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41.
Transfer of hygromycin resistance into Brassica napus using total DNA of a transgenic B. nigra line 总被引:3,自引:0,他引:3
The successful transfer of a marker gene (hpt gene) from Brassica nigra into B. napus via direct gene transfer was demonstrated. Total DNA was isolated from a hygromycin-resistant callus line, which contained three to five copies of the hpt gene. This line had been produced via direct gene transfer with the hygromycin resistance-conferring plasmid pGL2. The treatment of B. napus protoplasts with genomic DNA of B. nigra (HygR) resulted in relative transformation frequencies of 0.1–0.4%. Similar transformation rates were obtained in direct gene transfer experiments using B. napus protoplasts and plasmid pGL2. 相似文献
42.
K. Peng A. J. W. G. Visser A. van Hoek C. J. A. M. Wolfs J. C. Sanders M. A. Hemminga 《European biophysics journal : EBJ》1990,18(5):277-283
Fluorescent probes located in heterogeneous environments give rise to anomalous time-resolved fluorescence anisotropy. A simple analytical expression of anisotropy has been derived for the case of a small difference in local fluorescence lifetimes. The expression has the diagnostic advantage that the time dependence of the fluorescence anisotropy can be predicted from the differences in fluorescence lifetimes and residual anisotropies of the probes located in different sites. Using this model, the local fluorescence anisotropy parameters and the relative contributions of the lipid probe octadecyl rhodamine B in a lipid environment and in the vicinity of bacteriophage M13 coat protein reconstituted in phospholipid bilayers, composed of 80% 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 20% 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol have been determined experimentally. At 40°C, the correlation times for bound and free probes are 2.3 and 3.0 ns, respectively, while the corresponding order parameters are 0.85 and 0.62, respectively.Abbreviations ESR
electron spin resonance
- DMPC
1,2-dimyristoyl-sn-glycero-3-phosphocholine
- DMPC
1,2-dimyristoyl-sn-glycero-3-phosphoglycerol
- L/P ratio
phospholipid to coat protein molar ratio
- <>
average fluorescence lifetime
-
r(0)
initial anisotropy
-
r()
residual anisotropy
On leave of Shanghai Medical Equipment Research Institute, 77 Jiang Ning Rd. Shanghai, People's Republic of China
Offprint requests to: M. A. Hemminga 相似文献
43.
Cytochalasin B (CB) is known to interfere reversibly with the cytoplasmic contractile filamental network of mammalian cells. The role of the microfilament system in the mechanism of the reactive oxygen intermediates release of polymorphonuclear leukocytes (PMNL) was studied for different kinds of stimuli. PMNL from fresh human blood were treated with CB and stimulated by adherence on plastic surfaces, by opsonized zymosan, by phorbol myristate acetate and by N-formylmethionyl-phenylalaline. The production of reactive oxygen species were monitored by simultaneous detection of native, luminol-independent, luminescence (NL) and luminol-dependent luminescence (LDL) using a method of spectral discrimination. Different influences of CB on NL with respect to LDL as well stimuli-dependent influences of CB on the luminescence response of PMNL were observed. Especially phagocytosis-associated activation of PMNL was strongly inhibited by CB, whereas LDL was reduced to a much greater extent in comparison with NL. A firm involvement of the microfilament system is indicated, but it depends on the kind of stimulus engaged. 相似文献
44.
A chemiluminescent assay for hepatitis-B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra-assay CV, 1.96-2.45%; inter-assay CV, 5.26-8.11% and has a lower detection limit for hepatitis-B surface antigen of 1.3U/I. 相似文献
45.
Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na+-dependent l -[3H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated l -[3H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC50 value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in Vmax with no change in Km. PMA also increased the transport of the nonmetabolizable analogue, d -[3H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na+-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α-phorbol 12,13-didecanoate, did not stimulate l -[3H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2+ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na+/H+ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na+-dependent l -[3H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS. 相似文献
46.
In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 M) and higher concentrations (1.5 M and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 g/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure. 相似文献
47.
Some novel transcription attenuation mechanisms used by bacteria 总被引:2,自引:0,他引:2
48.
The content of reduced glutathione and of glutathione disulfide as well as the activities of glutathione reductase, glutathione peroxidase, glutathione S-transferases, catalase and superoxide dismutases were determined in human hepatoma Hep 3B cells in relation to free-radical toxicity in order to appreciate the defense capacities of these cells compared to data on normal hepatocytes. When Hep 3B cells were exposed to lindane, a known inducer of free-radical production, superoxide dismutase activity appeared as the best-adapted cellular parameter for early detection of the resulting free-radical toxicity.Abbreviations AAS
atomic absorption spectrometry
- CDNB
1-chloro-2,4-dinitrobenzene
- DMEM
Dulbecco's modified Eagle medium
- GPx
glutathione peroxidase
- G.Red
glutathione reductase
- GSH
reduced glutathione
- GSSG
glutathione disulfide
- GST
glutathione S-transferases
- Prot
proteins
- SOD
superoxide dismutase 相似文献
49.
本文比较了用HSV-1(IEA)和B病毒(IFA)的方法检查猴B病毒相关抗体和B病毒抗体的结果:IEA检查出的B病毒相关抗体阴性猴,经IFA检查,全部为B病毒抗体阴性。用IFA检查了B病毒相关抗体阴性的恒河猴,在单笼隔离饲养六个月后,有98.3%的动物B病毒相关抗体仍为阴性,表明IRA的阴性结果有较好的一致性。本文讨论了建立无B病毒感染猴群的可行性。 相似文献
50.
To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK–) and leaky (M-CK1) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK– and M-CK1 mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels. 相似文献