Mutation induction by thymine deprivation in Escherichia coli B/R. II. Mutation in the repressed and derepressed tryptophan operon.

True Trp⁺ reversions are induced by thymine deprivation in cells with repressed trp operons as efficiently as in derepressed cells. At least part of the mutations are fixed during thymine starvation, i.e. in the absence of net DNA synthesis. The hypothesis is put forward that thymineless mutagenesis is due to repair-replication under limited concentrations of 5′-dTTP, performed by an inducible error-prone “DNA-polymerizing activity” on single-strand gaps.     

Mutation induction by thymidine deprivation in Escherichia coli B/r. I. Influence of caffeine.

The addition of caffeine to the plating medium after thymine deprivation of E. coli WP2 uvr+ thyA or WP2 uvrA thyA had no influence on survival. Caffeine, however, reduced the frequency of mutants. The hypothesis is presented that the reduced mutagenesis is due to the sensitivity to caffeine of an inducible error-prone repair mechanism operating during thymine deprivation and after the re-addition of thymine.     

A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

Banding patterns of the chromosomes ofCercopithecus neglectus: Comparison withMiopithecus talapoin andErythrocebus patas

The G, Q, and C bands and the location of the nucleolar organizing regions (NORs) of the chromosomes of two male Cercopithecus neglectusare described. The diploid number of the species is 2n =62. Comparison with the karyotypes of Miopithecus talapoin (2n =54), and Erythrocebus patas (2n =54)showed the presence of total banding homeology for only 10 chromosome pairs.     

The role of glucagon in the regulation of blood glucose: Model studies

Mathematical models afford a procedure of unifying concepts and hypotheses by expressing quantitative relationships between observables. The model presented indicates the roles of both insulin and glucagon as regulators of blood glucose, albeit in different ranges of the blood glucose concentrations. Insulin secretion is induced during hyperglycemia, while glucagon secretion results during hypoglycemia. These are demonstrated by simulations of a mathematical model conformed to data from the oral glucose tolerance test and the insulin infusion test in normal control subjects and stable and unstable diabetic patients. The model studies suggest the parameters could prove of value in quantifying the diabetic condition by indicating the degree of instability. Presented at the Society for Mathematical Biology Meeting, University of Pennsylvania, Philadelphia, August 19–21, 1976.     

Cellulose acetate electrophoresis of protein kinases: Detection of the active forms using various substrates

Electrophoresis on cellulose acetate strips was used to analyze protein kinases from normal rat liver. In addition to already well-characterized cAMP-dependent protein kinases type I and II and cAMP-independent casein kinases I and II, this method enabled the detection of several supplementary bands corresponding to kinases which were investigated according to their substrate specificity, activation by cAMP, and inhibition by the specific inhibitor of the catalytic subunit of cAMP-dependent protein kinases or by heparin. Using this rapid, sensitive, and resolutive electrophoretic method, different isozyme patterns could be obtained starting from minute amounts of different types of biological material.     

Conformational change of turkey-gizzard caldesmon induced by specific chemical modification with carbodiimide

Water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to internally cross-link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86,974 Da of chicken-gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook. R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13,873-13,879). Comparative circular dichroism analysis indicated a decrease of the alpha-helix content from 43% to 36% resulting from the chemical modification. The 1H-NMR spectra of the native and modified caldesmon showed that the covalent cross-linking affected mainly the central and N-terminal parts of the molecule. The C-terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90-kDa proteolytic N-terminal fragment to give an internally cross-linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F-actin bundles. These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F-actin bundles or weaker interactions between actin filaments.     

Metabolic utilization of muscular L-proline in 24-hr starved rats.

1. The aim of this paper was to study the in vivo skeletal muscle L-proline related to its destination to other key tissues such as liver and intestine as well as to give some insight into the role of blood cells in proline handling. 2. L-U-[14C]Proline was injected intramuscularly and following by sampling of blood, liver, intestine and contralateral muscle at 20 and 30 min after injection. 3. The distribution of radioactivity between blood cells and plasma and in total and individual amino acids, protein and glycogen fractions was determined in the above tissues. 4. The pattern of well fed rats was compared with those submitted to 24-hr complete starvation. 5. During starvation a minor degree of proline oxidation occurs. 6. The main destruction of proline in the liver seem to be the synthesis of proteins. 7. The radioactivity recovered in the blood proline fraction of starved rats is twice that of the fed rats and that it could be attributed mainly to plasma protein. 8. We have obtained in vivo evidence for the role of erythrocyte in the interorgan proline transport.