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Biomedical subjects

M Urabe

Publications and source records attributed to M Urabe.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1998

Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro.

Glial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively; P < 0.01). Furthermore, the dopaminergic neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinson's disease.

Animals↗
PubMed1998

Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors.

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.

Animals↗
PubMed1998

Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblasts.

We investigated the feasibility of an inducible apoptosis system to regulate cells genetically engineered for ectopic cytokine production. In a previous study, cDNA encoding the ligand-binding domain of the rat estrogen receptor was fused to the sequence for murine Fas transmembrane and cytoplasmic regions, and expression of the fusion protein (MfasER) in L929 fibroblasts resulted in estrogen-dependent apoptosis. We applied this MfasER/estrogen strategy to apoptosis-mediated regulation of cytokine production, using the human granulocyte colony-stimulating factor (G-CSF) as a model. Upon estrogen treatment, the G-CSF producers expressing MfasER showed an apoptotic phenotype and died in several hours, with termination of G-CSF production. This estrogen-induced apoptosis was not influenced by whether the target cells were proliferating or resting, unlike a conventional suicide system involving the herpes simplex virus 1 thymidine kinase (HSVtk). That is, estrogen induced prompt and extensive apoptosis in the resting cells which expressed MfasER, while ganciclovir treatment induced only partial reduction of the resting cells which expressed HSVtk. These results imply the feasibility of apoptosis-mediated regulation of cytokine production by genetically modified cells for supplement gene therapy.

Animals↗
PubMed1998

The use of heterologous promoters for adeno-associated virus (AAV) protein expression in AAV vector production.

Although adeno-associated virus (AAV) vectors are potentially useful gene transfer vehicles for gene therapy, the vector production system is currently at the developmental stage. We constructed AAV helper plasmids (Rep and Cap expression plasmids) by replacing a native AAV promoter, p5, with various heterologous promoters to examine whether the efficiency of AAV vector production was influenced by modulating the AAV protein expression pattern. The helper plasmids containing heterologous promoters (EF, CMV, SV40, B19p6, and CAG promoters, respectively) expressed Rep78/68 more efficiently than a conventional helper plasmid (pIM45), but the expression of Rep52/40 and Cap decreased, resulting in a significant reduction in AAV vector production. Furthermore, the efficiency of vector production never fully recovered even if the Cap proteins were supplied by an additional expression plasmid. A large amount of Rep78/68 and/or a reduced level of Rep52/40 may have deleterious effects on AAV vector production. The present findings will aid in the development of a more efficient AAV vector production system.

Capsid↗
PubMed1998

Fas and mutant estrogen receptor chimeric gene: a novel suicide vector for tamoxifen-inducible apoptosis.

Several cancer gene therapy strategies involve suicide genes to kill the neoplasm, or to regulate effector cells such as lymphocytes. We have developed an inducible apoptosis system with a Fas-estrogen receptor fusion protein (MfasER) for rapid elimination of transduced cells. In the present study, we further improved this molecular switch for estrogen-inducible apoptosis to overcome concerns with the wild-type estrogen receptor and its natural ligand, 17beta-estradiol (E2). The ligand-binding domain of MfasER was replaced with that of a mutant estrogen receptor which is unable to bind estrogen yet retains affinity for a synthetic ligand, 4-hydroxytamoxifen (Tm). The resultant fusion protein (MfasTmR) and MfasER were expressed in L929 cells for examination of their ligand specificities. Tm induced apoptosis in MfasTmR-expressing cells (L929MfasTmR) at 10(-8) M or higher concentrations, but induced no apoptosis in MfasER-expressing cells (L929MfasER) at up to 10(-6) M. On the other hand, E2 induced apoptosis in L929MfasER at concentrations as low as 10(-10)-10(-9) M, while it did so partially in L929MfasTmR at concentrations greater than 10(-7) M. Thus, L929MfasTmR cells were highly susceptible to Tm, but refractory to E2, with 100-1,000 times more tolerance than L929MfasER. These results suggest that the MfasTmR/Tm system would induce apoptosis in the target cells more safely in vivo, working independently of endogenous estrogen.

Animals↗
PubMed1998

Echocardiographic evaluation in long-term continuous ambulatory peritoneal dialysis compared with the hemodialysis patients.

In dialysis patients, the prevalence of severe left ventricular (LV) hypertrophy and systolic failure, important predictors of cardiovascular morbidity and mortality, has been reported to be very high. Therefore, we investigated cardiac function in 17 long-term CAPD patients (dialysis duration: 76.5 +/- 13.2 months; L-CAPD) by echocardiography and pulsed Doppler, and then compared with 16 short-term CAPD patients (dialysis duration: 28.9 +/- 11.9 months; S-CAPD), 21 long-term hemodialysis patients (dialysis duration: 165.1 +/- 52.7 months; L-HD), and 22 short-term hemodialysis patients (dialysis duration: 71.3 +/- 28.9 months; S-HD), except for the cases with diabetes mellitus, ischemic heart disease, cardiac surgery or overt congestive heart failure. We selected 13 normotensive patients with normal kidney function as normal control group matched for sex and age (Control). Concerning with L-CAPD, S-CAPD, L-HD, and S-HD, these four groups were matched for age and original diseases. We examined blood pressure (BP), cardiothoracic rate (CTR), antihypertensive (AHT) drugs and laboratory data. Wall thickness, left atrium, ventricular chamber size, ejection fraction (EF) and left ventricular mass (LV mass) [Devereux et al. 1986] were measured by echocardiograph. Peak early diastolic flow velocity (E), peak atrial filling velocity (A), A/E ratio and deceleration time of peak early diastolic flow velocity (DT) were calculated by analyzing transmitral flow, recorded by pulsed Doppler. BP control, CTR and EF were significantly worse in L-CAPD than in other patient groups. A/E as one of parameters for cardiac diastolic function was significantly higher in L-CAPD than in HD patients. LVMI (LV mass index: LV mass/body surface area) was significantly higher in L-CAPD than in other groups. LVMI in CAPD patients was shown to be significantly worse as time goes. Volume control by itself without AHT drugs could achieve good BP control in the long-term CAPD patients who were changed to maintenance hemodialysis because of peritoneal sclerosis. We concluded that LV hypertrophy and systolic dysfunction tend to progress in CAPD patients as time goes on. Also it is suggested that the cause of cardiac dysfunction in CAPD patients was mainly based on poor BP control probably due to overhydration, and therefore, appropriate volume control in CAPD patients is especially important.

Antihypertensive Agents↗
PubMed1997

Development of a novel selective amplifier gene for controllable expansion of transduced hematopoietic cells.

To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)-dependent murine cell line, Ba/F3, the cells showed IL-3-independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Delta(5-195)GCRER, whose product lacks the extracellular G-CSF-binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Delta(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

Animals↗