In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.     

Determination of the shape of the operculum by the bithorax complex in Drosophila melanogaster

Summary We have studied the course of the operculum line in the larval hypoderm of several bithorax complex mutants of Drosophila melanogaster. The bifurcation of the line, a characteristic of the first abdominal segment in wild-type (A₁), can also appear in the metathoracic (T₃) and other abdominal segments (A₂, A₃) depending on mutations within the bithorax complex. Therefore, we concluded that the course of the operculum line and thus the shape of the operculum is not determined by a suprasegmental gradient of positional information but by the functional state of the genes of the bithorax complex in each metamere. The dorsal and ventral branches of the operculum line react differently, the dorsal branch being more sensitive to the effect of loss of function mutations (bxd, iab-2 ^k), the ventral branch more affected by gain of function mutations (Hab). In some cases the effects of the mutations on the operculum line differed from those in the adult, suggesting a difference in sensitivity of larval hypodermal cells and histoblast cells to the functional gene products of the bithorax complex.     

The effect of papain upon proline and sodium transport of rat renal brush-border membrane vesicles

Treatment of renal brush-border membrane vesicles with papain resulted in the removal of the activity of maltase, gamma-glutamyl transpeptidase and leucine aminopeptidase by 85, 50 and 75%, respectively. Stripping of these membrane enzyme activities constituted about 2% of the total membrane proteins and resulted in a widespread diminution in the ability of a variety of amino acids and sugars to be taken up by the membrane vesicles which remained osmotically responsive. Kinetic analysis of the uptake of proline, which was shown previously to be transported by both sodium-dependent and sodium-independent systems, revealed that the Vmax for the sodium-dependent system and Km for the sodium-independent system were halved, but other parameters were not affected indicating that the papain treatment altered sodium-gradient-stimulated entry and the affinity of the sodium-gradient-independent system for proline. Experiments on sodium entry and efflux demonstrate a marked enhancement of flux, so that equilibration of the sodium gradient occurred about 5-times more rapidly than in untreated vesicles. This occurred without any change in the osmotic properties of the vesicle with regard to sodium or amino acid uptake. Studies of fluorescence polarization suggest that incubation with papain does not alter the lipid domains of the membrane.     

Rapid determination of [guanidino-15N]arginine in plasma with gas chromatography--mass spectrometry: application to human metabolic studies

A rapid gas chromatographic-mass spectrometric method for the determination of 15N in the guanidino nitrogens of arginine is described. The method is based on formation of the N-tetratrifluoroacetyl-arginine derivative. Approximately 0.15 mol% excess 15N can be detected in as little as 50 microliters of plasma with an average coefficient of variation of 8.8%. The possible fragmentation pattern of the N-tetra-trifluoroacetyl-arginine derivative is described. The method was applied to determine the appearance of 15N enrichment in plasma arginine in a healthy adult volunteer during a constant infusion of 15NH4Cl. A plateau level of 0.7 atom% excess was observed 2 h after the 15NH4Cl infusion was started.     

Rabbit kidney function in vitro: the effect of colloids, energy substrate, a vasodilator, perfusion pressure, and bovine serum albumin

An in vitro perfusion system at 37 degrees C for the assessment of rabbit kidney function is described. The purpose of this assay system is to evaluate the effects of cryobiological manipulation on kidney function. The effect of the colloids dextran (MW = 70,000, 80,000, and 180,000) in the perfusate at 110 mm Hg were compared to a reduced perfusion pressure, colloid-free perfusate. Better function was obtained at lower perfusion pressure with the colloid-free perfusate. Less damage was noted histologically on light and electron microscopy. Investigation of energy substrates on rabbit kidney function demonstrated that butyrate, or lactate, in addition to glucose resulted in increased sodium and glucose reabsorption over glucose alone. Substrate-free perfused kidneys exhibited depressed Na transport. Lactate, and to some extent butyrate, decreased net glucose utilization. An alpha-adrenergic blocking agent, isoxsuprine, in the initial flush solution did not appear to be beneficial. An increase of perfusion pressure from 50 to 75 mm Hg resulted in an increase in GFR. Tubular function was enhanced by inclusion of small amounts of BSA in the perfusate.     

Renal brush-border-membrane vesicles prepared from newborn rats by free-flow electrophoresis and their proline uptake.

A method for the isolation of brush-border membranes from newborn-rat kidney, employing centrifugation and free-flow electrophoresis, is described. The composition and purity of the preparation was assessed by determination of enzyme activities specific for various cellular membranes. Free-flow electrophoresis resolves the newborn-rat renal membrane suspension into two populations of alkaline phosphatase-enriched brush-border membranes, designated 'A' and 'B', with the A peak also showing activity of (Na+ + K+)-stimulated ATPase, the basolateral membrane marker enzyme, whereas those of the B peak were enriched 11-fold in alkaline phosphatase and substantially decreased in (Na+ + K+)-stimulated ATPase activity. Membranes in the A peak showed a 7-fold enrichment of alkaline phosphatase, and (Na+ + K+)-stimulated ATPase activity similar to that of the original homogenate. Proline uptake employed to assess osmotic dependency revealed 7% binding of proline to the B vesicles and 31% to the A vesicles. This contrasts with 60% proline binding to vesicles prepared by centrifugation alone. Unlike vesicles from adult animals, proline uptake by B vesicles did not show an Na+-stimulated overshoot, but did exhibit an Na+-gradient enhanced rate of early proline entry. proline entry.     

Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.     

Kinetics of Utilization of Organic Compounds in the Growth of Mycobacterium tuberculosis.

Bowles, Jean A. (University of Colorado School of Medicine, Denver), and William Segal. Kinetics of utilization of organic compounds in the growth of Mycobacterium tuberculosis. J. Bacteriol. 90:157-163. 1965.-To obtain a workable system for a study of the kinetics of nutrient utilization (based on specific quantitative assay) by Mycobacterium tuberculosis, several cultural refinements were introduced: the use of shake culture, a 40-fold increase in the size of inoculum, substitution of glutamate for asparagine as nitrogen source, and elimination of glucose from the medium with glycerol remaining as carbon source. These modifications resulted in reduction to a tenth of the lag phase of glycerol utilization (from 40 to 4 days), and in a greatly increased rate of growth. Both coordinate and sequential patterns of nutrient utilization were in evidence, except in the case of citrate, which was never utilized under a variety of conditions of culture. The coordinate pattern of glucose-glutamate and glucose-glycerol utilization would appear to rule out catabolite repression by glucose. However, elimination of glucose from the medium resulted in elimination of the 4-day lag period before glutamate utilization was initiated, leaving open to question the role of glucose in this system. Evidence is presented for the hypothesis that the sequential pattern of glutamate-glycerol utilization is a function of glutamate repression of glycerol oxidation in the growth of M. tuberculosis, although no diauxie effect is apparent. In a determination of which nutrient-utilization systems were regulated by induction, only in the case of glycerol was evidence obtained for an inducible system. The enzymatic mechanisms underlying these patterns of nutrient utilization are presently being investigated.