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81.
Analysis of Escherichia coli TonB membrane topology by use of PhoA fusions. 总被引:14,自引:8,他引:6 下载免费PDF全文
Alkaline phosphatase (PhoA) fusions to TonB amino acids 32, 60, 125, 207, and 239 (the carboxy terminus) all showed high PhoA activity; a PhoA fusion to TonB amino acid 12 was inactive. The full-length TonB-PhoA fusion protein was associated with the cytoplasmic membrane and retained partial TonB function. These results support a model in which TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder of the protein, including its hydrophobic carboxy terminus, extending into the periplasm. 相似文献
82.
83.
84.
Keith Paige Melanie Palomares Patricia A. D’Amore Susan J. Braunhut 《In vitro cellular & developmental biology. Animal》1991,27(2):151-157
Summary The growth of the endothelial cell (EC) is tightly regulated throughout the body. Many factors have been implicated in modulating
EC growth including diffusible compounds, cell-to-cell interactions, and the extracellular matrix (ECM). Retinol, or vitamin
A alcohol, has recently been shown to inhibit the growth of bovine capillary ECs, in vitro. Retinoids are known to modify
ECM in other cell systems, and pure ECM components have been shown to effect EC growth rates. We, therefore, examined the
role of the matrix in the retinol-induced inhibition of ECs. Cell-free matrices from control and vitamin A-treated ECs were
prepared by removing cells with EGTA treatment after 7 d of culture. Matrix proteins were analyzed by solubilizing the matrices
in 5M quanidine-HCl and performing Western blot analysis using specific antibodies to matrix proteins. In isolating the ECM, we
observed that retinol-treated cultures of ECs were resistant to EGTA removal; retinol-treated ECs required twice the exposure
time to EGTA to detach from their matrix than did controls cells. Western blot analysis of matrix proteins derived from control
and retinol-treated EC cultures demonstrated a 1.6-fold increase in lamininβ chains and a 2.5-fold increase in fibronectin in the ECM of retinol-treated EC compared to control cell matrix. Functional
properties of these matrices were assessed by plating control and Day 6 retinol-treated ECs onto the matrices and measuring
attachment and growth by determining cell numbers at 24, 72, and 144 h. These studies revealed that control cells attached
in greatest numbers to a control matrix whereas retinol-treated ECs preferentially attached to a matrix derived from retinol-treated
cells. Furthermore, control ECs which grew rapidly on a control matrix were growth inhibited on a retinol-derived matrix.
These data indicate that vitamin A treatment of ECs effects both their phenotype and influences the composition and the functional
properties of their underlying ECM. These studies also demonstrate that alterations of the matrix are at least in part responsible
for the growth inhibition of EC by retinol. 相似文献
85.
M J Gauthier B Lafay R Christen L Fernandez M Acquaviva P Bonin J C Bertrand 《International journal of systematic bacteriology》1992,42(4):568-576
On the basis of phenotypical characteristics and analysis of 16S rRNA sequence, a new species belonging to a new genus is described, and the name Marinobacter hydrocarbonoclasticus is proposed. This organism, isolated from Mediterranean seawater near a petroleum refinery, is a gram-negative, aerobic, rod-shaped bacterium. It grows at NaCl concentrations of 0.08 to 3.5 M and uses various hydrocarbons as the sole source of carbon and energy. Its DNA has a guanine-plus-cytosine content of 52.7 mol%. The 16S rRNA analysis shows a clear affiliation between M. hydrocarbonoclasticus and the gamma group of the phylum Proteobacteria. A close phylogenetic relationship appears among the species Marinomonas vaga, Oceanospirillum linum, Halomonas elongata, and Pseudomonas aeruginosa. Because of the impossibility of finding a single most closely related species, we suggest that this bacterium be assigned to a new genus, at least temporarily. The possibility of a revision of this status when new data appear is, however, not excluded. The type strain is M. hydrocarbonoclasticus SP.17 (= ATCC 49840). 相似文献
86.
Incorporation of radiolabeled thymidine is commonly used to investigate DNA damage. Using a filter-binding assay, we observed that the addition of various doses of [methyl-3H]thymidine (0.2 and 2 microCi/ml) or [2-14C]thymidine (0.02 and 0.2 microCi/ml) in the culture medium for 2 days, a standard method for cell-labeling, induces DNA fragmentation in HL-60 human promyelocytic cells. This effect was dose- and time-dependent and the DNA fragments were not protein-linked since the levels of DNA fragmentation were identical in the presence and in the absence of proteinase K (0.5 mg/ml). Radiolabeled thymidine-induced DNA fragmentation was associated with an inhibition of cell growth, but cells remained able to exclude trypan blue, suggesting that plasma membrane integrity was conserved, except at very high doses of [methyl-3H]thymidine (2 microCi/ml). By agarose-gel electrophoresis, the DNA-fragmentation was demonstrated to be internucleosomal with a typical ladder pattern. Addition of unlabeled thymidine to the culture medium prevented DNA fragmentation in a dose-dependent manner, indicating that radiolabeled thymidine incorporation in DNA was directly responsible for DNA fragmentation. We conclude that radiolabeling of DNA using thymidine incorporation can induce DNA fragmentation in some cell lines such as HL-60. This observation must be taken into account in methods using radiolabeling to study DNA damage in these cells. 相似文献
87.
Claire Zehnacker Thomas W. Becker Akira Suzuki Elisa Carrayol Michel Caboche Bertrand Hirel 《Planta》1992,187(2):266-274
Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C16) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C35) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C16 as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C35 was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp
base pairs
- Fd-GOGAT
ferredoxin-dependent glutamate synthase
- GS
glutamine synthetase
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate
The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48. 相似文献
88.
Thomas W. Becker Michel Caboche Elisa Carrayol Bertrand Hirel 《Plant molecular biology》1992,19(3):367-379
89.
Kim B. Saunders Patricia A. D’Amore 《In vitro cellular & developmental biology. Animal》1992,28(7-8):521-528
Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues.
In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting
a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial
cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition.
However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the
individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane,
permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle
cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through
pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited
endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated
internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact
or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study
of cell-cell interactions in a variety of systems. 相似文献
90.
Modulation of collagen production by fibroblasts. Effects of chronic exposure to agonists that increase intracellular cyclic AMP. 总被引:3,自引:0,他引:3 下载免费PDF全文
Cultured human lung fibroblasts were evaluated for their responsiveness to isoprenaline (isoproterenol) or prostaglandin E2 before and after chronic incubation with the agonist. Cells incubated for 6 h with either agonist were suppressed in terms of collagen production and exhibited increased intracellular cyclic AMP. Cells incubated for 72 h with the agonist and then re-challenged for 6 h with the same agonist did not demonstrate suppressed collagen production or increased cyclic AMP. Cells incubated for 72 h with isoprenaline still responded to prostaglandin E2 when challenged for 6 h; however, when the order of agonist exposure was reversed, cells incubated with prostaglandin E2 did not respond to a challenge by isoprenaline. If cells were allowed to recover for 48 h without the agonist after a 72 h chronic incubation, they recovered their responsiveness to the agonist. The results indicate that, although cultured fibroblasts may become desensitized to one agonist, they may retain their sensitivity to a second agonist and chronic suppression of collagen production may be achieved by alternate exposure to isoprenaline and prostaglandin E2. 相似文献