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Ricardo Gonzalez-Mendez David Wemmer George Hahn Norma Wade-Jardetzky Oleg Jardetzky 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(3):274-280
A continuous-flow NMR culture system for mammalian cells has been developed on which 31P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by 31P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques. 相似文献
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Active oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves-tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K+ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K+ channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of the K+ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K+ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K+ currents. © 1993 Wiley-Liss, Inc. 相似文献
5.
Characterization of FP22, a large streptomycete bacteriophage with DNA insensitive to cleavage by many restriction enzymes 总被引:1,自引:0,他引:1
Bacteriophage FP22 has a very broad host range within streptomycetes and appeared to form lysogens of Streptomyces ambofaciens ATCC 15154. FP22 shared strong cross-immunity and antibody cross-reactivity with bacteriophage P23, but not with seven other streptomycete bacteriophages. FP22 particles had a head diameter of 71 nm and a tail length of 307 nm. The FP22 genome was 131 kb, which is the largest bacteriophage genome reported for streptomycetes. The G + C content of the genome was 46 mol% and restriction mapping indicated that FP22 DNA had discrete ends. NaCl- and pyrophosphate-resistant deletion mutants were readily isolated and the extent of the deletions defined at least 23 kb of dispensable DNA in two regions of the genome. The DNA was not cleaved by most restriction endonucleases (or isoschizomers) which have been identified in the streptomycetes, including the tetranucleotide cutter MboI (GATC). 相似文献
6.
Aromatic L-amino acid decarboxylase (AADC) is responsible for the conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are important neurotransmitters. We characterized genomic clones derived from the rat AADC locus by Southern blot and nucleotide sequencing analyses to explore the exonal organization of the gene. Our results suggest that the rat AADC gene is relatively large, containing at least 12 exons and spanning at least 40 kb in the rat genome. In this study, nine exons corresponding to 71% of the published cDNA sequence were identified, the smallest of which was as short as 20 base pairs (bp). In the Drosophila dopa decarboxylase (DDC) gene, the sequences homologous to these nine exons are all present in the fourth exon. This implies that either multiple intron sequences have been added to the vertebrate AADC gene or alternatively, deleted from the invertebrate gene after the divergence of vertebrates and invertebrates during evolution. 相似文献
7.
Assay of DNA-RNA hybrids by S1 nuclease digestion and adsorption to DEAE-cellulose filters. 总被引:11,自引:1,他引:10
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A fast and accurate assay procedure for DNA-RNA hybrids is described in which exhaustive digestion of unhybridized DNA with S1 nuclease is followed by binding of hybrids to filter discs of DEAE-cellulose. The digested DNA can be efficiently washed from the filters so that background levels of 0.1-0.2% of input tracer DNA can be achieved, in contrast to the much higher (approximately 1-5%) backgrounds obtained using TCA precipitation procedures. Short duplexes, as small as 36 nucleotides in length, which are inefficiently bound to hydroxyapatite, are quantitatively bound to the DEAE-cellulose filters. 相似文献
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Monoclonal antibodies generated by immunization with a plasma-membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. were used in combination with fluoresceinor rhodamine-labeled goat anti-mouse immunoglobulins to identify heterokaryons in protoplast fusion procedures. Antibody labeling did not inhibit callus formation nor plantlet regeneration. The antibodies are non-invasive and surface labeling provides clear optical discrimination of true heterokaryons from unfused aggregates as well as from parental protoplasts and homokaryons. Labeling is stable throughout fusion and hence by pre-labeling parental protoplast populations the strategy is both versatile and of general applicability. 相似文献
9.
Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O2 diffusion. Here we describeexperiments using O2 specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O2 concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O2 concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O2 concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O2 concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O2 fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O2 concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N2or air. Key words: Oxygen, root nodules, air spaces 相似文献
10.
Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications 总被引:1,自引:0,他引:1
Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of
individuals representing 54 species of frogs, two species of salamanders, a
caecilian, and a lungfish. Eight of these sites were present in all species
examined, and two were found in all but one species. Alignment of these
conserved restriction sites revealed, among anuran 28S rRNA genes, five
regions of major length variation that correspond to four of 12 previously
identified divergent domains of this gene. One of the divergent domains
(DD8) consists of two regions of length variation separated by a short
segment that is conserved at least throughout tetrapods. Most of the
insertions, deletions, and restriction-site variations identified in the
28S gene will require sequence-level analysis for a detailed reconstruction
of their history. However, an insertion in DD9 that is coextensive with
frogs in the suborder Neobatrachia, a BstEII site that is limited to
representatives of two leptodactylid subfamilies, and a deletion in DD10
that is found only in three ranoid genera are probably synapomorphies.
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