Study of the inhibition of mitotic activity in the rat liver]

When affecting the rat liver cells by the alkylating agent dipin or X-ray irradiation in combination with genome induction by phenobarbital, a considerable decrease was observed in the mitotic activity of hepatocytes after hepatoectomy. The decrease in the mitotic activity is preserved for 90 days between introduction of mutagene and phenobarbital. When increasing the time interval to 150 days the inhibition in the mitotic activity is not observed.     

Formaldehyde-induced mutations in Drosophila melanogaster in dependence of the presence of acids

Summary The mutagenic activity of various combinations of formaldehyde, formic acid, acetic acid and hydrochloric acid was investigated by a sex-linked lethal test. All combinations were mutagenic and showed a mutation pattern from which it is concluded that in feeding experiments spermatocytes I are especially sensitive to the pairs of chemicals tested. In vapour experiments all germ cell stages were found to be susceptible.The presence of volatile acids was found to be necessary for the mutagenic activity of formaldehyde in the vapour state. Mutagenic effects were also observed in larvel feeding experiments, in which only these acids were added to the medium. Experiments with stabilized pH at 7.5 did not show a significant mutagenic effect of formaldehyde.It is postulated that the tested agents are catalase inhibitors, which promote the formation of peroxides or free radicals which interfere with DNA replication, thus producing mutations.

---

Zusammenfassung Mit Hilfe des Muller-5-Tests wird bei Drosophila melanogaster die mutagene Aktivität von Formaldehyd, Ameisensäure, Essigsäure und Salzsäure allein und in verschiedenen Kombinationen von Formaldehyd mit den Säuren geprüft. Alle Kombinationen sind mutagen. Da keine Mutagenität mehr nachweisbar ist, wenn die Fliegen mit Formaldehyd mit einem pH 7 behandelt werden, ist anzunehmen, daß die mutagene Wirkung von Formaldehyd von einem sauren Milieu abhängig ist. Die untersuchten Substanzen werden den Tieren entweder in der Gasphase oder durch Beimischung zum Nährboden verabreicht. Im ersten Fall werden alle Keimzellstadien beeinflußt, während die Applikation im Nährboden nur die Spermatocyten I angreift. Der Wirkungsmechanismus der untersuchten Stoffe wird als eine Katalase-Hemmung gedeutet, die zur Bildung von u. a. freien Radikalen und damit — über Störung der DNS-Synthese — zum Entstehen von Mutationen führt.

     

Differences in the degrees of stability of colchicine-resistant clones of murine hepatoma XXIIa

Cells resistant to colchicine in the parental line of mouse hepatoma XXIIa could be revealed with a frequency of 4-4.5 per 10(5) cells when selected at the drug concentration as high as 0.05 mkg/ml. MNNG as a mutagene was shown to increase the number of resistant cells by 5-6 times. 6 clones of independent origin differed in the level of resistance and in the stability to retain it under non-selective conditions. Multistep selection from the stable clones via 0.25, 1.0, 4.0, 5.0, 10.0 and 20.0 mkg/ml resulted in the appearance of some highly resistant subclones. Stable clones of all the steps of selection appeared to be resistant to ethidium bromide. Genetical polymorphism of colchicine-resistance is suggested in the line of mouse hepatoma XXIIa.     

Effect of the chaotropic anions thiocyanate and perchlorate on the entry of ricin into Vero cells.

The effect of different anions on the sensitivity of Vero cells to ricin was investigated. The cells were equally sensitive to ricin when NaCl was replaced by NaBr, NaI, Na2SO4 or with iso-osmotic concentrations of mannitol. In contrast, NaSCN and NaClO4 strongly protected against ricin at pH 7.2, but not at pH 7.6. The possibility that the protective effect is due to the ability of chaotropic anions to decrease the pH close to the membrane is discussed.     

Interrelationship between sodium cyanate and pH in the regulation of tumor cell division

Previous studies have suggested that the selective inhibitory effects of sodium cyanate on tumor metabolism in vivo may be related to a lower interstitial pH in tumors. In the present work, the influence of extracellular pH on the actions of sodium cyanate was studied with one rat hepatoma cell line (HTC) and two human colon tumor cell lines (HT29 and LS174T) and with rat hepatocytes to determine if the effects are accompanied by changes in intracellular pH. With some tumor cells, an inhibition of cell proliferation was observed when the cells were exposed to an acidic medium (pH 6.6). However, the LS174T line of human tumor cells divided at pH 6.6 essentially as fast as at pH 7.4. In the concentration range of 0.02-0.1 mg/ml, a greater inhibitory effect of cyanate on cell proliferation was observed at the lower pH. Intracellular pH was found to be influenced by the sodium ion concentration of the medium to a similar degree in the three tumor lines that were examined. The intracellular pH was found to be significantly affected by cyanate in rat hepatocytes and in two of the tumor cell lines (HT29 and LS174T). The data suggested that not only does extracellular pH influence the inhibitory effect of cyanate on tumor cell proliferation but also that cyanate can affect the regulation of intracellular pH in normal and neoplastic cells.     

Effects of procaine on the intracellular pH of Chinese hamster ovary cells heated at 42.0 or 45.0 degrees C

The local anesthetic procaine greatly sensitizes cells to hyperthermia. Though it is generally accepted that procaine is a membrane-active agent that increases membrane fluidity in cells, the mechanism by which it potentiates heat killing is unknown. In this paper we report changes in intracellular pH (pHi) of Chinese hamster ovary (CHO) cells heated at 42.0 or 45.0 degrees C in the presence of procaine. The pHi was measured with flow cytometry using the dye 1,4-diacetoxy-2,3-dicyanobenzene (ADB). Studies were carried out using cells grown at normal pH (7.3) or cells placed in low-pH (6.6) medium 4 h prior to and during heating (acute low-pH treatment). Low-pH-adapted cells (PHV2), which were obtained previously by continuous culture in pH 6.6 medium, were also used. Normal cells heated in the presence of procaine at pH 7.3 underwent a large decrease in pHi compared to cells heated without procaine. Procaine had little additional effect on the intracellular pH of cells in medium with a pH of 6.6 for 4 h before and during 30 min of heating. PHV2 cells exposed to chronic low-pH conditions were resistant to acidification when heated with or without procaine. The surviving fraction of cells heated with procaine was significantly lower under all pH conditions than that of cells heated without procaine. Cells heated at 42.0 degrees C with procaine also became greatly acidified and their survival was reduced. These data suggest that the reduction in pHi caused by procaine may be part of the mechanism of heat sensitization, but cannot account for it entirely. Furthermore, the degree of procaine sensitization and intracellular acidification is dependent on the extracellular pH, with a larger effect occurring at pH 7.3 than at pH 6.6.     

Role of pH in fibroblast proliferation

Secondary cultures of human diploid fibroblasts were used to study the effect of pH on cellular proliferation. In nonconfluent cultures, the growth rate at pH 7.1 was similar to that at pH 7.7 regardless of serum concentration. However, the saturation density achieved at pH 7.7 at any serum concentration was always 2–4 times that achieved at pH 7.1, although the greatest differences in saturation density were observed at the higher serum levels. The results suggest that the effect of pH on saturation density is due to two factors. One, cells at pH 7.1 seem to have a greater ability to undergo contact-inhibition than at pH 7.7, independent of any serum functions; and, two, confluent cells in medium at pH 7.1 are somewhat less sensitive to growth stimulation by increasing serum concentration than are confluent cells raised in medium at pH 7.7.     

Characteristics of yeast cell flocculation by Lactobacillus fermentum

The effects of different metal ions, carbohydrates, heat and enzymatic treatments on the flocculation of yeast cells caused by a flocculant type of Lactobacillus fermentum were investigated. Calcium ion was required at pH 3.0, 4.5 and 6.2 for complete flocculation. Some flocculation was detected at pH 4.5 even if no calcium was added to the system. Manganese and magnesium ions were capable of partly replacing calcium at pH 6.2. Mannose had an inhibitory effect on flocculation, while other sugars had no effect. Protease is capable of inhibiting the flocculating ability of bacterial cells. Heat treatment of bacterial cells also destroyed the flocculating ability and the effectiveness of this treatment was pH dependent. No effect of protease or heat treatment on yeast cells was found. The results suggest that a cell wall component of L. fermentum, mannan residues of yeast cells and divalent ions were involved in this phenomenon.     

Iron uptake by Caco-2 cells following in vitro digestion: Effects of heat treatments of pork meat and pH of the digests

The present in vitro studies report on iron uptake by Caco-2 cells from pepsin and pepsin + pancreatin-digested pork meat proteins at pH values between 4.6 and 7 mimicking conditions in the duodenum and the proximal jejunum, respectively. Heat treatment of the pork meat resulted in increased iron uptake from pepsin-digested samples to Caco-2 cells at pH 4.6. The major enhancing effects on iron uptake by Caco-2 cells were observed after pepsin digestion in the pH range 4.6–6.0, whereas the pepsin + pancreatin-digested samples resulted in negligible iron uptake in Caco-2 cells at pH 7. Thus, the results emphasize the importance of separating pepsin-digested and pepsin + pancreatin-digested proteins during in vitro studies on iron availability. Furthermore, the present results showed the pH dependency of iron uptake anticipated. The enhancing effect of ascorbic acid was verified by increased iron uptake from pepsin-digested pork meat samples at pH 4.6, while no effect of ascorbic acid was observed at pH 7 in pepsin + pancreatin-digested samples.     

Studies of genetic effects in the D7 strain of Saccharomyces cerevisiae under different conditions of pH

The genetic effects of variation in pH in culture media and in suspension tests were examined in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. Deviation from the normal pH of 6.24 in the liquid culture medium, has a significant effect on cellular growth and on mitotic gene conversion at the trp5 locus. Frequencies of reversion at the ilv I-92 locus and of mitotic crossing-over at the ade2 locus are not significantly influenced. Suspension tests, performed using phosphate buffer (pH 5.8), strongly confirm the original results. Our data suggest that the increase in mitotic gene conversion under various conditions of pH is due to a specific effect of pH itself on the cells of S. cerevisiae. In fact, increases were obtained using the same pH in both cellular growth and non-growth conditions. The maximum effect detected with both procedures was obtained at pH 5.8; in the growth test, at this pH, gene conversion frequency appeared to be most pronounced, being about 10 times higher than that of the control. These results suggest that pH exerts its specific action both on growing and non-growing yeast cells, and the difference in induction of genetic effect between these two conditions is probably due to a time factor.     

Energetics of Helicobacter pylori and its implications for the mechanism of urease-dependent acid tolerance at pH 1

In the presence of urea the neutrophilic human pathogen Helicobacter pylori survives for several hours at pH 1 with concomitant cytoplasmic pH homeostasis. To study this effect in detail, the transmembrane proton motive force and cytoplasmic urease activity of H. pylori were determined at various pH values. In the absence of urea, the organism maintained a close-to-neutral cytoplasm and an internally negative membrane potential at external pH values greater than 4 to 5. In the presence of urea, H. pylori accomplished cytoplasmic pH homeostasis down to an external pH of 1.2. At this external pH, the cytoplasmic pH was 4.9 and the membrane potential was slightly negative inside. The latter finding is in contrast to the situation in acidophiles, which develop inside-positive membrane potentials under similar conditions. Measurements of the time course of the membrane potential confirmed that addition of urea to the cells led to hyperpolarization. Most likely, this effect was due to electrogenic export of ammonium cations from the cytoplasm. The urease activity of intact cells increased nearly exponentially with decreasing external pH. This activation was not due to enhanced gene expression at low external pH values. In cell extracts the pH optimum of urease activity was dependent on the buffer system and was about pH 5 in sodium citrate buffer. Since this is the cytoplasmic pH of the cells at pH 1 to 2, we propose that cytoplasmic pH is a factor in the in vivo activation of the urease at low external pH values. The mechanism by which urease activity leads to cytoplasmic pH homeostasis in H. pylori is discussed.     

The Synthesis of Cyclotene and Its Related Compounds by the Acyloin Condensation

A mutant which required glutamate for growth as the sole nitrogen source was derived from alkalophilic Bacillus No. 8–1 by UV irradiation. The relationship was examined between cell growth and glutamate transport into cells.

Cell growth and glutamate transport into cells were dependent on extracellular pH in the presence of Na⁺, and both were maximum between pH 9 and 10. The quantitative relation between specific growth rate and glutamate uptake rate indicated that the amount of glutamate required for growth at pH 7 and 9 was consistent with that of glutamate transported at pH 7 and 9, respectively. But the amount of glutamate transported at pH 7 was not sufficient to support growth at pH 9. The glutamate transport system of this mutant strain evidently had an effect on growth.     

Effect of Na+ and K+ ions on the luminescence of intact Vibrio harveyi cells at different pH values

The bioluminescent activity of intact Vibrio harveyi cells loaded with different concentrations of NaCl and KCl at different pH values was studied. In the pH range of 6.5-8.5, the effect of Na+ was significantly higher than that of K+ at all concentrations studied. Maximum luminescent activity was observed in cells loaded with 0.68 M NaCl. When Na+ was nonuniformly distributed on the plasma membrane, the cell luminescence kinetics was nonstationary in the 20-min range: during incubation, the luminescence intensity increased at pH 6.5 and decreased at pH 8.5. The activation and damping rate constants depended on the Na+ gradient value. The maximum of luminescent activity shifted during incubation from pH 8.5 to 6.5-7.0. The luminescence kinetics in the systems with KCl was stationary; the maximum level of luminescence was observed in the pH range of 7.0-7.5. Under Na(+)-controlled conditions, the cell respiration and luminescence changed in synchronism. The protonophore CCP at a concentration of 20 microM completely inhibited luminescence at pH 6.5 and was ineffective at pH 8.5.     

Dextransucrase secretion in Leuconostoc mesenteroides depends on the presence of a transmembrane proton gradient.

The relationship between proton motive force and the secretion of dextransucrase in Leuconostoc mesenteroides was investigated. L. mesenteroides was able to maintain a constant proton motive force of -130 mV when grown in batch fermentors at pH values 5.8 to 7.0. The contribution of the membrane potential and the transmembrane pH gradient varied depending on the pH of the growth medium. The differential rate of dextransucrase secretion was relatively constant at 1,040 delta mU/delta mg (dry weight) when cells were grown at pH 6.0 to 6.7. Over this pH range, the internal pH was alkaline with respect to the external pH. When cells were grown at alkaline pH values, dextransucrase secretion was severely inhibited. This inhibition was accompanied by an inversion of the pH gradient as the internal pH became more acidic than the external pH. Addition of nigericin to cells at alkaline pH partially dissipated the inverted pH gradient and produced a fourfold stimulation of dextransucrase secretion. Treatment of cells with the lipophilic cation methyltriphenylphosphonium had no effect on the rate of dextransucrase secretion at pH 5.5 but inhibited secretion by 95% at pH 7.0. The reduced rate of secretion correlated with the dissipation of the proton motive force by this compound. Values of proton motive force greater than -90 mV were required for maximal rates of dextransucrase secretion. The results of this study indicate that dextransucrase secretion in L. mesenteroides is dependent on the presence of a proton gradient across the cytoplasmic membrane that is directed into the cell.     

Decrease of mutagenic action of cyclophosphamide by buckwheat melanin preparation

Melanins are polyphenolic pigments of plants, animals and microbes with antioxidant and antiradiation activity. Water-soluble melanin from buckwheat is experienced as antimutagenic means (0.01-10 mg/kg per os) for cyclophosphamide (20 mg/kg i.p.) in the 3 series of experiments. The frequency of chromosome aberrations in the cells of mice bone marrow after mutagene is reduced 2-6 times once under influence of melanin.     

A study of the aggregation of human red blood cells induced by picric acid

The effect of picric acid on the aggregation of human erythrocytes was studied. It was shown that the addition of picric acid to a suspension of washed erythrocytes leads to a decrease in pH of medium to 1.5-2 and the formation of echinocytes. Stirring the suspension of echinocytes at low pH values results in a strong aggregation of cells. Increasing the pH value to 7.4 leads to a desaggregation of echinocytes. It was found that picric acid does not induce the aggregation of cells fixed by glutaraldehyde. A substantial decrease in the aggegation of spheric erythrocytes obtained after heating the cells at 50 degrees C was observed.     

Effects of pH and type of sugar in the medium on tyrosinase activity in cultured melanoma cells.

The tyrosinase (EC 1.14.18.1) activity of cultured mouse melanoma cells B16 in the stationary phase of growth, depends greatly on the pH of the medium and the kind of sugar present. The enzyme activity of a homogenate of cells grown at pH 7.2 in Eagles's MEM supplemented with 10% new born calf serum and con taining galactose in place of glucose, was about ten times that of a homogenate of cells cultured at pH 6.3 in the same medium. The tyrosinase activity changed reversibly on changing the pH of the culture medium. When cultured at a constant pH of 7.2, cells grown with 1 mM galactose had about five times higher tyrosinase activity than cells grown with 1 mM glucose. Only a small amount of lactate accumulated in cultures with glucose and it had little effect on the enzyme activity. These two findings explain the very low tyrosinase activity of cells cultured in medium with 5 mM glucose: the low activity is due to the presence of glucose and to the low pH resulting from conversion of glucose to lactic acid.     

Effect of pH on cea gene expression in cells irradiated with UV-light and unirradiated Escherichia coli cells

The effect of pH on the expression of the cea gene encoding colicin EI in Escherichia coli was investigated by measuring the beta-galactosidase activity in the UV-irradiated growing cells carrying the cea-lacZ fusion. Maximum activity was observed at pH 7, and inhibition of expression was observed at pH 6 and pH 8. Treatment of the irradiated cells with 50-mM acetate increased inhibition at pH 6.0-7.5. No correlation between cea expression and the rate of cell growth was observed at different pH levels. Preliminary treatment with acetate at pH 7 reduced the expression of the recA gene, which participates in the regulation of the cea gene to 33% in irradiated cells and to 25% in nonirradiated cells.     

The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells.

1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.