Chemotactic factor causes rapid decreases in phosphatidylinositol,4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils

Stimulation of rabbit neutrophils prelabeled with 32P by the synthetic chemotactic peptide f-Met-Leu-Phe induces a rapid decrease in the radioactivity in both phosphatidylinositol, 4,5 bis phosphate and phosphatidylinositol 4-monophosphate. The mean +/- standard error of the mean values of the maximum decrease in phosphatidylinositol, 4,5 bis phosphate occurred at 10 seconds following stimulation and is equal to 19 +/- 3% of the control value. The corresponding value for phosphatidylinositol 4-monophosphate occurred at 60 seconds following stimulation and is equal to 37 +/- 7% of the control value. On the other hand, the radioactivity in phosphatidic acid and lysophospholipids increased continuously with time following stimulation. The relationship of these changes to calcium release and neutrophil activation is discussed.     

Aminoacyl-tRNA synthetases: inter-site interaction as a possible proofreading mechanism

Smooth muscle cell energetics of taenia caeci during relaxation, activity and maximal contraction were investigated using ³¹P-NMR. In relaxed muscle obtained in calcium-free medium, [ATP], [phosphocreatine] and [sugar phosphate] were 4.4 mM, 7.7 mM and 2.8 mM, respectively. There was only a small difference in the energetics of spontaneously active and maximally contracted muscles, but under both conditions substantial changes occurred as compared with relaxed muscles. The internal pH in relaxed muscle was found to be 7.05, which acidified to 6.5 during contraction. The level of sugar phosphates was found to be not a limiting factor in energetics.     

A stimulating effect of guanyl nucleotides on the rat-liver soluble cyclic GMP high-affinity phosphodiesterase activity

The high affinity (low Km) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis-resistant analogue, guanylylimidodiphosphate (GppNHp), display a half-maximal stimulating effect at almost the same concentration (5 X 10(-6) M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high Km) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAE-cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.     

Purification and characterization of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B

The membrane (Na⁺ + Mg²⁺)-ATPase of Acholeplasma laidlawii B has been solubilized with a Brij-58/sodium deoxycholate mixture and purified by a combination of gel filtration and ion-exchange chromatography. The purified, partially delipidated ATPase has a specific activity of 195 μmol P_i/mg protein per h, which could be enhanced by 25% upon the addition of exogenous phospholipids. The kinetic properties of the purified enzyme are similar to those of the native membrane-bound enzyme, suggesting that it has not been substantially altered during the purification procedure. The enzyme is an assembly of five polypeptide species and its kinetic properties suggest that it is dissimilar to other known ATPases.     

Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

Effect of TPA on the mutagenicity of caffeine in the soybean mutation test

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a well-known tumor promoter in mouse-skin carcinogenesis. Its effects on mutagenesis in a soybean test system were examined, and the effects were judged from the appearance of spots of various colors on the leaves. When soybean seeds were treated with TPA plus 0.03% caffeine, the frequency of spots per leaf decreased significantly and in proportion to the concentration of TPA. TPA alone at concentrations of 1–20 μg/ml did not induce any mutations. Mutations induced by γ-rays were not affected by administration of TPA either before or after exposure to γ-rays. The mechanism of suppression by TPA of mutations induced by caffeine is discussed.     

Physiological changes in, and phosphate uptake by potato plants during development of, and recovery from phosphate deficiency

The development of phosphate deficiency (P-stress) was observed in rooted sprouts of Solanum tuberosum L. cv. Desiree growing in solutions without phosphate. Shoot growth was inhibited by P-stress within 3 to 5 days of terminating the phosphate supply, while significant effects on root growth were not recorded until 7 to 9 days. Thus, the shoot:root dry weight ratio decreased from 4.3 to 2.6 over a 10-day period. Growth in the absence of an exogenous phosphate supply progressively diluted the phosphorus in the plant. The proportional decrease in concentration was similar in roots and shoots over a 7-day period, even though the former were growing more quickly. The potential for phosphate uptake per unit weight of root increased rapidly during the first 3 days of P-stress. When the plants were provided subsequently with a labelled, 1 mol m^?3 phosphate solution, the absorption rate was 3 to 4-fold greater than that of control plants which had received a continuous phosphate supply. The increased rate of uptake by P-stressed plants was accounted for by an increase (3-fold) in the V_max of system 1 for phosphate transport and by a marked increase in the affinity of the system for phosphate (decrease in K_m). In the early stages of P-stress, before marked changes in growth were measured, the proportion of labelled phosphate translocated to the shoots increased slightly relative to the controls when a phosphate supply was restored. In the later stages of stress a greater proportion was retained in the root system of P-stressed plants than in that of controls. In plants with roots divided between solutions containing or lacking a phosphate supply, the increased absorption rate was determined by the general demand for phosphate in the plant and not by the P-status of the particular root where uptake was measured. By contrast, the poportion translocated was strongly dependent on the P-status of the root. The restoration of a phosphate supply to P-stressed plants was marked by a rapid increase in the P concentration in snoots and roots which returned to levels similar to unstressed controls within 24 h. The enhanced uptake rate persisted for at least 5 days, resulting in supra-normal concentrations of P in both shoots and roots, and in the formation of extensive necrotic areas between the veins of mature leaves. Autoradiographs showed accumulations of ³²P in these lesions and at the points where guttation droplets formed on leaves.     

Complex organization of a cryptic satellite DNA in the genome of the marine invertebrate Rapana thomasiana Grosse (Gastropoda)

Isopicnic centrifugation in Cs₂SO₄-Ag⁺ gradients at pH 7.0 reveals that the genome of the marine snail Rapana thomasiana Grosse (Gastropoda) contains an AT-rich satellite fraction comprising 5% of the DNA. Restriction enzyme analysis shows that the satellite DNA is composed of a number of related subsets arranged in tandem arrays. They have evolved from the segmental amplification of an 1460 bp long monomer unit with a complex inner organization. Most probably, the present basic repeat originates from an ancestral 400–500 bp long sequence in which some insertions and/or deletions have occurred.     

Estimation of pyruvate decarboxylation in perfused rat skeletal muscle

A total of 46 E. coli strains showing mannose-resistant, P-blood-group independent hemagglutination of human erythrocytes were tested for binding to neuraminic acid. Nine of the strains completely lost their hemagglutination activity after the erythrocytes were treated with neuraminidase. To characterize the receptor structure, different neuraminic acid containing glycoproteins, their desialylated derivatives and neuraminyl oligosaccharides were tested for hemagglutination inhibition. These studies showed that the nine strains had binding specificity for alpha 2-3 linked neuraminic acid.