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Photo-bleaching and photon saturation in flow cytometry. 总被引:2,自引:0,他引:2
In flow cytometry, small particles travel at a high speed through a bright light spot. The high light intensity at the point of measurement causes measurable photon saturation. This observation indicates that the rate at which individual dye molecules emit photons is close to the maximum emission rate. Despite the short exposure time, individual molecules may go through a few hundred excitation cycles while they are in the light beam. The absorbed light dose causes significant dye destruction. This article presents experimental procedures to determine the extent of photon saturation and photo-bleaching of dyes bound to cell nuclei in a flow cytometer. Measurements of Hoechst and propidium iodide bound to chromatin show that the amount of dye bleached per emitted photon is the same at low and high illumination intensities. This finding indicates that photon emission and dye destruction are both the result of the absorption of single excitation photons. The experimental observations allow rough estimates of the lifetime of the excited state and the lifetime of the molecule. The lifetime of the Hoechst 33258 bound to DNA is estimated to be 100 excitation-relaxation cycles. The average propidium iodide molecule lasts approximately 200 excitation-relaxation cycles. The theoretical considerations show that the optimal illumination conditions are different for bleaching and nonbleaching dyes. An optical arrangement for high precision measurements of bleaching dyes is presented. 相似文献
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R G Allen P L Toy R K Newton K J Farmer R S Sohal 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1985,82(2):399-402
Effects of varied levels of glutathione, an intracellular redox buffer, were examined in the adult male housefly in order to study the inter-relationship between enzymic and non-enzymic antioxidant defenses. An increase of over 100% in the concentrations of glutathione was induced by the administration of 3 mM L-2-oxothiazolidine-4-carboxylate (LOC), which increases the intracellular level of cysteine. A decrease in glutathione concentration of up to 85% was achieved by the administration of L-buthionine-SR-sulfoximine (BUS), which irreversibly inhibits glutamylcysteine synthetase. Life spans of houseflies were shortened by a decrease in the glutathione concentration, but were not prolonged by augmentation of glutathione. Metabolic rate and superoxide dismutase activity were independent of glutathione concentration. H2O2 was increased by both experimental regimes, whereas catalase activity was decreased by BUS. Results suggest that catalase activity is influenced by glutathione concentration. 相似文献
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Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation. 总被引:8,自引:1,他引:7 下载免费PDF全文
H A Bledsoe J A Carroll T R Whelchel M A Farmer D W Dorward F C Gherardini 《Journal of bacteriology》1994,176(24):7447-7455
In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures. 相似文献
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Veronica J. Farmer Marija Zanic 《BioEssays : news and reviews in molecular, cellular and developmental biology》2023,45(1):2200081
Almost 40 years since the discovery of microtubule dynamic instability, the molecular mechanisms underlying microtubule dynamics remain an area of intense research interest. The “standard model” of microtubule dynamics implicates a “cap” of GTP-bound tubulin dimers at the growing microtubule end as the main determinant of microtubule stability. Loss of the GTP-cap leads to microtubule “catastrophe,” a switch-like transition from microtubule growth to shrinkage. However, recent studies, using biochemical in vitro reconstitution, cryo-EM, and computational modeling approaches, challenge the simple GTP-cap model. Instead, a new perspective on the mechanisms of microtubule dynamics is emerging. In this view, highly dynamic transitions between different structural conformations of the growing microtubule end – which may or may not be directly linked to the nucleotide content at the microtubule end – ultimately drive microtubule catastrophe. 相似文献
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Cell surface carbohydrates play an important role in the regulation of neurite outgrowth during neuronal development. We have investigated the actions of the plant lectin concanavalin A (Con A), a carbohydrate-binding protein, on neurite outgrowth from hippocampal pyramidal neurons in primary cell culture. Neurons plated in culture medium containing nanomolar concentrations of Con A have a larger number of primary neurites arising directly from the cell soma than do neurons plated in culture medium alone. Furthermore, Con A causes counterclockwise turning of neurites in over 70% of the cultured neurons. Both of these effects of Con A are blocked by the hapten sugar alpha-methyl-D-mannopyranoside, suggesting that they result from the interaction of Con A with a cell surface carbohydrate. Another lectin with a different sugar specificity, wheat germ agglutinin, does not modulate neurite outgrowth. Analysis of neurite outgrowth using video-enhanced microscopy reveals that the counterclockwise turning is accompanied by directionally biased extension of filopodia from the growth cones of growing neurites. Treatment of the neurons with cytochalasin, which disrupts actin polymerization, eliminates the neurite turning induced by Con A, suggesting that actin microfilaments are involved in directional control of neurite outgrowth. 相似文献
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Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain 13C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D 15N/15N-separated NOESY, as many main-chain and side-chain 1HN/15N resonances as possible must be assigned. Traditionally, only backbone amide 1HN/15N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain 1HN/15N resonances to side-chain 13C resonances with high sensitivity: NH2-filtered 2D 1H-15N HSQC (H2N-HSQC), 3D H2N(CO)C/ and 3D H2N(COC/)C/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N/)C/ and H(N/C/)C/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated 13C-/15N-labeled human carbonic anhydrase II (2H-HCA II). Because more than 95% of all side-chain 13C resonances in 2H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain 1HN/15N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain HN protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only 1HN-1HN NOE restraints. In these studies, the inclusion of NOE restraints to side-chain HN protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed. 相似文献
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