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441.
Ageyama N Hanazono Y Shibata H Ohto K Ono F Nagashima T Ueda Y Donahue RE Hasegawa M Ozawa K Yoshikawa Y Terao K 《Comparative medicine》2002,52(5):445-451
We have established safe and efficient methods for autologous hematopoietic stem cell (HSC) transplantation in cynomolgus monkeys (Macaca fascicularis) that include regimens of supportive care to ensure survival during hematopoietic reconstitution following otherwise lethal total body irradiation. Eleven young adult cynomolgus monkeys were studied. Bone marrow was aspirated from the ilium and/or tuber ischiae after administration of recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF). Using the immunomagnetic selection method, CD34+ cells were then isolated (90 to 95% pure) as a fraction containing HSCs. Just prior to transplantation, the animals received myeloablative total body irradiation-500 to 550 cGy daily for two days. The monkeys re-infused with CD34+ cells developed moderate to severe myelosuppression, with some animals requiring intravenous hyperalimentation, antibiotic administration, and blood transfusion. Hematopoiesis was restored in all animals after transplantation. It took 12 days, on average, until the peripheral white blood cell count reached more than 1,000 cells/microl. Up to two years after transplantation, signs of radiation-induced pneumonitis or other radiation-related disorders were not evident at the aforementioned dose of irradiation. This transplantation model will be useful for testing new approaches using HSCs for therapy of many diseases and will offer unique insights into the biology of these cells. 相似文献
442.
Nagashima T Ueda Y Hanazono Y Kume A Shibata H Ageyama N Terao K Ozawa K Hasegawa M 《Biochemical and biophysical research communications》2003,303(1):170-176
We previously developed "selective amplifier genes (SAGs)" which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34(+) cells than the GCR-type SAG. One mutant Mpl-type SAG (Delta GCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence. 相似文献
443.
Direct mechanical force measurements during the migration of Dictyostelium slugs using flexible substrata 下载免费PDF全文
We use the flexible substrate method to study how and where mechanical forces are exerted during the migration of Dictyostelium slugs. This old and contentious issue has been left poorly understood so far. We are able to identify clearly separate friction forces in the tip and in the tail of the slug, traction forces mostly localized in the inner slug/surface contact area in the prespore region and large perpendicular forces directed in the outward direction at the outline of contact area. Surprisingly, the magnitude of friction and traction forces is decreasing with slug velocity indicating that these quantities are probably related to the dynamics of cell/substrate adhesion complexes. Contrary to what is always assumed in models and simulations, friction is not of fluid type (viscous drag) but rather close to solid friction. We suggest that the slime sheath confining laterally the cell mass of the slug experiences a tension that in turn is pulling out the elastic substrate in the direction tangential to the slug profile where sheath is anchored. In addition, we show in the appendix that the iterative method we developed is well adapted to study forces over large and continuous fields when the experimental error is sufficiently low and when the plane of recorded bead deformations is close enough to the elastomer surface, requirements fulfilled in this experimental study of Dictyostelium slugs. 相似文献
444.
Furuta T Mori C Suzuki A Shibasaki H Yokokawa A Kasuya Y 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,801(2):165-171
This study describes a high-performance liquid chromatographic (HPLC) method for the simultaneous determination of 6beta-hydroxycortisol (6beta-OHF) and cortisol in human urine using either methylprednisolone or beclomethasone as internal standard. Separation was achieved on a reversed-phase phenyl column by a gradient elution of 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH with acetonitrile (4:6, v/v). 6beta-Hydroxycortisol and cortisol were monitored by UV absorption at 239 nm. The lower quantitation limits of the present HPLC method were 21.5 ng/ml for 6beta-OHF and 5.0 ng/ml for cortisol in urine. The within-day reproducibilities in the amounts of 6beta-OHF and cortisol determined were in good agreement with the actual amounts added, the relative error being less than 1.59%. The inter-assay precisions (R.S.D. values) were less than 7.91% for 6beta-OHF and cortisol. The method was compared with the GC/MS method by measuring 6beta-OHF in the same urine samples. A good correlation was found between the amounts determined by the two methods. The regression equations for the HPLC (y) and GC/MS (x) methods were: y=1.0701x+17.389 (r=0.9772) for methylprednisolone as internal standard and y=1.0827x+6.1364 (r=0.9794) for beclomethasone as internal standard. 相似文献
445.
Mizuno K Sawa M Harada H Tateishi H Oue M Tsujiuchi H Furutani Y Kato S 《Bioorganic & medicinal chemistry letters》2004,14(24):5959-5962
A series of tryptamine-based 2-thiophenesulfonamide derivatives were prepared and their agonistic activity for the beta-adrenergic receptors (ARs) was evaluated. Compound 54, containing 7-methanesulfonyloxy tryptamine, was found to be a highly potent beta3-AR agonist (EC50=0.21 nM, IA=97%) with excellent selectivity for the beta3-AR over the beta1- and beta2-ARs (210- and 86-fold, respectively). 相似文献
446.
Masuo N Ito K Yoshimune K Hoshino M Matsushima K Koyama Y Moriguchi M 《Protein expression and purification》2004,38(2):272-278
We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote. 相似文献
447.
Cell type proportioning in Dictyostelium slugs: lack of regulation within a 2.5-fold tolerance range
Ismael Ràfols · Yasuji Sawada · Aiko Amagai · Yasuo Maeda · Harry K. MacWilliams 《Differentiation; research in biological diversity》2001,67(4-5):107-116
The proportion of prestalk and prespore cells in Dictyostelium discoideum slugs is often cited as an example of "almost perfect" regulation. The pattern is similar over a very wide range of cell number; furthermore, removal of either of the cell types leads to compensatory transdifferentiation. Several studies of Dictyostelium fruiting bodies, however, have suggested that proportioning in Dictyostelium differs systematically from true constancy. We have confirmed this in the slug stage using a short-lived beta-galactosidase as a reporter of the prestalk specific ecmA gene expression: the prestalk proportion decreases from 24+/-5% in slugs of 10(3) cells to 10+/-3% when 10(5) cells are present. Regeneration experiments suggest that this difference is not due to a modulation of the proportioning set-point by size, as one might have expected; instead there appears to be a regulatory "tolerance zone" at all sizes. After amputation of the whole posterior region, transdifferentiation stops after the fraction of prestalk has been reduced from 100% to 28+/-20%, well above the initial value of 10+/-3%, while after anterior removal the transdifferentiation endpoint is about 10%. Most strikingly, we find no regulation at all after partial amputations of the prespore region. It seems that any prestalk proportion is stable between a approximately 10% lower threshold and a approximately 30% upper threshold. To explain this, we propose a regulation mechanism based on a negative feedback plus cell type bistability. In both intact and regenerating slugs we find that the slug morphology is regulated so that the length-to-width ratio of the anterior region is constant. 相似文献
448.
The relative importance of the liver and kidney for glycine conjugation of ortho-substituted benzoic acids was investigated. Glycine conjugation of ortho-substituted benzoic acids was investigated in mouse liver and kidney mitochondria. The extent of glycine conjugation of benzoic acids with the halogen group decreased in the order F > Cl > Br > I. The conjugation of salicylic acid with glycine took place in only the kidney. 2-Methoxybenzoic acid exhibited no activity in the liver and kidney. The difference in glycine conjugation of ortho-substituted benzoic acids was observed between liver and kidney. The kidney was more active in glycine conjugation of ortho-substituted acids than the liver. In addition, the relationship between glycine conjugation and the chemical structure of ortho-substituted acids was examined in the liver and kidney. The size of the substituent had a far greater influence over glycine conjugation in the liver and kidney. Glycine conjugation was also dependent on the substituent electronegativity. It may be important that the substrates undergoing glycine conjugation contain a flat region coplanar to the carboxylate group. 相似文献
449.
This study describes a new approach using stable isotope methodology in evaluating 11beta-HSD activities in vivo based on urinary excretion of cortisol, cortisone, and their A-ring reduced metabolites. The method involved the measurement of deuterium-labeled cortisol and its deuterium-labeled metabolites by GC/MS simultaneously with endogenous cortisol, cortisone, and their A-ring reduced metabolites after oral administration of deuterium-labeled cortisol to normal human subjects. This stable isotope approach offered unique advantages in assessing the appropriateness of measuring unconjugated and total (unconjugated + conjugated) cortisol, cortisone, and their A-ring reduced metabolites in urine as indices of renal 11beta-HSD2 activity in man. Our results strongly support that the measurement of urinary unconjugated cortisol and cortisone is a significant advance in assessing 11beta-HSD2 activity. 相似文献
450.