and apoptosis induction were estimated by lactate dehydrogenase (LDH) assay, Hoechst 33342 staining, or the TUNEL method. To study Hsp70, cytochrome c, and caspase 3, Western blot analyses were performed. RESULTS: The AxSHEwt-transduced cells escaped apoptosis, as revealed by the LDH assay, Hoechst 33342 staining, and the TUNEL method. A massive amount of the tagged Hsp70 was demonstrated in the AxSHEwt-transduced chondrocytes but not in control cells. Hsp70 did not affect the cytosolic cytochrome c level, but appeared to have obstructed the activation of caspase 3. CONCLUSION: Experimentally overexpressed Hsp70 almost completely inhibited NO- or staurosporine-induced apoptosis in primary chondrocytes.

Adenoviruses, Human↗

PubMed2003

Evaluation of sensory function after median nerve decompression in carpal tunnel syndrome using the current perception threshold test.

The recovery level for sensory function after carpal tunnel release for the treatment of idiopathic carpal tunnel syndrome (CTS) was assessed with the current perception threshold (CPT) test. Seventeen CTS patients (21 hands) were followed, and the CPTs at the index finger of each patient was measured preoperatively and at 1, 3, and 6 months postoperatively. After carpal tunnel release, there was significant recovery of CPT at all stimulation frequencies, indicating improvement of all sensory functions including sensations of temperature, pain, touch, and vibration.

Adult↗

PubMed2003

Effect of heat stimulation on viability and proteoglycan metabolism of cultured chondrocytes: preliminary report.

Thermotherapy has been applied to various joint diseases and injuries, but its direct effects on articular cartilage have remained unclear. The present study examined the effects on cell viability and metabolism by using the chondrocyte-like cell line HCS-2/8. The temperatures and durations of heat stimulation were 39 degrees, 41 degrees, 43 degrees, and 45 degrees C for 15 or 30 min. After heat stimulation of 41 degrees C or lower for 15 or 30 min, cell viability increased and proteoglycan metabolism was accelerated, whereas after stimulation at 43 degrees C or higher for 30 min the viability and metabolism decreased. These results indicate that appropriate heat stimulation positively affects cell viability and the proteoglycan metabolism of articular cartilage, whereas too much heat stimulation produces negative effects. Clinical efficacy is therefore determined by the overall thermal dose. When the appropriate combination of temperature and duration is found, thermotherapy for diseases and injury of articular cartilage can be highly useful in clinical practice.

Cell Survival↗

PubMed2003

Genetic analysis of steroid-induced osteonecrosis of the femoral head.

With advanced organ transplantation technology, steroid-induced osteonecrosis of the femoral head (ONF) is one of the most troublesome complications. Steroid sensitivity varies among individuals, and the involvement of polymorphism of various genes relating to steroid metabolism is suggested. The present study investigated the relation between single nucleotide polymorphism (SNP) on the DNA sequence of cytochrome p(450) and ONF development. The subjects were 80 renal transplant patients. Genome DNA was obtained from the peripheral blood, and SNP analyses for CYP3A4, CYP2D6, and CYP2C19 were conducted using various methods: direct sequencing, polymerase chain reaction restriction fragment length polymorphism, and DNA Chip. The relation between ONF development and SNP was statistically analyzed. It is useful if the generating risk judged according to SNPs to prevention of steroid-induced ONF is possible. This time, SNPs, which are clearly related to ONF, were not accepted, although the possibility that SNP related to steroid metabolism is involved in ONF development is important. We think that it is necessary to examine this area more closely.

Aryl Hydrocarbon Hydroxylases↗

PubMed2003

Coverage of the femoral head by the acetabular labrum in dysplastic hips: quantitative analysis with radial MR imaging.

Hip dysplasia is an important cause of osteoarthrosis of the hip, especially in Japanese. Recently, the role of the acetabular labrum in hip diseases has attracted the attention of researchers, but the condition of the labrum in the dysplastic hips has not been described. We used radial MRI to measure the degree of coverage of the femoral head by the acetabulum and the labrum, corresponding to 7 positions on the acetabular rim at every 15 degrees from anterosuperior 45 degrees via midsuperior to posterosuperior 45 degrees. 38 Japanese patients (51 hips) with dysplasia, and 11 healthy controls 122 hips) were studied. In the dysplastic hips, the labrum was larger than in the controls. We found no correlation between the coverage of the acetabulum in the anterosuperior positions and the size of the labrum among the dysplastic hips.

Acetabulum↗

PubMed2003

Evaluation of descending spinal cord tracts in patients with thoracic cord lesions using motor evoked potentials recorded from the paravertebral and lower limb muscles.

We evaluated the function of the descending spinal cord motor tracts in patients, with and without spinal cord lesion, using motor evoked potentials. We studied 50 normal volunteers and 15 patients with thoracic lesions. The onset latency of the negative waves of motor evoked potentials for the thoracic spines was obtained, and the descending spinal cord conduction time was measured for the thoracic segments. In normal subjects, motor evoked potentials of paravertebral muscles recorded from T1-T2 to T5-T6 initially appeared as negative waveform with transcutaneous electrical stimulation over occipitocervical junction, although those from T6-T7 to T8-T9 were initially positive and those from more caudal sites were flatter. The motor evoked potential waveforms of tibialis anterior muscles evoked by electrical stimulation over the occipitocervical junction were markedly similar to those over the L1-L2. In patients with upper thoracic lesions, descending spinal cord conduction time from T2-T3 to T5-T6 was prolonged (p < 0.01). The descending spinal cord conduction time from T5-T6 to T11-T12 was also prolonged (p < 0.01) in patients with lower thoracic lesion. The descending spinal cord conduction time from T2-T3 to T11-T12 in patients with smaller motor function scores (<2) was significantly prolonged (p < 0.01) compared with normal subjects and patients with larger function scores. The methods of recording motor evoked potentials from paravertebral muscles with transcutaneous electrical stimulation over occipitocervical junction were useful for evaluating the level and motor function of thoracic cord lesions.

Adolescent↗

PubMed2002

Successful genetic transduction in vivo into synovium by means of electroporation.

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.

Animals↗

PubMed2002

Significant antitumor effects obtained by autologous tumor cell vaccine engineered to secrete interleukin (IL)-12 and IL-18 by means of the EBV/lipoplex.

The EBV/lipoplex is a nonviral gene delivery system composed of a cationic lipid and Epstein-Barr virus (EBV)-based plasmid vector that carries the EBV oriP and EBV nuclear antigen 1 (EBNA1) gene. Because the EBNA1 supports retention, nuclear localization, and transcriptional upregulation of the oriP-bearing plasmid, cells transfected with the EBV/lipoplex express the transgene at a very high level. We hypothesized that tumor cells genetically manipulated with the EBV/lipoplex may be used as a tumor vaccine without drug selection, strongly contributing to immunotherapy of patients with malignancies. The cytokines interleukin (IL)-12 and IL-18 exert a variety of immune-regulatory functions including interferon (IFN)-gamma production and cytotoxic T lymphocyte (CTL) and natural killer (NK) activation. Here, we investigated the possible therapeutic effects of an autologous tumor cell vaccine in the B16 melanoma model. The vaccine was engineered to secrete IL-12 and IL-18 by means of the EBV/lipoplex. B16 cells were subcutaneously implanted into syngenic mice followed by repetitive immunization with irradiated B16 cells that had been transfected 3 days earlier by TFL2-3, a novel cationic lipid, with EBV-plasmid vectors encoding IL-12 and/or IL-18 genes (B16/mIL-12, B16/mIL-18, and B16/mIL-12+mIL-18). The mice vaccinated with B16/mIL-12 underwent strong tumor suppression accompanied by a high IFN-gamma production. Both CTL and NK activities were significantly elevated in these mice. When the tumor cell vaccine was prepared by means of conventional (non-EBV) plasmid vectors combined with the same cationic lipid, the therapeutic outcome was not as good, suggesting the superiority of the EBV-based plasmid in engineering these types of tumor vaccines. Vaccination with B16/mIL-18 was not effective in suppressing tumors, whereas B16/mIL-12+mIL-18 showed comparable antitumor therapeutic validity as B16/mIL-12 did. When IFN-gamma mutant (IFN-gamma(-/-) mice were treated, B16/mIL-12 vaccine did not show any therapeutic activity, suggesting the necessity of IFN-gamma in the anti-melanoma immune responses. In contrast, the antitumor effect was not affected by NK depletion in mice that received repetitive injections with anti-asialo GM1 antibody. Furthermore, vaccination with B16/mIL-12 significantly suppressed pulmonary metastases in mice that had been intravenously injected with parental B16. Our results suggest that the EBV/lipoplex is quite useful in generating an autologous tumor cell vaccine and that IL-12 is an important component of the vaccine.

Animals↗

PubMed2002

The 2001 revised criteria for diagnosis, classification, and staging of idiopathic osteonecrosis of the femoral head.

The 2001 revised criteria for the diagnosis, classification, and staging of idiopathic osteonecrosis of the femoral head were proposed in June 2001, by the working group of the Specific Disease Investigation Committee under the auspices of the Japanese Ministry of Health, Labor and Welfare, to establish criteria for diagnosis and management of idiopathic osteonecrosis of the femoral head. Five criteria that showed high specificity were selected for diagnosis: collapse of the femoral head (including crescent sign) without joint-space narrowing or acetabular abnormality on x-ray images; demarcating sclerosis in the femoral head without joint-space narrowing or acetabular abnormality; "cold in hot" on bone scans; low-intensity band on T1-weighted MRI (bandlike pattern); and trabecular and marrow necrosis on histology. Idiopathic osteonecrosis of the femoral head is diagnosed if the patient fulfills two of these five criteria and does not have bone tumors or dysplasias. Necrotic lesions are classified into four types, based on their location on T1-weighted images or x-ray images. Type A lesions occupy the medial one-third or less of the weight-bearing portion. Type B lesions occupy the medial two-thirds or less of the weight-bearing portion. Type C1 lesions occupy more than the medial two-thirds of the weight-bearing portion but do not extend laterally to the acetabular edge. Type C2 lesions occupy more than the medial two-thirds of the weight-bearing portion and extend laterally to the acetabular edge. Staging is based on anteroposterior and lateral views of the femoral head on x-ray images. Stage 1 is defined as the period when there are no specific findings of osteonecrosis on x-ray images, although specific findings are observed on MRI, bone scintigram, or histology. Stage 2 is the period when demarcating sclerosis is observed without collapse of the femoral head. Stage 3 is the period when collapse of the femoral head, including crescent sign, is observed without joint-space narrowing. Mild osteophyte formation in the femoral head or acetabulum may be observed in stage 3. Stage 3 is divided into two substages. In stage 3A, collapse of the femoral head is less than 3 mm. In stage 3B, collapse of the femoral head is 3 mm or greater. Stage 4 is the period when osteoarthritic changes are observed.

Femur Head Necrosis↗

PubMed2002

A possible intermediate step during apoptotic execution.

Many proteases are known to be involved in apoptosis. Among them, interleukin-1beta converting enzyme (ICE) and its family proteases, which are called caspases, play critical roles in the execution stage of apoptosis. We previously reported that a proteasome-inhibitor, benzyloxycarbonyl Leu-Leu-leucinal (ZLLLal), induced apoptosis in MOLT-4 cells. In the present study, in order to analyze the detailed mechanism of ZLLLal-induced apoptosis, we examined the effect of a caspase-inhibitor, acetyl(Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (AcYVADcmk), on ZLLLal-induced apoptosis in the cells. Agarose gel electrophoresis revealed that low concentrations of AcYVADcmk efficiently suppressed apoptotic DNA fragmentation. However, the cells presented morphology different from normal, apoptotic or necrotic cells, although DNA fragmentation was suppressed. The same examination was performed on the cells with anti-Fas antibody-induced apoptosis, and the same results were obtained. Some cells with a similar morphology were found even without the caspase-inhibitor in the early stage of anti-Fas antibody-induced physiological apoptosis. In addition, apoptotic cascade was reactivated by washing out the caspase inhibitor from the DNA degradation-suppressed cells. Therefore, this newly found morphological feature shows the presence of a step prior to caspase activation in the cells, and this is the first report presenting the pre-caspase-activated step in the apoptotic cascade.

Amino Acid Chloromethyl Ketones↗

PubMed2002

Power spectrum analysis of compound muscle action potential in carpal tunnel syndrome patients.

The objective of using wave-form analysis to assess compound muscle action potential (CMAP) in entrapment neuropathy had not been fully developed. We applied the power spectrum analysis to patients with carpal tunnel syndrome (CTS) for this purpose. 24 patients with CTS were divided into three stages according to Mackinnon s classification, and 50 normal volunteers were examined. CMAP was obtained from the abductor pollicis brevis with supramaximal stimulation to median nerve. Mean and peak frequencies were measured by power spectrum analysis. The distal latencies of CMAP and the sensory nerve conduction velocities showed some prolongation in CTS patients. Integral values of CMAP were also decreased in CTS patients. Mean and peak frequencies of power spectrum of CMAP in volunteers were 134 Hz and 98 Hz, respectively. These values shifted into lower frequencies in CTS patients, namely 102 Hz and 61 Hz. Regardless of clinical stage, distal latency of CTS patients correlated with mean frequency.

Action Potentials↗

PubMed2000

Predictive value of magnetic resonance imaging in avascular necrosis following talar fractures.

Ischemic bone necrosis following talar fractures is a problematic complication and its early diagnosis is important. Patients with Hawkins Type II and III talar fractures received internal fixation using titanium alloy screws, and chronological bone changes were observed with Magnetic Resonance Imaging (MRI). With the Type II patient, Hawkins' sign was radiographically confirmed 2 months after the surgery. Furthermore, there were no changes of MR images for 2 years and a good clinical outcome was obtained. However, in the Type III patient, Hawkins' sign was negative and MRI revealed a low signal-intensity band on the talus 2 months after the surgery and then necrosis was radiographically confirmed; pain appeared 10 months later. These 2 cases suggest that MRI is a useful means for detecting bone necrosis with talar fractures in the early post-operative period.

Journal Article↗

PubMed2000

Anterior cruciate ligament reconstruction using the double bundle method.

Clinical results of our reconstruction technique for anterior cruciate ligament using the double bundle, i.e., the combination of bone-tendon-bone (BTB) from the patellar tendon and semitendinosus tendon (ST), were evaluated. BTB was fixed in the tunnels produced on the isometric points on the tibia and femur, and ST, on the tibial tunnel through the same route as BTB. Throughout the observation period, no patients developed pain, limited range of motion, and/or instability of the operated knee. All patients were able to return to previous sports activities within 12 months. No apparent changes occurred on the reconstructed ligament. In 4 of 14 patients, knee stability was quantitatively examined, and a good result was obtained. The double bundle was found to be a useful method for patients who require physiologically more durable reconstruction.

Journal Article↗

PubMedDate not supplied

Development of the acetabulum and the acetabular labrum in the normal child: analysis with radial-sequence magnetic resonance imaging.

The degree of acetabular and labral coverage was clarified in children at different positions using radial-sequence magnetic resonance imaging. Scans were performed on 40 hips in 20 normal children (group A, 6-8 years old; group B, 9-11 years old; group C, 12-13 years old) and on 19 hips of 10 healthy adults as controls. The degrees of coverage of the femoral head by acetabulum, acetabular labrum, and both were measured at seven positions at every 15 degrees from anterosuperior 45 degrees via midsuperior to posterosuperior 45 degrees. Coverage of the femoral head by the acetabulum in young children was less than in adults at all positions, but the total coverage including the labrum was greater than in adults. Development of the acetabulum and the acetabular labrum showed differences by position.

Acetabulum↗