Influence of extracellular pH on intracellular pH and cell energy status: relationship to hyperthermic sensitivity

The effect of variable extracellular pH on intracellular pH, cell energy status, and thermal sensitivity was evaluated in CHO cells over the extracellular pH range of 6.0 to 8.6. Extracellular pH was adjusted with either lactic acid, HCl, or NaOH. Regardless of the method of pH adjustment, the results obtained were similar. The relationship between extracellular and intracellular pH was dependent upon the pH range examined. Intracellular pH was relatively resistant to a change in extracellular pH over the pHe range of 6.8 to 7.8 (i.e., delta pHi congruent to delta pHe X 0.33). Above and below this range, delta pHi congruent to delta pHe X 1.08 or X 0.76, respectively. Cellular survival after a 30-min heat treatment at 44 degrees C remained constant over the extracellular pH range of 7.0 to 8.4, but varied substantially over a similar intracellular pH range. The cellular concentration of the high energy phosphate reservoir, phosphocreatine, decreased with decreasing pH. However, the cellular concentrations of ATP, ADP, and AMP remained constant over the entire pH range examined. It is concluded that increased thermal sensitivity resulting from a change in extracellular pH is not due to cellular energy depletion. Furthermore, intracellular pH is a more accurate indicator of thermal sensitivity than is extracellular pH.     

Lonidamine-induced, pH dependent inhibition of cellular oxygen utilization

The inhibitory effect of lonidamine 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid on oxygen utilization by Chinese hamster ovary (CHO) and murine fibrosarcoma (FSa-II) cells was evaluated with a Clark oxygen electrode. The drug produced a small but statistically significant inhibition of oxygen uptake at normal pH (7.4) in CHO and FSa-II cells of 16 and 11%, respectively. However, at low pH (6.65) the inhibitory effect of lonidamine increased dramatically to 60% in both CHO and FSa-II cells. Because of the potential difference between tumor and normal tissue pH, lonidamine and similar drugs may be effective for selectively modifying oxygen utilization and concentration in tumor tissue which might lead to increased radiation and hyperthermic sensitization in tumors compared to normal tissue, resulting in an improvement in the therapeutic ratio.     

Factors influencing the oxidation of cysteamine and other thiols: implications for hyperthermic sensitization and radiation protection

Some of the factors influencing the oxygen uptake and peroxide formation for cysteamine (MEA) and other thiols in serum-supplemented modified McCoy's 5A, a well-known medium used to cultivate a variety of cells in vitro, have been studied. The oxidation of MEA and cysteine in modified McCoy's 5A has been compared with that in Ham's F-12, MEM, and phosphate-buffered saline. All of the growth media were supplemented with 10% calf serum and 5% fetal calf serum. The rate of oxygen uptake for all of the studied thiols was greatest in McCoy's 5A. The data indicate that this medium may contain more copper than the other preparations. MEA and cysteine were found to be more effective at 0.4 mM at producing peroxide than dithiothreitol (DTT). N-acetylcysteine was the least reactive. The ability to produce peroxide is dependent upon the temperature, the concentration of thiol, the presence of copper ions, and pH of the medium. MEA and other thiol oxidation is inhibited by the copper chelator diethyldithiocarbamate. Catalase also reduces the oxygen uptake for all thiols. This inhibition involves the recycling of peroxide to oxygen. Superoxide dismutase (SOD) was found to stimulate the oxygen uptake in the case of MEA and cysteine, but had little or no effect with DTT and glutathione. The combined presence of SOD and catalase resulted in less inhibition of oxygen uptake than that obtained by catalase alone. Alkaline pH was found to enhance the oxidation of cysteine and MEA. An important observation was the inhibition of MEA oxidation at 0 degrees C and the stimulation at 42 degrees C. The results indicate that many problems may arise when thiols are added to various media. A major consideration is concerned with the production of peroxide, superoxide, and reduced trace metal intermediates. The presence of these intermediates may result in the production of hydroxyl radical intermediates as well as the eventual oxygen depletion from the medium. Oxygen depletion may alter the results of radiation sterilization and carcinogen activation. Radical production will cause cell damage that is temperature dependent. Therefore, careful consideration must be given to changes in oxygen tension when thiols are added to cells growing in complicated growth medium to protect against either chemical or radiation damage.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.      

DNA methylation-associated colonic mucosal immune and defense responses in treatment-na?ve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.     

Variability in the kinetics of thermotolerance decay in three cell lines

The magnitude of thermotolerance and rate of tolerance decay has been evaluated in three cell lines following isotime and isoeffect initial heat treatments at 44 degrees C. Following the development of maximal thermotolerance, all cell lines lost tolerance at an exponential rate. However, for the three cell lines examined, V-79, CHO, and A-7, differences were apparent in the time required for maximal tolerance development, magnitude of induced tolerance, and rate of tolerance decay. This variability in the thermotolerance response of cells suggests difficulties which might be encountered if in vivo fractionation protocols are to be developed to minimize damages to normal tissue and maximize damages to tumor tissue.     

Flora Europaea Notulae Systematicae ad Floram Europaeam spectantes No. 20 Corrigenda et Addenda

Following the publication of the last of the series of Flora Europaea Notulae, No. 20 in the Botanical Journal of the Linnean Society , 76: 297–384 (1978), a number of additions or alterations have been drawn to our attention. These are published in continuation.