Fixation and repair of radiation-induced potentially mutagenic damage sensitive to hypertonic treatment in human diploid fibroblasts

The effect of hypertonic salt treatment on the repair of potentially lethal damage and potentially mutagenic damage in X-irradiated asynchronous and synchronous human diploid fibroblasts (IMR91) have been studied. Resistance to 6-thioguanine was used for the mutagenic end point. When cells in late-S-phase were treated with hypertonic salt solution immediately after X-irradiation, both cell killing and mutation induction were enhanced, as compared to X-irradiation alone. This suggests that X-irradiation of cells in late S phase induces both potentially lethal damage and potentially mutagenic damage and that both are sensitive to hypertonic salt solution. When cells were allowed 2 h for repair after exposure to X-rays, both types of damage were completely repaired. Almost the same results were obtained with asynchronous cells. These results are discussed in terms of the relationship between radiation damage leading to cell lethality and mutagenesis.     

Thermal denaturation of DNA in concentrated salt-free solutions: comparison of microcalorimetric and spectrophotometric data

Scanning microcalorimetry and spectrophotometry were used to study the dependence of melting enthalpy (delta Hm) and temperature (Tm) on DNA concentration in salt free solutions and on NaCl concentration in solutions with constant DNA concentration. This data is used to calculate the Manning's charge density parameter which is found to be equal 1.8. The linear dependence of Tm on the logarithm of DNA concentration in salt free solution was obtained. An approximate evaluation of dissociation degree in native DNA at different concentrations was made by comparison of straight lines in the Tm = f(lg CNaCl) and Tm = f(lg Cp) coordinates.     

EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M₄PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H₂O₂, which is the recombination product of ·OH, and OCl^- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.     

Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography

Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.     

NaCl对小麦苗叶片脯氨酸氧化酶活性和游离脯氨酸累积的影响

在含NaCl营养液中培养的小麦幼苗较之无NaCl营养液中的幼苗。其脯氨酸氧化酶活性降低,而游离脯氨酸含量则升高;培养液的渗透势越低,培养时间越长,则脯氨酸氧化酶的活性越低,且游离脯氨酸的含量越高。去除胁迫后酶活性恢复,脯氯酸含量下降。不同渗透剂对氧化酶活性抑制强弱顺序为MgCl_2>NaCl>甘露醇,引起脯氨酸累积效应的强度顺序为MgCl_2>NaCl>甘露醇。超微结构显示,高NaCl浓度下部分线粒体结构受损伤,膜和嵴部分消失。     

Quantitative and Qualitative Analyses of the Antibody Response of Adult Mice Tolerized with Deaggregated Bovine γ-Globulin

The antibody response of mice to bovine γ-globulin(BGG) was suppressed either specifically by an intravenous injection of deaggregated soluble BGG (sBGG) or nonspecifically by X-irradiation. Immunization with the subcutaneous injection of BGG in Freund's incomplete adjuvant was given to mice either various days after sBGG injection or immediately after X-irradiation. Antigen-elimination (AE) test and passive hemagglutination(PHA) test were employed for estimating the immune status. The AE test indicated that the induction of tolerance was accomplished in the first 2 days after sBGG injection and that the tolerant state was stable at least for about 30 days thereafter. The degree of suppression by 1000 μg of sBGG corresponded to that obtained by X-irradiation at the dose of 400 R or more, and 100 μg of sBGG was equivalent to 300 R X-irradiation. The PHA test indicated, however, that such a correspondence as mentioned above between the dose of tolerogen and that of X-irradiation was not so stable as was seen by the AE test. Thus, the PHA titers of tolerized animals tended to recover up to the level of untolerized animals during the period of time from 10 days to 20 days after the tolerogen injection. Such discrepancies between the features in the AE test and those in the PHA test seemed attributable to a low avidity antibody formation in the tolerized animals, as judged by the hemagglutination-dissociation test. Hemagglutination by means of the sera from tolerized animals was seen to be reversed by the addition of free antigen more easily than the hemagglutination achieved by the sera of control animals or X-irradiated animals. The relationship between PHA titers and AE capacities of antibodies was investigated by the passive immunization of normal mice previously given the antigen. The result showed that the PHA titer did not always correlate with the AE capacity.     

On the application of polyelectrolyte “limiting laws” to the helix-coil transition of DNA. I. Excess univalent cations

A general theory of polyelectrolyte solutions is here used to calculate the differences in Gibbs free energy, enthalpy, and entropy between the coil and helix forms of DNA at any temperature and salt concentration. The salt has univalent cations and is assumed present in excess over the base concentration. The results are restricted to sufficiently dilute solutions. It is shown that the salt concentrations effect is entirely entropic in origin. When applied to the melting temperature, the calculations yield a relation between the enthalpy difference at the melting temperature and the slope of the plot of melting temperature vs. the logarithm of the salt concentration. In accord with observation, both the Gibbs free energy difference at any fixed temperature and the melting temperature are predicted to be linear functions of the log of the salt concentration. However, the theory is not in quantitative agreement with enthalpy data. Data on various colligative and transport properties of both helix and coil forms are reviewed in the text and in Appendix B, and good agreement is found with theory for both forms. No attempt is made to explain why the theory is quantitative for these properties but not for heat measurements. Finally, in Appendix A, an approximate calculation is made of the free energy contributions due to ionic effects not associated with the salt concentration.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters.

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Linoleic and oleic acids alter the licking responses to sweet, salt, sour, and bitter tastants in rats

The free fatty acids (FFAs), linoleic and oleic acids, commonly found in dietary fats can be detected by rats on the basis of gustatory cues following conditioned taste aversion pairings. FFAs depolarize the membrane potential of isolated rat taste receptor cells by inhibiting delayed rectifying potassium channels. This study examined the licking response of rats to sweet, salt, sour, and bitter taste solutions when 88 muM linoleic acid, 88 muM oleic acid, or an 88 muM linoleic-oleic acid mixture was added to the solutions. The presence of linoleic, oleic, and the linoleic-oleic acid mixture in sweet solutions produced increases in the licking responses, whereas adding linoleic, oleic, and the linoleic-oleic acid mixture to salt, sour, or bitter taste solutions produced decreases in licking responses when compared with the licking responses to the solutions in the absence of the FFAs. We conclude that FFAs may act in the oral cavity to depolarize taste receptor cells and therefore to increase the perceived intensity of concomitant tastants, thus contributing to the enhanced palatability associated with foods containing high dietary fat.     

The metabolites of catecholamines in urine of patients irradiated therapeutically.

The metabolites of catecholamines were determined in 24-hour urine samples of patients with genital carcinoma and treated by radio therapy. The patients were irradiated first with gamma-rays of radium and then with X-rays. The radium sources (80 mCi) were placed intracavitarily for 46 hours twice within 2 weeks. X-irradiation (800 R daily), applied 1 month after radium treatment, was delivered on four abdominal fields over 15 days. The quantities of excreted catecholamine metabolites during irradiation were compared with control values (obtained before irradiation) in the same patients. Gamma-irradiation provoked a significant increase in the excretion of 3-methoxy-4-hydroxy-mandelic acid, metadrenaline and normetadrenaline, as well as of homovanillic acid, whereas X-irradiation provoked only a significant increase in the excretion of free 3-methoxy-4-hydroxy-phenylglycol. The increased excretion might be explained: (1) in the case of radium application, by direct radiation-induced release of catecholamines from the peripheral symphathetic nerves; (2) in the case of X-irradiation, by putting in the motion the complex of early neuroendocrine reactions via irradiated adrenal medulla.     

Cytogenetic analysis of X-ray-induced chromosome aberrations in spontaneously leukaemic AKR mice.

The increased frequency of numerical and structural chromosomal aberrations in spontaneously leukaemic AKR mice, compared with the values of healthy control CBA/H-T6T6 mice, induced by X-irradiation, migh be connected with the predisposition to malignant growth, probably indirectly helping the virus activation, or acting together with the immune deficiency, by creating a weaker system that is more sensitive to carcinogenic agents.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.     

Association of endogenous retroviruses with radiation-induced leukemias of BALB/c mice.

X-irradiation of BALB/c mice in the second month of life induced a high incidence of generalized lymphatic leukemia of T-cell origin, beginning at 7 months of age. Infectious ecotropic murine leukemia virus (B-tropic predominant over N-tropic) was isolable from all tumor extracts but exhibited a wide titer range among individual leukemias. Detection of infectious xenotropic virus usually required extensive amplification on indicator cells. Dual-tropic (mink cell focus-forming) virus has not been found in the leukemias. Expression of ecotropic virus in tail extracts prepared at 6.5 months of age, although greatly enhanced compared with unirradiated controls, was not found to be prognostic of tumor development in individual mice. We conclude that leukemogenesis does not show a simple dependence on infectious murine leukemia virus expression in these mice.     

Protein-protein interactions in concentrated electrolyte solutions

Protein-protein interactions were measured for ovalbumin and for lysozyme in aqueous salt solutions. Protein-protein interactions are correlated with a proposed potential of mean force equal to the free energy to desolvate the protein surface that is made inaccessible to the solvent due to the protein-protein interaction. This energy is calculated from the surface free energy of the protein that is determined from protein-salt preferential-interaction parameter measurements. In classical salting-out behavior, the protein-salt preferential interaction is unfavorable. Because addition of salt raises the surface free energy of the protein according to the surface-tension increment of the salt, protein-protein attraction increases, leading to a reduction in solubility. When the surface chemistry of proteins is altered by binding of a specific ion, salting-in is observed when the interactions between (kosmotrope) ion-protein complexes are more repulsive than those between the uncomplexed proteins. However, salting-out is observed when interactions between (chaotrope) ion-protein complexes are more attractive than those of the uncomplexed proteins.     

Interactions of Oxygen at High Pressure and Radiation in Drosophila

Oxygen at high pressure (OHP) and X-irradiation can interact in the fruit fly Drosophila melanogaster to potentiate toxic actions characteristic of one agent alone. 1000 kvp X-irradiation in doses of 30, 60, and 75 kr accelerated the acute immobilization of young male Drosophila by oxygen at 7.8 atm, up to rates twice that observed with such oxygen pressure alone. X-irradiation alone in these dosages did not acutely immobilize the Drosophila. X-irradiation during exposure to 7.8 atm pO₂ was more effective and consistent in producing this potentiation than was X-irradiation that preceded exposure to OHP. Acute OHP toxicity in young female Drosophila was not potentiated by 75 kr of X-irradiation. On the other hand, shortening of the life span of young male Drosophila by the above doses of X-irradiation was augmented significantly by a concurrent 40 min exposure to OHP (which alone did not significantly decrease life span). This shows, for the first time, that oxygen can affect not only the acute effects of radiation, but also the residual irreversible effects indicated by the life span shortening.     

THE COLLOIDAL BEHAVIOR OF EDESTIN

1. It has been shown by titration experiments that the globulin edestin behaves like an amphoteric electrolyte, reacting stoichiometrically with acids and bases. 2. The potential difference developed between a solution of edestin chloride or acetate separated by a collodion membrane from an acid solution free from protein was found to be influenced by salt concentration and hydrogen ion concentration in the way predicted by Donnan''s theory of membrane equilibrium. 3. The osmotic pressure of such edestin-acid salt solutions was found to be influenced by salt concentration and by hydrogen ion concentration in the same way as is the potential difference. 4. The colloidal behavior of edestin is thus completely analogous to that observed by Loeb with gelatin, casein, and egg albumin, and may be explained by Loeb''s theory of colloidal behavior, which is based on the idea that proteins react stoichiometrically as amphoteric electrolytes and on Donnan''s theory of membrane equilibrium.     

Effect of parenteral nitrogen feeding on the free amino acid composition of the blood serum and liver tissue in irradiated rats

A considerable change in the free amino acid composition of blood serum and hepatic tissue was noted on the 7th and 14th days following total-body X-irradiation of rats with a dose of 2.9 Gy. The total free amino acid content of blood serum increased and that of hepatic tissue decreased by 85% (on an average) as compared to the intact controls. Quantitative changes in the content of individual amino acids were analysed. Polyamine injected enterally for 7 days and parenterally for 3 days after irradiation aids the elimination of the postirradiation changes in the amino acid balance.     

Baculovirus-mediated gene transfer is attenuated by sodium bicarbonate

BACKGROUND: Baculovirus transduction of cultured mammalian cells is typically performed by incubating the cells with virus using culture medium (e.g. Dulbecco's modified Eagle's medium (DMEM)) as the surrounding solution. However, we previously uncovered that DMEM hinders the baculovirus-mediated gene transfer. METHODS: In this study, we systematically explored the influences of promoter and medium constituents on the transduction efficiency by using different recombinant viruses and surrounding solutions for transduction, followed by flow cytometric analyses. Whether the key medium component impeded baculovirus binding to the cells and subsequent virus entry was investigated by immunofluorescence/confocal microscopy and quantitative real-time polymerase chain reaction (Q-PCR). RESULTS: We demonstrated that the poorer transduction by using DMEM as the surrounding solution is independent of the promoter. Examination of the medium constituents group by group revealed that the balanced salt solution suppresses the baculovirus transduction. By omitting individual salt species in the balanced salt solution, we surprisingly uncovered that NaHCO(3), a common buffering agent, exerts the inhibitory effects in a concentration-dependent manner. Intriguingly, NaHCO(3) did not debilitate the baculovirus, nor did it inhibit virus binding to the cells. Instead, NaHCO(3) inhibited baculovirus transduction by reducing the intracellular virus number. CONCLUSIONS: To our best knowledge, this is the first report unraveling the significance of NaHCO(3) in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO(3) from the medium during the transduction period.