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1.
Sheri L. Holmen Matt W. Vanbrocklin Robert R. Eversole Susan R. Stapleton Leonard C. Ginsberg 《In vitro cellular & developmental biology. Animal》1995,31(5):347-351
Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids,
however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available
cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study
include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium
methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic
(EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these
vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio.
Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current
theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection
studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes
comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions
for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration
of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical,
easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes. 相似文献
2.
M. A. Lelu K. Klimaszewska G. Pflaum C. Bastien 《In vitro cellular & developmental biology. Plant》1995,31(1):15-20
Summary Cotyledonary somatic embryos ofLarix × leptoeuropaea that developed after various maturation times on media containing abscisic acid showed different frequencies of conversion
into plants. Drying of these somatic embryos under high relative humidity (RH) before germination improved plantlet recovery
and eliminated differences in the performance of somatic embryos matured for different times. However, dehydration of somatic
embryos under 98% RH to a water content below that of zygotic embryos excised from mature seeds (0.97 and 1.36 g H2O/g dry weight, respectively) showed a strong positive correlation between longer maturation time and desiccation tolerance.
Drying somatic embryos at 4° C under 59% RH for 1 wk resulted in desiccation to a water content of 0.30 g H2O/g dry weight, which was the closest to the hydration state of zygotic embryos in dried, stored seeds (0.20 g H2O/g dry weight). Under this condition, only somatic embryos matured for 5 wk germinated and produced plantlets at a relatively
high frequency (73 and 41%, respectively). 相似文献
3.
Occasionally a mutation occurs in liquid cultures ofAnacystis nidulans, spreading quickly through the population and causing cells to adhere together in clumps. This phenotype is stable indefinitely and is an inherited characteristic of all cells within a clumping culture. Inoculation with a few living cells from a clumping culture quickly produces the clumping genotype in a majority of cells within a previously non-clumping culture. Killed cells, broken cell extracts, or media from clumping cultures do not produce aggregation in non-clumping cultures. Actively growing cells in clumping cultures do not affect non-clumping cultures when separated by 0.4 μm Millipore filter. Apparently transfer of the clumping trait requires direct contact between living cells. Pili-like projections connect individual cells within clumps, but no slime layer or capsule is seen. Clumps can be dispersed without cell damage; reaggregation requires photosynthesis. 相似文献
4.
Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli 总被引:14,自引:0,他引:14
S H Shen P Chrétien L Bastien S N Slilaty 《The Journal of biological chemistry》1991,266(2):1058-1063
A 2.7-kilobase fragment of DNA from Oerskovia xanthineolytica containing the gene for a beta-1,3-glucanase has been isolated and its complete nucleotide sequence determined. The sequence was found to contain two large open reading frames. Purification of the mature native enzyme and subsequent amino-terminal sequencing defined the glucanase gene in one reading frame which potentially encodes a protein of 548 amino acids. We have expressed this glucanase gene in Escherichia coli under control of the lacUV5 promoter and found the product to be secreted into the periplasm as a mature enzyme of about the same molecular weight as that of the native protein. The recombinant enzyme was purified to near homogeneity by a single step of high performance liquid chromatography. The ability of the recombinant enzyme to digest beta-glucan substrates and to lyse viable yeast cells was found to be indistinguishable from that of the native protein. Deletion of the cysteine-rich carboxyl-terminal 117 amino acids of the enzyme, which also contain two duplicated segments, abolished the lytic activity but did not significantly affect the glucanase function of the protein. The possible involvement of this domain in interaction with the yeast cell wall is discussed. 相似文献
5.
Effects of acute and chronic restraint and estrus cycle on pituitary-adrenal function in the rat 总被引:1,自引:0,他引:1
Female Long-Evans hooded rats with 5-day estrus cycles were subjected to 4 hr of continuous restraint for either 1 or 20 days. On the last day of the stress regimen, plasma and adrenal corticosterone concentrations were determined and classified according to the stage of the estrous cycle. The results indicated that acute stress produced greater plasma corticosterone concentrations than controls only during estrus, whereas in response to chronic stress significant stress-induced increments were observed during estrus and proestrus. The results suggest that the estrous cycle influences the magnitude of the stress-induced increments for both acute and chronic stress. In addition, the pituitary-adrenal system did not show habituation to repeated administration of this stress, but sensitization was observed during proestrus. 相似文献
6.
Identification of an arsenic-sensitive block to primate lentiviral infection of human dendritic cells
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Dendritic cells are central to the early events of human immunodeficiency virus type 1 (HIV-1) transmission, but HIV-1 infects dendritic cells inefficiently in vitro compared to activated CD4(+) T cells. There is a strong postentry restriction of HIV-1 infection in dendritic cells, partly mediated by the cellular restriction factor APOBEC3G. Here, we reveal that arsenic trioxide markedly increases HIV infection of immature and mature dendritic cells as well as blood-derived myeloid dendritic cells in an APOBEC3G- and TRIM5alpha-independent way. Our data suggest the presence of powerful, arsenic-sensitive antiviral activities in primary human immune cells of the dendritic cell lineage. 相似文献
7.
Bastien Polizzi Andrea Fanesi Filipa Lopes Magali Ribot Olivier Bernard 《PLoS computational biology》2022,18(4)
We present a spatial model describing the growth of a photosynthetic microalgae biofilm. In this 2D-model we consider photosynthesis, cell carbon accumulation, extracellular matrix excretion, and mortality. The rate of each of these mechanisms is given by kinetic laws regulated by light, nitrate, oxygen and inorganic carbon. The model is based on mixture theory and the behaviour of each component is defined on one hand by mass conservation, which takes into account biological features of the system, and on the other hand by conservation of momentum, which expresses the physical properties of the components. The model simulates the biofilm structural dynamics following an initial colonization phase. It shows that a 75 μm thick active region drives the biofilm development. We then determine the optimal harvesting period and biofilm height which maximize productivity. Finally, different harvesting patterns are tested and their effect on biofilm structure are discussed. The optimal strategy differs whether the objective is to recover the total biofilm or just the algal biomass. 相似文献
8.
9.
The authors have studied the in vitro conversion of 18 hydroxycorticostérone to aldosterone (18 oxidation) by duck adrenal subcellular fractions. Considering the new hypothesis about the mechanism of this step (hydroxylation mechanism) the authors have investigated a possible relationship between this reaction and cytochrome P450. With experimental conditions described, data show that metyrapone, a cytochrome P450 competitive inhibitor does not inhibit 18 oxidation. In contrast, 18 oxidation is inhibited by spirolactones (spironolactones, canrenone, potassium canrenoate). These compounds act at the cytochrome P450 level but have also an uncoupling effect which has been recently discovered. The effects of metyrapone and spirolactones on 18 oxidation as well as the different behaviour between biologicaly and organically synthetised 18 hydroxycorticosterone allow us to propose hypotheses for the mechanism of this step. 相似文献
10.
Victoire Cardot‐Ruffino Vronique Chauvet Cassandre Caligaris Adrien Bertrand‐Chapel Nicolas Chuvin Roxane M. Pommier Ulrich Valcourt David Vincent Sylvie Martel Sophie Aires Bastien Kaniewski Pierre Dubus Philippe Cassier Stphanie Sentis Laurent Bartholin 《Genesis (New York, N.Y. : 2000)》2020,58(5)
Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype. 相似文献