Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

Electrophoretic mobility of irradiated erythrocytes and Ehrlich ascites tumour cells.

The subunits of RNA polymerase I are partially resolved during density gradient centrifugation. An analysis of the relative subunit composition with respect to specific catalytic activity shows that the molar ratio of the 24,000 dalton subunit directly correlates with polymerase activity. Since this polypeptide is found also in polymerases II and III, it may be required for activity of all yeast nuclear RNA polymerases.     

Continuous intracellular recording from mammalian neurons exposed to hyperbaric helium, oxygen, or air.

We developed a hyperbaric chamber for intracellular recording in rat brain stem slices during continuous compression and decompression of the tissue bath with the inert gas helium. Air, rather than helium, was also used as the compression medium in some cases to increase tissue nitrogen levels. An important feature is the chamber door, which opens or closes rapidly at 1 atmosphere absolute (ATA) for increased accessibility of the microelectrode. The door also closes and seals smoothly without disrupting the intracellular recording. Hyperbaric oxygen was administered during helium compression using a separate pressure cylinder filled with perfusate equilibrated with 2. 3-3.3 ATA oxygen. Measurements of tissue/bath PO(2) and pH confirmed that the effects of compression using helium or air could be differentiated from those due to increased PO(2). One hundred and thirteen neurons were studied during 375 compression cycles ranging from 1 to 20 ATA (mode 3.0 ATA). We conclude that it is technically feasible to record intracellularly from the same mammalian neuron while changing ambient pressure over a physiologically important range. These techniques will be useful for studying how various hyperbaric environments affect neurophysiological mechanisms.     

Electrophoretic mobility of human erythrocytes. On the applicability of the charged layer model.

The limitations of previous linear electrokinetic theories are discussed. A special model of the surface charge distribution, based on the minimum condition of the interfacial electrostatic free energy, is introduced. The model describes the electrophoretic mobility, taking into account the electroosmotic flow through the surface macromolecular layer and the surface conductivity. This nonlinear electrophoretic theory describes experimental data obtained with human erythrocytes. Numerical results for an uniformly distributed space charge are also presented.     

Electrophoretic mobility and hydrophobicity as a measured to predict the initial steps of bacterial adhesion

The relationship between physiochemical surface parameters and adhesion of bacterial cells to negatively charged polystyrene was studied. Cell surface hydrophobicity and electrokinetic potential were determined by contact angle measurement and electrophoresis, respectively. Both parameters influence cell adhesion. The effect of the electrokinetic potential increases with decreasing hydrophobicity. Cell surface characteristics determining adhesion are influenced by growth conditions. At high growth rates, bacterial cells tend to become more hydrophobic. This fact can be of ecological significance for controlling the spread of bacteria throughout the environment.     

Electrophoretic mobility as a function of pH for disaggregated amphibian gastrula cells

To explore possible chemical differences between the cell surfaces of endoderm, chordamesoderm, and inner ectoderm cells of the Rana pipiens gastrula, their electrophoretic mobilities have been measured as a function of pH. In this communication, we present evidence for an electrophoretic hierarchy consistent over a wide pH range, which suggests quantitative differences in surface components between the tissues of the gastrula.     

Electrophoretic mobility of supercoiled,catenated and knotted DNA molecules

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.     

Electrophoretic mobility and hydrophobicity as a measured to predict the initial steps of bacterial adhesion.

The relationship between physiochemical surface parameters and adhesion of bacterial cells to negatively charged polystyrene was studied. Cell surface hydrophobicity and electrokinetic potential were determined by contact angle measurement and electrophoresis, respectively. Both parameters influence cell adhesion. The effect of the electrokinetic potential increases with decreasing hydrophobicity. Cell surface characteristics determining adhesion are influenced by growth conditions. At high growth rates, bacterial cells tend to become more hydrophobic. This fact can be of ecological significance for controlling the spread of bacteria throughout the environment.     

Lipid peroxidation is not the cause of lysis of human erythrocytes exposed to inorganic or methylmercury

Exposure of human erythrocytes in a 50% hematocrit to 0.5-1 mM Hg2+ initiated immediate hemolysis which proceeded at a constant rate without any formation of lipid hydroperoxides. Treatment of 0.03% hematocrits with 0.4 ppm of Hg2+ or 40 ppm of methylmercury caused rapid hemolysis after a short lag period. The kinetics of the process were unaltered by saturation of the cell suspensions with oxygen, by its replacement with He or CO, or by variation in the level of vitamin E in the membranes. The results show that peroxidation of erythrocyte membrane lipids is not the cause of hemolysis induced by either Hg2+ or methylmercury.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Seasonal activity of frog erythrocytes by data of electrophoretic mobility

The peculiarities of frog erythrocyte electrophoretic mobility, coupled to the seasonal course of temperatures, have been studied. At the periods of anabiosis and of burst of hemopoiesis, in the vascular bed there increases the portion of functionally young erythrocytes (up to 22%) with increased values of the cell membrane surface charge. Preparation to winter is accompanied by a rise of the number of circulating functionally worn-down blood cells (up to 60%) with low values of the superficial charge and low mobility in electrical field. Use of the cell microelectrophoresis method of evaluation of seasonal activity of frog erythrocytes allows obtaining objective data about the cellular surface charge and its depending functional cell activity without submitting the erythrocytes to modifying actions.     

Swimming impairment and acetylcholinesterase inhibition in zebrafish exposed to copper or chlorpyrifos separately,or as mixtures

Pesticides such as chlorpyrifos (CPF) and metals such as copper can impair swimming behavior in fish. However, the impact to swimming behavior from exposure to mixtures of neurotoxicants has received little attention. In the current study, we analyzed spontaneous swimming rates of adult zebrafish (Danio rerio) to investigate in vivo mixture interactions involving two chemical classes. Zebrafish were exposed to the neurotoxicants copper chloride (CuCl, 0.1 μM, 0.25 μM, 0.6 μM, or 6.3, 16, 40 ppb), chlorpyrifos (CPF, 0.1 μM, 0.25 μM, 0.6 μM, or 35, 88, 220 ppb) and binary mixtures for 24 h to better understand the effects of Cu on CPF neurotoxicity. Exposure to CPF increased the number of animals undergoing freeze responses (an anti-predator behavior) and, at the highest CPF dose (0.6 μM), elicited a decrease in zebrafish swimming rates. Interestingly, the addition of Cu caused a reduction in the number of zebrafish in the CPF exposure groups undergoing freeze responses. There was no evidence of additive or synergistic toxicity between Cu and CPF. Although muscle AChE activity was significantly reduced by CPF, there was a relatively poor relationship among muscle AChE concentrations and swimming behavior, suggesting non-muscle AChE mechanisms in the loss of swimming behavior. In summary, we have observed a modulating effect of Cu on CPF swimming impairment that appears to involve both AChE and non-AChE mechanisms. Our study supports the utility of zebrafish in understanding chemical mixture interactions and neurobehavioral injury.     

Formation of modified DNA bases in cells exposed either to gamma radiation or to high-LET particles

The aim of the present study was to measure the formation of eight base modifications in the DNA of cells exposed to either low-LET ((60)Co gamma rays) or high-LET ((12)C(6+) particles) radiation. For this purpose, a recently optimized HPLC-MS/MS method was used subsequent to DNA extraction and hydrolysis. The background level of the measured modified bases and nucleosides was shown to vary between 0.2 and 2 lesions/10(6) bases. Interestingly, thymidine glycols constitute the main radiation-induced base modifications, with an overall yield of 0.097 and 0.062 lesion/10(6) bases per gray for gamma rays and carbon heavy ions, respectively. Both types of radiations generate four other major degradation products, in the following order of decreasing importance: FapyGua > 5-HmdUrd > 5-FordUrd > 8-oxodGuo. The yields of formation of FapyAde and 8-oxoAde are one order of magnitude lower than those of the related guanine modifications, whereas the radiation-induced generation of 5-OHdUrd was below the limit of detection of the assay. The efficiency for both types of radiation to generate base damage in cellular DNA is low because the highest yield per gray was 0.097 thymine glycols per 10(6) DNA bases. As a striking observation, the yield of formation of the measured DNA lesions was found to be, on average, twofold lower after exposure to high-LET radiation ((12)C(6+)) than after exposure to low-LET gamma radiation. These studies show that the HPLC-MS/MS assay provides an accurate, reliable and sensitive method for measuring cellular DNA base damage.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.