Biomedical subjects

Kenji Kawamura

Publications and source records attributed to Kenji Kawamura.

At least 19 recordsLinked to original sources

PubMed2006

Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor.

A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H- and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors.

Animals↗

PubMed2006

Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication.

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

Animals↗

PubMed2006

Oberlin partial ulnar nerve transfer for restoration in obstetric brachial plexus palsy of a newborn: case report.

An 8 month old male infant with Erb's birth palsy was treated with two peripheral nerve transfers. Except for rapid motor reinnervations, elbow flexion was obtained by an Oberlin's partial ulnar nerve transfer, while shoulder abduction was restored by an accessory-to-suprascapular nerve transfer. The initial contraction of the biceps muscle occurred two months after surgery. Forty months after surgery, elbow flexion reached M5 without functional loss of the ulnar nerve. This case demonstrates an excellent result of an Oberlin's nerve transfer for restoration of flexion of the elbow joint in Erb's birth palsy. However, at this time partial ulnar nerve transfer for Erb's birth palsy is an optional procedure; a larger number of cases will need to be studied for it to be widely accepted as a standard procedure for Erb's palsy at birth.

Case Reports↗

PubMed2006

A protein kinase signal-responsive gene carrier modified RGD peptide.

We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using a cationic polymer. However, this polymer alone cannot deliver any gene into living cells. In the present work, we modified the signal-responsive polymer to the RGD peptide for the introduction of a polymer/DNA complex into living cells and succeeded in regulating the gene expression responding to intracellular PKA activation.

Cell Line, Tumor↗

PubMed2006

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C.

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.

Amino Acid Motifs↗

PubMed2006

Experimental study of vascularized tissue-engineered bone grafts.

BACKGROUND: Vascularized bone grafting has become a useful method for treatment of large bone defects and infected nonunions that lack adequate blood supply. This method has disadvantages, however, such as donor-site complications. To overcome these disadvantages, the authors developed a new method for creating vascularized tissue-engineered bone grafts in an experimental model. METHODS: Fisher rat bone marrow mesenchymal stem cells were cultured for 2 weeks in fully opened, interconnected, porous hydroxyapatite ceramic. The composites of mesenchymal stem cells and hydroxyapatite were implanted in the medial calf region together with the saphenous vascular bundle in syngeneic rats. Two weeks after implantation, the vascular pedicle with the implants was exposed and covered with silicone rubber to prevent vascular invasion through surrounding tissues (vascularized mesenchymal stem cell/hydroxyapatite implants). In addition, nonvascularized mesenchymal stem cell/hydroxyapatite implants were created with a ligated vascular pedicle. Implants of vascularized hydroxyapatite alone were prepared as a control. All implants were retrieved 4 weeks after surgery. RESULTS: Histologically, de novo bone formations were observed in the vascularized implants. This was in contrast with only necrotic tissue observed in the nonvascularized implants. Bone formation was not observed entirely in the vascularized hydroxyapatite-alone implants. For biochemical analysis, alkaline phosphatase activity and osteocalcin content of the vascularized mesenchymal stem cell/hydroxyapatite implants were significantly higher than those of the nonvascularized mesenchymal stem cell/hydroxyapatite and vascularized hydroxyapatite-alone implants. CONCLUSION: The results of this study indicated that the vascularized tissue-engineered bone grafts could be transferred as free vascularized grafts to lesions that lack adequate blood supply.

Alkaline Phosphatase↗

PubMed2006

Diffusive separation of the lower atmosphere.

The separation of atmospheric constituents by gravity has been proposed theoretically for almost two centuries. However, turbulent mixing has prevented the detection of this phenomenon in the lower atmosphere. By using precise measurements of the Ar/N2 ratio of air samples taken under strong nocturnal inversions, we have detected such separation in near-surface layers. The effect is shown to be consistent with combined influence of thermal and gravimetric separation, with the thermal contribution being more important.

Journal Article↗

PubMed2006

Treatment of Kienböck's disease with cultured stem cell-seeded hybrid tendon roll interposition arthroplasty: experimental study.

BACKGROUND: Several procedures that consist of excision arthroplasty, intercarpal arthrodesis with or without excision of the lunate, proximal row carpectomy, and joint leveling procedures are used to treat advanced Kienböck's disease. However, no reconstructive surgery for a collapsed lunate has been established. The aim of this study was to develop a method of tendon roll interposition arthroplasty using a tissue-engineering technique to improve the clinical results of the tendon roll interposition arthroplasty for treating advanced Kienböck's disease. METHODS: Twenty-four Japanese white rabbits were used for this study as three treatment models of Kienböck's disease. The lunate of the right paw was excised, and then one of three kinds of tendon roll was interposed into the excision space as follows: group A (controls), a conventional tendon roll made of autologous Achilles tendon; group B (cored tendon roll) a tendon roll with a core of the collagen-ceramic composite; and group C (hybrid tendon roll), a scaffold seeded with cultured cells was rolled in the Achilles tendon. In all groups the right wrist was immobilized with cast 6 weeks after surgery. Wrist radiography was evaluated 0, 4, 6, and 12 weeks after surgery; specimens were sectioned and evaluated histologically 12 weeks after surgery. RESULTS: In group C the small shadow of the hydroxyapatite granule gradually increased until 12 weeks after surgery, whereas in group B the shadow of the core gradually decreased. The deficit spaces of the lunate remained radiographically lucent for 12 weeks after surgery in group A. Histological findings revealed new bone formation at the center and stained cartilage matrix at the peripheral of the hybrid tendon roll group (group C) but not in group A or B. CONCLUSIONS: Radiological and histological examinations proved that it is possible to make new hybrid tendon rolls using this method, as osteogenesis in the center and cartilage matrix in the peripheral of the tendon roll were revealed.

Animals↗

PubMed2006

Fixation choices for closed simple unstable oblique phalangeal and metacarpal fractures.

Percutaneous K-wire fixation is still a useful technique for closed oblique phalangeal and meta-carpal fractures when an adequate closed reduction can be achieved. Lag screw fixation may be the best choice for open fixation of long oblique phalangeal and metacarpal fractures. For short oblique fractures, plating or tension band wiring is recommended. Plating provide rigid fixation to allow early mobilization; however, one may encounter frequent complications such as extensor lag, stiffness, or joint contracture when plating technique is used in phalangeal fractures. Tension band wiring technique at the phalangeal location may reduce such complications. Overall, successful outcomes of treating phalangeal and metacarpal fractures require a clear appreciation of fracture anatomy and pattern. It is mandatory for the treating surgeon to be familiar with all the treatment techniques discussed in order to tailor a specific technique for a particular injury and patient type.

Biomechanical Phenomena↗

PubMed2006

Giant-cell tumors of the carpus.

Giant cell tumor (GCT) of bone is a rare, benign tumor with some aggressive characteristics such as a high recurrence rate. The tumor usually occurs in the distal radius although it has been reported in the carpus. We reviewed 63 manuscripts published from 1935 to 2005 and report on the treatment of GCT of carpal bone in 29 cases. Intralesional procedures, such as curettage, were associated with a high incidence of recurrence (24%), whereas cases treated with an excisional procedure did not recur.

Amputation, Surgical↗

PubMed2006

Outcomes using an internal osteotomy and distraction device for corrective osteotomy of distal radius malunions requiring correction in multiple planes.

PURPOSE: Many different methods have been proposed for correction of distal radius malunions; however, precise correction of a severe malunion that requires simultaneous adjustment of displacement, angulation, and rotation in multiple planes is difficult. This prospective study measured radiographic and functional outcomes using an adjustable osteotomy, distraction, and fixation system for distal radius malunions that required correction in multiple planes. METHODS: Five consecutive patients with symptomatic distal radius malunions requiring correction in multiple planes were recruited into this study. All patients had correction of the malunion with an adjustable osteotomy, distraction, and fixation device. Radiographic parameters and objective and subjective functional outcomes were measured before surgery and at 3 months, 6 months, and 1 year after surgery. RESULTS: All radiographic measurements improved after surgery. Grip strength, pinch strength, and the Jebsen-Taylor test showed only marginal improvement. Ulnar deviation of the wrist and forearm pronation-supination improved, but the wrist extension-flexion arc did not. The Michigan Hand Outcomes Questionnaire showed improvement in all domains, and significant improvement was seen in the overall, work, aesthetic, and satisfaction domains. The Michigan Hand Outcomes Questionnaire, however, also indicated a substantial amount of residual impairment in all domains, including activities of daily living and pain. CONCLUSIONS: Corrective osteotomy with the adjustable osteotomy, distraction, and fixation device resulted in improvement in distal radius anatomy and function. However, not all improvements were statistically significant, and anatomy and function did not returned to baseline levels. Despite residual disability, patients noted substantial improvement in subjective outcomes. This system is useful for improving anatomy and function in distal radius malunions that require correction in multiple planes, but patients should be informed that they cannot expect to regain normal anatomy or function. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Activities of Daily Living↗

PubMed2006

Post-traumatic big toe reconstruction using free flaps.

The big toe is of great importance for good stability and gait, but few reports have documented reconstruction of big toe defects. In this study, seven male patients, aged 17 to 59 years at surgery (average: 35 years), were treated for big toe defects. The metatarsophalangeal (MTP) joints of the big toe were intact in all patients. Five patients were treated with free peroneal flaps (including one perforator flap), and two with free scapular flaps; flap sizes ranged from 9 x 4 to 24 x 6 cm. Follow-up periods ranged from 10 to 29 months, (average: 16.6 months). The iliac was used as the grafted bone in four patients and the scapula in one. Six flaps survived completely, and bone unions were achieved within 3 months. One flap became partially necrotic due to arterial thrombosis. All patients returned to their original jobs, and the cosmetic appearances of all toes were acceptable.

Adolescent↗

PubMed2006

Intracellular signal-responsive artificial gene regulation.

In gene therapy, in order to avoid serious side effects due to the unexpected expression of the transgene in non-target cells, transgenes have to be delivered only to the target cells. In response to this issue, many researchers have aimed at developing target cell-selective gene carriers using active targeting strategies. However, such methodology does not always work, because an ideal molecular marker, which is specific to the target disease cells, is not always available. In this study, we introduce a new concept regarding target disease cell-selective gene therapy (D-RECS). Here, we use intracellular signals, which are activated to an extraordinary degree only in the target disease cells, as a trigger for transgene expression using polymer-peptide conjugates. This strategy could actually activate gene expression in the target signal-activated cells only. Hyper-activation of certain intracellular signals has been reported in many diseases. Thus, this new strategy is expected to provide a powerful methodology for future gene therapy. In this review, the basic concept, some examples, and the molecular design of D-RECS carriers are introduced.

Animals↗

PubMed2006

Induction of centrosome amplification in p53 siRNA-treated human fibroblast cells by radiation exposure.

Centrosome amplification can be detected in the tissues of p53(-/-) mice. In contrast, loss of p53 does not induce centrosome amplification in cultured human cells. However, examination of human cancer tissues and cultured cells has revealed a significant correlation between loss or mutational inactivation of p53 and occurrence of centrosome amplification, supporting the notion that p53 mutation alone is insufficient to induce centrosome amplification in human cells, and that additional regulatory mechanisms are involved. It has recently been shown that gamma irradiation of tumor cells induces centrosome amplification. However, the precise mechanism of radiation-induced centrosome amplification is not fully understood. In the present study, CCD32SK diploid normal human fibroblasts were transfected transiently with short interfering RNA (siRNA) specific for human p53 (CCD/p53i). There was a small increase in the frequency of centrosome amplification in CCD/p53i cells (4.0%) without irradiation. In contrast, CCD/p53i cells after 5-Gy irradiation showed a marked increase in abnormal nuclear shapes and pronounced amplification of centrosomes (46.0%). At 12 h after irradiation, irradiated CCD/p53i cells were arrested in G(2) phase. By laser scanning cytometry, abnormal mitosis with amplified centrosomes was observed frequently in the accumulating G(2)/M population at 48 h after irradiation. In the present study, we found that siRNA-mediated silencing of p53 in normal human fibroblasts, together with DNA damage by irradiation, efficiently induced centrosome amplification and nuclear fragmentation, but these phenomena were not observed with either siRNA-mediated silencing of p53 or irradiation alone.

Cell Division↗

PubMed2006

Tissue-engineered approach for the treatment of steroid-induced osteonecrosis of the femoral head: transplantation of autologous mesenchymal stem cells cultured with beta-tricalcium phosphate ceramics and free vascularized fibula.

Autologous mesenchymal stem cells (MSCs) cultured with beta-tricalcium phosphate (beta-TCP) ceramics and with a free vascularized fibula were transplanted into three patients with steroid-induced osteonecrosis of the femoral head. The average follow-up period was 34 months and the average patient age at the time of surgery was 28 years old. Fifteen milliliters of bone marrow was obtained from the patients 4 weeks before surgery, and was used for in vitro proliferation of MSCs. beta-TCP granules were immersed in the MSC suspension and the cells were further cultured for 2 weeks. Cultured MSCs/beta-TCP composite granules were implanted into the cavity that remained after curettage of necrotic bone; and finally, a free vascularized fibula was grafted. All hips showed preoperative collapse and radiographic progression was observed in two hips postoperatively. Osteonecrosis did not progress any further and early bone regeneration was observed. This tissue-engineered approach has potentials for the treatment of osteonecrosis. However, our results suggested that the present procedure could not be used for cases with severe preoperative collapse.

Adult↗

PubMed2006

[Gene delivery system responding to the intracellular signal].

We designed a peptide-polymer conjugate as a novel gene carrier, which could control gene-expression responding to intracellular cAMP-dependent protein kinase (PKA) or caspase-3 signal. Each polymer containing a substrate peptide (ALRRASLG for PKA or GGGDEVD GGRKKRRQRRRPPQ for caspase-3) could form a tight complex with plasmid DNA via electrostatic interaction, and in this state, the gene expression was totally suppressed. Once the grafted peptides become phosphorylated or hydrolyzed by the appropriate enzyme, the net charge of the peptide changes from cationic to neutral or anionic, respectively, and the polymer then releases the DNA, such that expression of the foreign gene should become activated. We expect that these systems can be applied to the novel highly cell specific gene delivery strategy.

Caspase 3↗

PubMed2005

Atmospheric methane and nitrous oxide of the Late Pleistocene from Antarctic ice cores.

The European Project for Ice Coring in Antarctica Dome C ice core enables us to extend existing records of atmospheric methane (CH4) and nitrous oxide (N2O) back to 650,000 years before the present. A combined record of CH4 measured along the Dome C and the Vostok ice cores demonstrates, within the resolution of our measurements, that preindustrial concentrations over Antarctica have not exceeded 773 +/- 15 ppbv (parts per billion by volume) during the past 650,000 years. Before 420,000 years ago, when interglacials were cooler, maximum CH4 concentrations were only about 600 ppbv, similar to lower Holocene values. In contrast, the N2O record shows maximum concentrations of 278 +/- 7 ppbv, slightly higher than early Holocene values.

Journal Article↗

PubMed2005

An intracellular kinase signal-responsive gene carrier for disordered cell-specific gene therapy.

We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using cationic polymer. This cationic polymer (PAK) was a graft-type polymer with an oligopeptide that is a substrate for PKA and could regulate gene-expression in a cell-free system. In the present study, we carried out a detailed characterization of the PAK-DNA complex (AFM observation and DLS measurement) and tried to apply this polymer to living cells. In the unstimulated NIH 3T3 cells, transfection of the PAK-DNA complex showed no expression of the delivered gene. This means that PAK formed a stable complex with DNA in the normal cells to totally suppress gene expression. In contrast, significant expression was seen when the PAK-DNA complex was delivered to forskolin-treated cells. Thus, activated PKA disintegrates the complexes even in living cells, resulting in gene expression. Our results indicate that this type of intracellular signal-responsive polymer will be useful for the cell-specific release of genes.

3T3 Cells↗