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121.
Electric shock-mediated transfection of cells. Characterization and optimization of electrical parameters. 总被引:4,自引:0,他引:4 下载免费PDF全文
D J Winterbourne S Thomas J Hermon-Taylor I Hussain A P Johnstone 《The Biochemical journal》1988,251(2):427-434
The effect of various parameters on the electric shock-mediated permeabilization and transfection of CHO cells has been investigated. Up to 70% of the cells can be maintained transiently permeable to erythrosin B for periods of at least 1 h at 20 degrees C. Electrical conditions optimal for transient permeabilization were also optimal for efficient DNA transfection by pSV2neo. However, the DNA must be present during exposure to the electric field for efficient transformation. The same requirement existed for voltage-induced DNA toxicity. The results suggest that DNA moves into the cells by electrophoresis, not by simple diffusion. Based on these observations a simple, rapid procedure for optimizing the conditions for electric shock-mediated DNA transfer into cells has been developed. 相似文献
122.
Thomas Jahns Alexandra Zobel Diethelm Kleiner Heinrich Kaltwasser 《Archives of microbiology》1988,149(5):377-383
Evidence for the existence of an energy-dependent urea permease was found for Alcaligenes eutrophus H16 and Klebsiella pneumoniae M5a1 by studying uptake of 14C-urea. Since intracellular urea was metabolized immediately, uptake did not result in formation of an urea pool. Evidence is based on observations that the in vivo urea uptake and in vitro urease activity differ significantly with respect to kinetic parameters, temperature optimum, pH optimum, response towards inhibitors and regulation. The K
m for urea uptake was 15–20 times lower (38 M and 13 M urea for A. eutrophus and K. pneumoniae, respectively) than the K
m of urease for urea (650 M and 280 M urea), the activity optimum for A. eutrophus was at pH 6.0 and 35°C for the uptake and pH 9.0 and 65°C for urease. Uptake but not urease activity in both organisms strongly decreased upon addition of inhibitors of energy metabolism, while in K. pneumoniae, potent inhibitors of urease (thiourea and hydroxyurea) did not affect the uptake process. Significant differences in the uptake rates were observed during growth with different nitrogen sources (ammonia, nitrate, urea) or in the absence of a nitrogen source; this suggested that a carrier is involved which is subject to nitrogen control. Some evidence for the presence of an energy-dependent uptake of urea was also obtained in Pseudomonas aeruginosa DSM 50071 and Providencia rettgeri DSM 1131, but not in Proteus vulgaris DSM 30118 and Bacillus pasteurii DSM 33.Non-standard abbreviations CCCP
Carbonylcyanide-m-chlorphenylhydrazone
- DCCD
dicyclohexylcarbodiimide
- DNP
2,4-dinitrophenole 相似文献
123.
A twenty-two-fold increase in the relative affinity of estrogen receptor to poly (dA-dC).poly (dG-dT) in the presence of polyamines. 总被引:7,自引:5,他引:2 下载免费PDF全文
We studied the relative efficacy of polyamines to facilitate the binding of estrogen receptor to poly(dA-dC).poly(dG-dT). In the absence of polyamines, 1,400 micrograms/ml of this polynucleotide eluted 50% of bound estrogen receptor from DNA-cellulose. In contrast, 50% estrogen receptor was eluted by 65 micrograms/ml of poly(dA-dC).poly(dG-dT) complexed with 150 microM spermidine. Putrescine and spermine also enhanced the ability of poly(dA-dC).poly(dG-dT) to elute estrogen receptor, but the magnitude of the effect was not as high as that of spermidine. Control experiments with calf thymus DNA and poly(dA-dT).poly(dA-dT) showed 6- and 3-fold increase, respectively in their affinity for estrogen receptor in the presence of spermidine. The dramatic increase in the affinity of poly(dA-dC).poly(dG-dT) for estrogen receptor in the presence of polyamines might be a result of the conversion of the polynucleotide to the left-handed Z-DNA form. These results show that polyamines are capable of participating in estrogenic regulation of gene expression by altering the affinity of the receptor for specific DNA sequences. 相似文献
124.
Marc-Henri Stern Fangrong Zhang Gilles Thomas Claude Griscelli Alain Aurias 《Human genetics》1988,81(1):18-22
Summary To delimit the 14q32.1 recurrent breakpoint of ataxia telangiectasia clones, we performed an in situ hybridization study with various probes located on the 14q32 band. We thus mapped this breakpoint between the D14S1 and Pi loci. Furthermore, an interstitial duplication including D14S1 and a part of the IgH locus was demonstrated on a t(14;14) clone. 相似文献
125.
Michèle Ramsay Stephen T. Reeders Peter D. Thomson Lawrence S. Milner L. Lazarou Thomas M. Barratt Adrian Yau Ordan J. Lehmann Trefor Jenkins 《Human genetics》1988,79(1):73-75
Summary The autosomal dominant form of polycystic kidney disease (ADPKD) has been linked to the -globin gene locus on 16p. Linkage studies between the autosomal recessive type (ARPKD) and the 3 HVR of the -globin gene cluster showed that the ARPKD and ADPKD are not allelic. 相似文献
126.
Seasonal variation in cell volume of epilimnetic bacteria 总被引:2,自引:0,他引:2
The relationship between bacterial cell volume and temperature was examined for field data collected over a 4-year period and through controlled chemostat incubations of aPseudomonas sp. Volumes of planktonic bacteria were found to decrease as water temperature increased. Changes in temperature accounted for 38% of the variation in average cell volume (P<0.001). Average planktobacterial cell volume fell 42% from 0.217m3 in mid-winter to 0.127m3 in mid-summer. Similar results were found for the size distribution of epibacterial cells. Controlled chemostat incubations of aPseudomonas sp. indicated that cell volume was significantly affected by temperature, growth rate, and the interaction of temperature and growth rate. The data suggest that a change in cell volume as a result of a change in temperature is an intrinsic property of planktonic bacteria. 相似文献
127.
128.
Dynamics of autotrophic picoplankton in Lake Constance 总被引:7,自引:0,他引:7
The vertical distribution, biomass concentrations and growthrates of autotrophic picoplankton (APP) were investigated duringthe growing season (March-October) in Lake Constance in differentdepths. Cell numbers determined by epifluorescence microscopyvaried between 1.0 x 103 and 1.6 times; 105 cells ml1depending on season and water depth. Highest concentrationswere recorded above the thermodine in late summer. Numerically,APP consisted almost exclusively of chroococcoid cyanobactena.During lake stratification several peaks of biomass concentrationsoccurred in epilimsietic waters at intervals of 68 weeks.In-situ experiments using a dilution technique and dialysisbags revealed that during summer APP population dynamics wereprimarily driven by combined changes of their growth and grazingrates, whereas temperature was less important. Gross growthrates varied between 0.006 and 0.051 h1, grazing ratesbetween 0.002 and 0.053 h1. On average APP productionwas completely removed by grazing within the microbial community.Ciliates, heterotrophic nanoflagellates and rotifers have beenidentified as the major consumers of APP cells. APP biomassis small compared to larger phytoplankton, ranging from ito5% of total phytoplankton biovolume. Due to its high gross growthrates, which are on the same level as those of free-living pelagicbacteria, APP contributes slightly more to overall primary productionwith maximum percentages of {small tilde}15% in late summer. 相似文献
129.
Oxidation of γ-Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide-Independent Enzyme 总被引:2,自引:0,他引:2
Elaine E. Kaufman Thomas Nelson David Miller Noam Stadlan 《Journal of neurochemistry》1988,51(4):1079-1084
An antibody that inhibits over 95% of the cytosolic NADP+-dependent gamma-hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1-14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1-14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADP+ and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+-dependent cytosolic oxidoreductase which catalyzes this reaction. 相似文献
130.