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101.
Su Y Zharikov SI Block ER 《American journal of physiology. Lung cellular and molecular physiology》2002,282(6):L1183-L1189
The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2-4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 microM) for 2-4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 microM) for 2-4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein. 相似文献
102.
Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors 总被引:18,自引:0,他引:18
Zolotukhin S Potter M Zolotukhin I Sakai Y Loiler S Fraites TJ Chiodo VA Phillipsberg T Muzyczka N Hauswirth WW Flotte TR Byrne BJ Snyder RO 《Methods (San Diego, Calif.)》2002,28(2):158-167
Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml. 相似文献
103.
104.
Zorbas YG Kakurin VJ Denogradov SD Luzhkov SH Neofitov AC 《Biological trace element research》2002,90(1-3):155-173
Hypokinesia (diminished movement) induces muscle mineral depletion. However, the mechanism of muscle mineral depletion during
hypokinesia (HK) remains unknown. Measuring electrolyte retention and electrolyte values in muscle, plasma, and urine during
and after HK, the aim of this study was to discover if HK could depress mineral retention and lead to muscle mineral depletion.
Studies were done on 204 13-wk-old male Wistar rats (370–390 g) during 10 d pre-HK period, 98 d HK period, and 15 d post-HK
period. Rats were equally divided into two groups: vivarium control rats (VCR) and hypokinetic rats (HKR). All hypokinetic
rats were kept for 98 d in small individual cages, which restricted their movements in all directions without hindering food
and water intakes. All control rats were housed for 98 d in individual cages under vivarium control conditions. Both groups
of rats were pair-fed.
During the HK period skeletal muscle sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), and water content and electrolyte
retention decreased significantly (p < 0.05), while urinary and plasma electrolyte levels increased significantly (p < 0.05) in HKR compared with their pre-HK values and their respective VCR. During the initial days of the post-HK period,
mineral retention increased significantly (p < 0.05), plasma and urinary electrolyte level decreased significantly (p < 0.05), while muscle electrolyte and water content remained significantly (p < 0.05) depressed in HKR compared with VCR. Muscle mineral and water content, electrolyte retention, plasma, and urinary
electrolyte values did not change in VCR compared with their pre-HK values.
It was concluded that during HK decreased muscle mineral content may suggest muscle mineral depletion, while increased urinary
electrolyte loss and muscle mineral depletion may demonstrate reduced mineral retention. Reduced electrolyte excretion and
depressed muscle mineral content during post-HK may indicate skeletal muscle mineral depletion during HK. Dissociation between
electrolyte retention and muscle mineral depletion may demonstrate the presence of decreased electrolyte retention as the
mechanism of muscle electrolyte depletion during prolonged HK. 相似文献
105.
Redundancy in tumor necrosis factor (TNF) and lymphotoxin (LT) signaling in vivo: mice with inactivation of the entire TNF/LT locus versus single-knockout mice
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Kuprash DV Alimzhanov MB Tumanov AV Grivennikov SI Shakhov AN Drutskaya LN Marino MW Turetskaya RL Anderson AO Rajewsky K Pfeffer K Nedospasov SA 《Molecular and cellular biology》2002,22(24):8626-8634
Homologous genes and gene products often have redundant physiological functions. Members of the tumor necrosis factor (TNF) family of cytokines can signal activation, proliferation, differentiation, costimulation, inhibition, or cell death, depending on the type and status of the target cell. TNF, lymphotoxin alpha (LTalpha), and LTbeta form a subfamily of a larger family of TNF-related ligands with their genes being linked within a compact 12-kb cluster inside the major histocompatibility complex locus. Singly TNF-, LTalpha-, and LTbeta-deficient mice share several phenotypic features, suggesting that TNF/LT signaling pathways may regulate overlapping sets of target genes. In order to directly address the issue of redundancy of TNF/LT signaling, we used the Cre-loxP recombination system to create mice with a deletion of the entire TNF/LT locus. Mice with a triple LTbeta/TNF/LTalpha deficiency essentially manifest a combination of LT and TNF single-knockout phenotypes, except for microarchitecture of the spleen, where the disorder of lymphoid cell positioning and functional T- and B-cell compartmentalization is severer than that found in TNF or LT single-knockout mice. Thus, our data support the notion that TNF and LT have largely nonredundant functions in vivo. 相似文献
106.
Gutkina N Varlakhanova NV Lysova MV Kovalenko SP 《Biochemical and biophysical research communications》2002,298(1):37-40
Lac(+)/Lac(-) selection of recombinant plasmids based on the insertional inactivation of LacZalpha gene cannot differentiate recombinant clones in some cases. Several fragments of exon 11 of human brca1 gene were cloned in LacZalpha-containing plasmids so that frameshift appeared at the 5(')-end of the fragments tested but these fragments were in frame with the part of LacZalpha situated downstream of the polylinker. All plasmids except one caused blue colonies formation after being transformed in Escherichia coli LacZDeltaM15 cells in spite of the frameshift. The fact may be explained by reinitiation of translation within the mRNA transcribed from the inserted DNA fragments at in-frame AUG, GUG, and UUG. The data demonstrated limitations on the Lac(+)/Lac(-) selection of LacZalpha-based recombinant plasmids. 相似文献
107.
Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure,conduction, and gating characteristics in liposomes
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Sukharev S 《Biophysical journal》2002,83(1):290-298
The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of approximately 200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20-60 mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (gamma). Po(gamma) curves had the midpoint at 5.5 +/- 0.1 dyne/cm and gave estimates for the energy of opening DeltaG = 11.4 +/- 0.5 kT, and the transition-related area change DeltaA = 8.4 +/- 0.4 nm(2) when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed. 相似文献
108.
Crofts AR Shinkarev VP Dikanov SA Samoilova RI Kolling D 《Biochimica et biophysica acta》2002,1555(1-3):48-53
Since available structures of native bc(1) complexes show a vacant Q(o)-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme-substrate complex at the Q(o)-site. This is formed by binding of both ubiquinol (QH(2)) and the dissociated oxidized iron-sulfur protein (ISP(ox)). A binding constant of approximately 14 can be estimated from the displacement of E(m) or pK for quinone or ISP(ox), respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme-product complex is also formed at the Q(o)-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E(m) of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E(m) reflects a binding constant of approximately 4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q(o)-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states. 相似文献
109.
Endostatin is a potential inhibitor of Wnt signaling 总被引:33,自引:0,他引:33
Hanai J Gloy J Karumanchi SA Kale S Tang J Hu G Chan B Ramchandran R Jha V Sukhatme VP Sokol S 《The Journal of cell biology》2002,158(3):529-539
110.
Secondary lymphoid organs provide the necessary microenvironment for the cooperation of antigen-specific T- and B-lymphocytes and antigen-presenting cells in order to initiate an efficient immune response. Remarkable progress in understanding of the mechanisms of lymphoid organogenesis was achieved due to the analysis of various gene-targeted mice. This review primarily focuses on the role of lymphotoxin (LT) in development, maturation and maintenance of secondary lymphoid organs. 相似文献