Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide

We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny.     

Interaction of aldolase with pigeon erythrocyte plasma membranes]

Osmotically hemolysed pigeon erythrocytes retain a considerable part of the total cell content of aldolase activity. After washing off the ghosts from hemoglobin and removing the nuclei, a considerable portion of aldolase activity is found in the supernatant. The retained part of aldolase is rather firmly bound to plasma membranes (PM), as evidenced by the fact, that double washing with a mixture of 0.3 M sucrose, 0.01 M tris-HCl (pH 7.4) and 0.004 M MgCL2, or with 0.15 M NaCl or H2O does not appreciably decrease the aldolase activity of PM. Only washing of PM with 0.5 M NaCl results in appreciable decrease of aldolase retention by PM. The binding of aldolase proved to be temperature sensitive: after heating the binding of aldolase to PM specifically decreased. These data suggest that the interaction of the enzyme with PM of pigeon erythrocytes occurs in the intact cell and may be of physiological significance.     

(Vinylaryloxy)acetic acids. A new class of diuretic agents. 3. ((2-Nitro-1-alkenyl)aryloxy)acetic acids

A series of [(2-nitro-1-alkenyl)aryloxy]acetic acids was synthesized and tested in dogs for saluretic and diuretic activity. A number of these compounds exhibit a high order of activity on iv or po administration; representative of these is (E)-[2,3-dichloro-4-(2-nitropropenyl)phenoxy]acetic acid (5). The most highly active compounds are qualitatively similar in action to [2,3-dichloro-4-(2-methylenebutyryl)phenoxylacetic acid (ethacrynic acid) in causing a prompt increase in the excretion of water and of sodium and chloride ions in approximately equimolar amounts but are three to five times as potent. Potassium ion excretion is increased but less markedly than sodium excretion.     

Cardiovascular profile of mixidine fumarate, a compound which attenuates myocardial chronotropic responses

The effect of mixidine fumarate on myocardial chronotropic responses to various stimulants was examined. Mixidine decreased elevated heart rate in the anesthetized dog to basal levels. It produced a dose-related decrease in heart rate elevated reflexly by aminophylline, by beta adrenergic stimulation induced by isoproterenol, by sympathetic nerve stimulation and by intravenous infusion of glucagon. Mixidine attenuated the increase in contractile force produced by sympathetic nerve stimulation but not that induced by isoproterenol. The compound antagonized the increase in rate of isolated guinea-pig atria induced by both isoproterenol and histamine. In the conscious dog, mixidine caused no decrease in resting heart rate, mean arterial pressure and cardiac output. It reduced atropine-induced sinus tachycardia as well as that induced by treadmill exercise. Experiments in the dog heart-lung preparation indicated that attenuation of an epinephrine-induced sinus tachycardia led to a decrease in myocardial oxygen consumption and an increase in myocardial efficiency. These studies suggest that mixidine fumarate induces an antichronotropic activity by a direct effect on the sinoatrial node and by attenuating sympathetic nervous system input to the heart.     

The localized corrosion of resistant alloys in chloride solutions

A potentiostatic scratching technique has been used to study the breakdown potentials of stainless steel 304, Incoloy, Inconel 600, Monel 400 and Hastelloy C in chloride solution at pH 8 as a function of chloride activity, temperature and metal cold work. For the first four alloys break-down occurs through pitting. For Hastelloy C there is no pitting process, and breakdown occurs by a transpassive reaction, probably involving oxidation of Mo and Cr in the film to soluble anions.