Biomedical subjects

M Urabe

Publications and source records attributed to M Urabe.

At least 37 records · Page 2Linked to original sources

PubMed2000

Morphogenetic behavior of periodontium on inorganic implant materials: an experimental study of canines.

Periodontal ligament derived cells have the potential to regenerate all the components of the periodontium on the surface of inorganic implants, as well as on dentin. This suggests the hypothesis that the nature of the material affects the migration, proliferation, and differentiation of the progenitor cells for periodontium formation. To clarify this hypothesis, we evaluated the material-specific morphogenetic potential of periodontium-derived cells using an animal model for inducing cell migration from the functioning periodontium onto bioactive (hydroxyapatite, HA) and bioinert (titanium alloy, TA) material. Histologically, total periodontium including calcified cementum-like tissue only formed on HA and not on TA. Morphometrically, however, the length of fibrous connective tissue formed on HA was the same as on TA. This suggests that the bioactivity of the material does not affect the migration of periodontium-derived cells but strongly influences cell differentiation.

Animals↗

PubMed2000

Gene therapy of Parkinson's disease using adeno-associated virus (AAV) vectors.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)]. The most appropriate gene-delivery vehicles for neurons are adeno-associated virus (AAV) vectors, which are derived from non-pathogenic virus. Therefore, TH and AADC genes were introduced into the striatum in the lesioned side using separate AAV vectors in parkinsonian rats, and the coexpression of TH and AADC resulted in better behavioral recovery compared with TH alone. Another strategy for gene therapy of PD is the protection of dopaminergic neurons in the substantia nigra using an AAV vector containing a glial cell line-derived neurotrophic factor (GDNF) gene. Combination of dopamine-supplement gene therapy and GDNF gene therapy would be a logical approach to the treatment of PD.

Animals↗

PubMed2000

Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: prospects for gene therapy of ischemia-induced neuronal death.

The proto-oncogene bcl-2 is known as an anti-apoptotic gene that confers the ability to block neuronal cell death after transient ischemia. In order to examine whether the bcl-2 gene can be used for protection of ischemic brain injury, we generated adeno-associated virus (AAV) vectors capable of expressing human bcl-2. Replication-defective AAV vectors were found effectively to transfer and express bcl-2 gene in the gerbil hippocampal neurons. Transduction with AAV bcl-2 5 days before forebrain ischemia prevented the DNA fragmentation in the CA1 neurons that is commonly associated with ischemia-induced cell death. Furthermore, the application of AAV bcl-2 as late as 1 h following an ischemic insult also prevented DNA fragmentation in CA1 neurons. These results suggest that the bcl-2 protein has neuroprotective functions that inhibit ischemic cell death and demonstrate the potential of AAV bcl-2 for use in post-ischemic gene therapy in the brain. Gene Therapy (2000) 7, 1244-1249.

Adenoviridae↗

PubMed2000

Long-term tracking of murine hematopoietic cells transduced with a bicistronic retrovirus containing CD24 and EGFP genes.

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy, but current gene transfer methodologies are inadequate for efficient HSC transduction and perpetual transgene expression. To improve gene transfer vectors and transduction protocols, it is vital to establish a system to evaluate transgene expression and the long-term behavior of transduced cells in vivo. For this purpose, we constructed a bicistronic retrovirus encoding the human CD24 (as the first cistron) and the enhanced green fluorescent protein (EGFP; as the second cistron). Murine bone marrow cells were transduced with this vector and the transgene expression was monitored along with hematopoietic reconstitution. Stable expression of CD24 and EGFP was demonstrated in the long-term repopulating cells for at least 6 months, and multi-parameter flow cytometry illustrated expression of both markers in all the lymphohematopoietic lineages examined (B and T lymphoid, erythroid and myeloid). Sustained expression was also shown in the secondary transplants for 6 months, suggesting that self-renewing HSCs were transduced by this vector. Overall, EGFP-tagged bicistronic retroviruses would provide powerful tools for detailed in vivo analysis of transduced hematopoietic cells, such as transgene expression in conjunction with lineage differentiation. Gene Therapy (2000) 7, 1193-1199.

Animals↗

PubMed2000

Haemorrhagic cerebral sinus thrombosis associated with ulcerative colitis: a case report of successful treatment by anticoagulant therapy.

A 29-year-old man with ulcerative colitis was admitted to our hospital because of convulsions and a headache. Before admission, oral prednisolone had been administered due to his ulcerative colitis relapse. Computed tomography revealed a low-density area in the right frontal pole suggestive of a venous infarction. Once his headache and convulsions improved after the administration of an antiepileptic drug, he began to complain of right arm numbness and right hemianopsia again. An urgent magnetic resonance imaging angiograph showed extensive thrombosis in the superior sagittal sinus. We finally used the anticoagulant agents, heparin and urokinase, which eased his complaints and prevented the development of bloody stools. He was discharged with no neurological symptoms 25 days after admission. This is a rare case of sinus thrombosis complicated by ulcerative colitis, in which anticoagulant therapy was successful. Magnetic resonance imaging angiography was useful for the diagnosis and for evaluating the therapeutic effect.

Adult↗

PubMed2000

Adeno-associated virus-mediated vascular endothelial growth factor gene transfer into cardiac myocytes.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that stimulates endothelial cell proliferation, increases endothelial permeability, and promotes collateral vessel formation. We transferred human VEGF gene into rat cardiac myocytes using adeno-associated virus (AAV) vectors and investigated whether VEGF secreted from the transduced cardiac myocytes promoted proliferation of endothelial cells. We produced VEGF-expressing AAV vectors (AAV-VEGF) by the adenovirus-free method. Immunoblotting revealed VEGF protein expression in AAV-VEGF-transduced rat cardiac myocytes. More than 60% of cardiac myocytes were stained positively on immunohistochemical staining using anti-VEGF antibody. Concentration of VEGF in the culture medium of AAV-VEGF-transduced myocytes was increased in a vector dose-dependent manner, and VEGF secretion from the transduced myocytes persisted for > or = 14 days. Thymidine incorporation into human vascular endothelial cells was significantly increased by incubation with the conditioned medium from AAV-VEGF-transduced myocytes. This increased thymidine uptake was significantly inhibited by anti-VEGF antibody. We demonstrated here that AAV-mediated VEGF gene transfer into cardiac myocytes induces the secretion of functional VEGF.

Animals↗

PubMed2000

Effect of estrogen and progesterone on gene expression of growth regulatory molecules and proto-oncogene in vascular smooth muscle cells.

We examined influences of estrogen and progestogen on gene expression of the growth regulatory molecules: platelet-derived growth factor (PDGF), interleukin-1 (IL-1), interleukin-6 (IL-6) and proto-oncogene c-myc in vascular smooth muscle cells (VSMC) by reverse transcription-polymerase chain reaction (RT-PCR) and Southern-blotting. VSMC were exposed to estrone-sulfate (E1-S) and medroxyprogesterone acetate (MPA) to induce differentiation. E1-S inhibited the expression of PDGF-A chain, IL-1, IL-6 and c-myc mRNA, whereas MPA had no effect. Inhibition by E1-S was not affected by treatment combined with MPA. These findings suggest that estrogen modulates these growth regulatory molecules and c-myc gene expression in VSMC but not progestogen. We concluded that estrogen may have a direct atheroprotective effect through inhibition of growth regulatory factors.

Arteriosclerosis↗

PubMed1999

A switching system regulating subcellular localization of nuclear proteins using a viral protease.

We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, beta-galactosidase with the nuclear localization signal of SV40 (nbeta-gal), within the cytoplasm, nbeta-gal was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor (G-CSFR), a membrane protein. To liberate the nbeta-gal portion from the fusion protein, we used a protease derived from a plant virus, whose recognition sequence was inserted between the G-CSFR and nbeta-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescence microscopy. In contrast, when expressed together with the protease, beta-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the protease, indicating that this protease is not cytotoxic. These results suggest that the viral protease-mediated alteration of subcellular localization can potentially regulate the function of nuclear proteins.

Biological Transport↗

PubMed1999

A G-CSF receptor-gyrase B fusion gene: A new type of molecular switch for expansion of genetically modified hematopoietic cells.

We have developed a novel system for expansion of transduced hematopoietic cells. This system involves "selective amplifier genes" encoding fusion proteins between the granulocyte colony-stimulating factor receptor (Gcr) and the estrogen receptor (Er). The GcrEr chimeric gene conferred estrogen-dependent growth ability on murine hematopoietic cells. Here, we constructed a modified "selective amplifier gene" to circumvent possible concerns with the Er/estrogen switching system. The bacterial gyrase B (Gyr) gene was fused to the Gcr gene, and the GcrGyr fusion construct was introduced into interleukin-3 (IL-3)-dependent Ba/F3 cells. The dimeric antibiotic coumermycin induced IL-3-independent growth in Ba/F3 cells expressing GcrGyr. This stimulatory effect was antagonized by an excess amount of novobiocin, a monomeric form of coumermycin. These results suggest the feasibility of using Gyr as a molecular switch to regulate a growth signal in hematopoietic cells.

Aminocoumarins↗

PubMed1999

Osteoporosis influences the late period of fracture healing in a rat model prepared by ovariectomy and low calcium diet.

To elucidate the influence of osteoporosis on the fracture healing, we produced a rat osteoporosis model by ovariectomy and by maintaining a low calcium diet; and monitored the healing process radiographically, histologically, and biomechanically for 12 weeks. Radiologic, histologic and biomechanical findings of the fracture areas 6 weeks after making the fractures were almost identical in both the osteoporosis group and the control group. However, 12 weeks after making the fractures, newly generated bones in the osteoporosis group showed histological osteoporotic changes and their bone mineral density on the fracture site decreased. These findings show that estrogen-deficient and low calcium conditions greatly affect the bone in the later period of the healing process, but do not affect remarkably the early healing period. This is clinically important when we consider fracture treatments for patients with osteoporosis due to menopause.

Animals↗

PubMed1999

Development of a modified selective amplifier gene for hematopoietic stem cell gene therapy.

We have proposed a novel concept, ie selective expansion of transduced cells, to overcome the low efficiency of gene transfer into hematopoietic stem cells. Previously, a fusion gene encoding a chimeric receptor (DeltaGCRER) between the mouse granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain of rat estrogen receptor was constructed as a 'selective amplifier gene'. Although the chimeric gene conferred estrogen-inducible proliferation on the transduced Ba/F3 cells, it also mediated differentiation of the retrovirally transduced 32D cells upon estrogen treatment. Since only a growth signal is required for our purpose, we further modified the DeltaGCRER gene to attenuate its differentiation signal. Based on the observation that tyrosine-703 in wild-type G-CSFR plays a pivotal role in transmitting the differentiation signal, phenylalanine was substituted for this residue in DeltaGCRER. When the resultant selective amplifier gene (DeltaY703F-GCRER gene) was expressed in 32D cells, sustained growth was supported by estrogen, while differentiation was suppressed. These cells ceased to grow upon estrogen withdrawal and differentiated with G-CSF treatment. The present findings suggested that DeltaY703F-GCRER may have desirable properties as a selective amplifier for hematopoietic stem cell expansion and gene therapy.

Animals↗

PubMed1999

Efficient production of adeno-associated virus vectors using split-type helper plasmids.

Adeno-associated virus (AAV) vectors are potentially useful vehicles for the delivery of therapeutic genes into human cells. To determine the optimal expression pattern of AAV proteins (Rep78, Rep68, Rep52, Rep40, and Cap proteins) for packaging the recombinant AAV genome, helper plasmids were split into two portions. In this study, two sets of split-type helper plasmids were prepared; i.e., 1) a Rep expression plasmid (pRep) and Cap expression plasmid (pCap), and 2) a large Rep expression plasmid (pR78/68) and small Rep plus Cap expression plasmid (pR52/40Cap). When AAV vectors were produced using these sets of split-type helper plasmids at various ratios, the optimal ratio of (large) Rep expression plasmid and Cap expression plasmid was 1 to 9 for both sets. More importantly, the titers were comparable to or even higher than that of a conventional helper plasmid (pIM45) (4.9+/-2.1x10(11) vector particles/10 cm dish for pRep and pCap; 2.9+/-1.6x10(11) vector particles/10 cm dish for pR78/68 and pR52/40Cap; and 1.8+/-0.16x10(11) particles/10 cm dish for pIM45). Western analysis of AAV proteins suggests that the expression of a relatively small amount of large Rep and a large amount of Cap is important for optimal vector production. The present study shows that the AAV helper plasmid can be split without losing the ability to package the recombinant AAV genome, and provides us with valuable basic information for the development of efficient AAV packaging cell lines.

Cell Line, Transformed↗

PubMed1999

Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein.

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.

Alanine↗

PubMed1999

Metastatic pulmonary epithelioid hemangioendothelioma with peculiar radiographic features.

We report a case of a pulmonary epithelioid hemangioendothelioma presenting as a solitary pulmonary nodule with subsequent systemic spread, including histologically documented gingival metastasis. Chest radiography and computed tomography at initial evaluation showed a solitary 2 cm pulmonary nodule, associated with pleural indentation and vascular convergence. The tumor later spread systemically, including multiple pulmonary nodules, bilateral pleural effusions, and gingival swelling and erosion. A biopsy of the gingival lesion showed histologic features identical to that of the lung biopsy.

Fatal Outcome↗

PubMed1999

Hematopoietic stem cell gene therapy: a current overview.

Retrovirus-mediated gene transfer into murine hematopoietic stem cells and reconstitution of syngeneic mice have demonstrated persistence and functioning of the transgenes over extended periods of time. In contrast, clinically relevant levels of gene transfer into large animal and human stem cells have not been widely achieved. Results of current clinical gene transfer studies have raised fundamental questions about the physiology of primitive human hematopoietic cells and gene therapy vectors. Efforts are being undertaken to answer these problems and to develop more efficient gene therapy strategies.

Animals↗

PubMed1999

[A patient uninformed about his illness became aware of his lung cancer when an anesthesiologist visited him for pain control].

Much has been said about the importance of informed consent in Japan, but informing cancer to the patient has not been popular so far. We, as anesthesiologists, often treat pain in cancer patients, who occasionally, are not informed about the cancer. And we sometimes have patients with whom cautious consulting is necessary. This report presents our experience with a patient uninformed about the cancer but suspicious of his lung cancer. We met him as anesthesiologists, and this made the patient convinced that he had a cancer and was about to die soon.

Adult↗

PubMed1998

Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of enzymes in the biosynthetic pathway for dopamine. Tyrosine hydroxylase (TH) catalyzes the synthesis of L-dopa, which must be converted to dopamine by aromatic L-amino acid decarboxylase (AADC). Since the endogenous AADC activity in the striatum is considered to be low, coexpression of both TH and AADC in the same striatal cells would increase the dopamine production and thereby augment the therapeutic effects. In the present study, the TH gene and also the AADC gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC. Immunostaining showed that TH and AADC were coexpressed efficiently in the same striatal cells in vitro and in vivo. Moreover, cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine (6-OHDA)-lesioned rats, compared with rats receiving AAV-TH alone (p < 0.01). These findings suggest an alternative strategy for gene therapy of PD and indicate that the simultaneous transduction with two AAV vectors can extend their utility for potential gene therapy applications.

Animals↗

PubMed1998

Early development of Epstein-Barr virus-associated T-cell lymphoma after a living-related renal transplantation.

We herein report a case of Epstein-Barr virus (EBV)-associated T-cell lymphoma that developed within a month after a kidney transplantation. The recipient was a 37-year-old man who had evidence of a previous EBV infection. Cyclosporine, methylprednisolone, and azathioprine were used for immunosuppression, and acute rejection was treated with high-dose methylprednisolone. The lactate dehydrogenase level started to increase on day 24 and thereafter peaked on day 37 while also demonstrating progressive jaundice and a bleeding tendency. A transplant nephrectomy was done on day 37; however, the patient could not recover and eventually died of respiratory failure as a result of diffuse pulmonary edema. A pathological examination of the resected kidney revealed a diffuse proliferation of large atypical lymphoid cells in the parenchyma. Immunohistochemically, the tumor cells were positive for CD45 and T-cell marker, CD45RO, but negative for B-cell markers. EBV-encoded RNA was demonstrated within the neoplastic cells by in situ hybridization.

Adult↗