Molecular basis for sequence-specific DNA alkylation by CC-1065

CC-1065 is a potent antitumor antibiotic that binds covalently to N3 of adenine in the minor groove of DNA. The CC-1065 molecule is made up of three repeating pyrroloindole subunits, one of which (the left-hand one or A subunit) contains a reactive cyclopropyl function. The drug reacts with adenines in DNA in a highly sequence-specific manner, overlapping four base pairs to the 5'-side of the covalently modified base. Concomitant with CC-1065 covalent binding to DNA is an asymmetric effect on local DNA structure which extends more than one helix turn to the 5'-side of the covalent binding site. The DNA alkylation, sequence specificity, and biological potency of CC-1065 and a select group of trimeric synthetic analogues were evaluated. The results suggest that (a) noncovalent interactions between this series of compounds and DNA do not lead to the formation of complexes stable enough to be detected by footprinting methods, (b) sequence specificity and alkylation intensity can be modulated by the substituents on the nonreactive middle and right-hand segments, and (c) biological potency correlates well with ability to alkylate DNA. In addition, the extent and the sequence specificity of covalent adduct formation between linear DNA fragments and three analogues comprised of the CC-1065 alkylating subunit linked to zero (analogue A), one (analogue AB), or two (analogue ABC) nonreactive indole subunits were compared.(ABSTRACT TRUNCATED AT 250 WORDS)     

A hemodynamic comparison of young and older endurance athletes during exercise

This study assessed the hemodynamic responses to exercise of master athletes (56 +/- 5 yr of age) who placed in the top 10% of their age groups in local 10-km competitive events, competitive young runners (26 +/- 3 yr), young runners matched in training and performance to the master athletes (25 +/- 3 yr), and healthy older sedentary subjects (58 +/- 5 yr). The maximal O2 consumption (VO2max) of the master athletes was 9 and 19% lower than that of the matched young and competitive young runners, respectively. When compared at the same relative submaximal work rates, these three groups had similar stroke volumes and arteriovenous O2 (aVO2) differences, though the master athletes had lower VO2, cardiac output, and heart rate, and higher vascular resistance. The older sedentary group had a lower stroke volume, aVO2 difference, and higher vascular resistance than the master athletes. Maximal stroke volume and estimated aVO2 difference were the same in the three groups of athletes; the lower maximal heart rate of the master athletes appears to account for their lower VO2max. The older sedentary subjects' VO2max was 47% lower than that of the master athletes; this difference was almost equally the result of a lower stroke volume and a lower a-VO2 difference. Thus these older athletes did not exhibit the decline in maximum stroke volume and aVO2 difference that occurs with aging in sedentary individuals; they also appear to have retained a greater peripheral vasodilatory response than their sedentary peers.     

Glucose tolerance in young and older athletes and sedentary men

Groups of endurance-trained masters athletes (60 +/- 2 yr), older untrained men (62 +/- 1 yr), lean older untrained men (61 +/- 2 yr), endurance-trained young athletes (26 +/- 1 yr), and young untrained men (28 +/- 1 yr) were studied to obtain information on the separate effects of age, physical activity, and body fatness on glucose tolerance and insulin sensitivity. Each subject underwent an oral 100-g glucose tolerance test. Skinfold thickness was determined at six sites. The trained groups had a higher maximum O2 uptake capacity and lower sum of skinfolds than their sedentary peers. The lean older untrained group had a sum of skinfolds similar to that of the young untrained group. The masters athletes, young athletes, and young untrained men exhibited similar glucose tolerance whereas the two older untrained groups had an almost twofold greater total area under the glucose curve (P less than 0.05). The masters and young athletes had significantly blunted plasma insulin responses compared with the other three groups (P less than 0.05). The young and the lean older untrained groups had similar plasma insulin responses with significantly lower insulin levels than the older untrained group (P less than 0.05). These results provide evidence that regularly performed vigorous exercise can, in some individuals, prevent the deterioration of glucose tolerance and insulin sensitivity with age.     

Identification and characterization of a fatty acid binding protein in bovine mammary gland

A fatty acid binding protein (FABP) was isolated from bovine mammary cytosol by gel filtration and ion exchange chromatography. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a mol. wt. of 12,000. Isoelectric focusing showed two bands at pH 5.6 and 5.8. FABP bound long chain fatty acids and their CoA thioesters, but not medium or short chain fatty acids. Affinity constant (Ka) for 18:1 was about 2 micromolar. Endogenously bound fatty acids included 16:0, 18:0 and 18:1, in both covalent and noncovalent association with FABP. Activities of microsomal phosphatidic acid phosphatase, fatty acid:CoA ligase or diacylglycerol acyltransferase were not affected by purified FABP in vitro.     

Immunohistological localization of IgG1, IgA and secretory component in the bovine mammary gland during involution

Summary Immunoperoxidase methods were used to localize secretory component, immunoglobulin A and immunoglobulin G1 in mammary tissue from dairy cows. In lactating tissue, immunostaining for immunoglobulin A and secretory component was observed primarily in the luminal contents of alveoli. By day 2 of involution, alveolar epithelial cells stained for both immunoglobulin A and secretory component. Staining of alveolar epithelial cells for immunoglobulin A and secretory component continued throughout the period of mammary involution. No staining for secretory component was observed in the interalveolar stromal area. Immunoglobulin G1 immunostaining was localized primarily in the interalveolar areas in lactating tissue, but was localized at the apical and basolateral surface of alveolar cells on day 2 of involution. In contrast to immunoglobulin A, immunoglobulin G1 staining of epithelial cells did not persist and was primarily in the interalveolar areas by day 4. These results suggest that an increased localization of immunoglobulin G1 in bovine mammary epithelial cells may occur transiently in early involution, while an increase in immunoglobulin A and secretory component localization in epithelial cells persists throughout involution.     

Actin of chara giant internodal cells: a single isoform in the subcortical filament bundles and a larger, immunologically related protein in the chloroplasts

Internodal cells of Chara corallina Klein ex. Wild have been studied to determine the number of actin isoforms they contain and whether actin occurs at locations in the cortical cytoplasm outside the filament bundles. A monoclonal antibody to chicken actin is specific for actin in numerous animal cells but binds to two Chara proteins after their separation by two-dimensional polyacrylamide gel electrophoresis. One protein resembles known actins in relative molecular mass (43,000-M_r) and isoelectric point (5.5) while the other is distinctly different (58,000-M_r, isoelectric point = 4.8). Because it is indetectable in cells whose actin bundles have been extracted, the 43,000-M_r protein is assigned to the bundles and concluded to be rare or absent in the remaining cortical cytoplasm. The 58,000-M_r protein, in contrast, does not extract with the actin bundles. It was localized within the chloroplasts by immunofluorescence and by the dependence of proteolysis on the permeabilization of the chloroplast envelope.     

Mitogenic activity of snake venom lectins

Five lactose-inhibitable lectins have been isolated from snake venoms. These five share certain biochemical properties but are not identical (Gartner, Stocker & Williams, 1980; Gartner & Ogilvie, 1984). In this study the lectins were tested for their ability to stimulate lymphocytes to undergo DNA synthesis. We found that three of the lectins were comparable in mitogenic activity to the T cell lectin, concanavalin A (Con A). The mitogenic activity was blocked by lactose, a sugar which also blocks the haemagglutination activity of these lectins. Although mitogenic response appeared to be due to T cells, it depended on the presence of accessory cells in the culture. This requirement for macrophages could be replaced by the phorbol ester tumour promoter, 12-o-tetradecanoylphorbol-13-acetate (TPA).     

6-Mercaptopurine-induced alterations in mineral metabolism and teratogenesis in the rat

The relationship between 6-mercaptopurine-induced alterations in mineral metabolism and the teratogenic effects of the drug were investigated. Pregnant Sprague-Dawley rats were fed diets containing 4.5, 100, or 1,000 micrograms Zn per 1 g diet. On day 11 of gestation, dams were given intraperitoneal injections of 6-mercaptopurine (27.5 mg/kg). At term, dams fed the 1,000-micrograms Zn per 1 g diet showed fewer drug-induced deleterious effects on reproduction and embryogenesis than did those fed lower levels of zinc. Mineral analysis of maternal and fetal tissues revealed pronounced effects of 6-mercaptopurine on metabolism of zinc, copper, iron, calcium, and magnesium. The results of this study indicate that 6-mercaptopurine teratogenesis may be due in part to drug-induced changes in mineral metabolism.     

Effect of 6-mercaptopurine on mineral and metallothionein metabolism in the mouse

The effect of the anticancer drug 6-mercaptopurine (6-MP) on mineral metabolism was investigated in mice. C57Bl/6J female mice were injected intraperitoneally with 6-MP at 100 mg/kg body wt for one, two, four, or five consecutive days. On d 6 of the study, liver, kidney, and intestine were removed, and concentrations of zinc, copper, iron, manganese, magnesium, and calcium were measured. Hepatic concentrations of zinc, copper, iron, and calcium became higher as the number of drug injections increased. To determine if the altered mineral metabolism was a function of a drug-induced, acute-phase response, liver metallothionein and plasma ceruloplasmin were measured. Metallothionein concentrations in the liver became higher with increased number of injections, correlating with the stepwise increase in hepatic zinc. Gel filtration chromatography showed that most of the increase in liver zinc concentration was associated with a protein of mol wt of 6000–8000, the approximate weight of metallothionein. Ceruloplasmin concentrations were not affected by 6-MP injection. These results suggested that 6-MP alters zinc metabolism by sequestering zinc into the liver via induction of metallothionein synthesis and that the drug may induce an acute-phase response with an atypical acute-phase protein profile.