The alkaline elution procedure developed by Kohn and co-workers was used with the RPMI-6410 cultured human lymphoblastoid cell line to examine the hypothesis that anthracycline-induced DNA strand scission is mediated by oxygen- or superoxide-derived free radicals. Hypoxia was induced by gassing with nitrogen containing 5% carbon dioxide and less than 4 ppm oxygen. Alkaline elution studies showed hypoxia was induced, as the oxygen enhancement ratios for DNA strand breaks was 2.4 and 2.6 for the 250 R +/- oxygen and the 500 R +/- oxygen (1 R = 2.58 x 10(-4) C/kg) experiments, respectively. The pattern of adriamycin-induced DNA strand breaks and cross-linking was not affected by hypoxia with 1-h adriamycin exposures between 0.05 and 1.0 microgram/ml. Similarly, 1-h exposures of N-trifluoroacetyladriamycin-14-valerate at 3 or 10 micrograms/mL gave essentially identical alkaline elution profiles in the presence or absence of oxygen. These results do not support the hypothesis that oxygen-derived radicals play a primary role in anthracycline-induced DNA strand breakage.
Tonin, a new serine protease, found in high concentration in rat submaxillary glands, leads to a significant activation of human amniotic fluid renin. The optimum pH on renin activation by tonin was found at pH 6.0. The reaction was time dependent and the initial rate of angiotensin I generation was constant up to 2 h. The two amniontic fluid samples studied showed an increase in renin activity after incubation with tonin to about five times the control level (268 to 1240 pmol x h-1 x mL-1 and 1490 to 7480 pmol x h-1 x mL-1).
The incubation of PE2 ((6,7-3H)-labelled 3-propyl ether of estra-1,3,5(10)triene-3, 17 beta-diol (estradiol)) with various subcellular fractions of rat liver indicated that the hepatic metabolism of this compound occurs mainly in the microsomal fraction. In addition to the formation of 3-propyl ethers of estra-1,3,5(10)triene-3-ol-17-one (estrone) and estra-1,3,5(10-triene-3, 16alpha, 17 beta-triol (estriol) directly deriving from PE2, the microsomal proteins carried out the deetherification of the propyl ether group leading to phenolic steroids; among them, estradiol, estrone, and estriol were characterized. Protein-bound and water-soluble metabolites were found; the effects of glutathione and of the incubation conditions were in agreement with the thioconjugation of these derivatives. The microsomal metabolism of PE2, and specially the deetherification reaction, required the presence of oxygen and of NADPH as cofactor, the optimum pH ranging from 7.4 to 8. The participation of cytochrome P450 in these metabolic pathways was shown by a partially inhibited catabolism with carbon monoxide and by a more active metabolism in males than in females and when animals were pretreated with phenobarbital. These results allowed us to conclude that the hepatic deetherification of PE2 is carried out by a microsomal oxidative system which is very similar to the system involved in the demethylation of methyl ethers of estrogens.
The pathways leading to the formation of phosphatidylethanolamine in isolated hamster hearts were investigated. The contributions of the CDP-ethanolamine and the base exchange pathways were studied by perfusion with [3H]ethanolamine. The radioactivity of ethanolamine in the heart reached a maximum at 5 min of perfusion and remained constant throughout the perfusion period. Maximum labeling of phosphoethanolamine occurred at 25 min of perfusion and labeling of CDP-ethanolamine did not reach a maximum over the 30-min-perfusion period. Incorporation of radioactivity into phosphatidylethanolamine was marked by a lag during the first 15 min of perfusion, after which a linear increase was observed. This initial lag suggests the minor contribution of the base exchange pathway, as compared with the CDP-ethanolamine pathway. The CDP-ethanolamine pathway was estimated to contribute 290 nmol x min-1 x g heart-1 to total phosphatidylethanolamine formation in hamster heart. Phosphatidylethanolamine formation via decarboxylation of phosphatidylserine was studied by perfusion of hamster hearts with labeled serine. The contribution of this pathway was estimated to be 9.0 nmol x min-1 x g heart-1. Hence, it was concluded that phosphatidylethanolamine was synthesized by all three known pathways and the CDP-ethanolamine pathway was the major pathway for phosphatidylethanolamine biosynthesis in the mammalian heart. The low activities of phosphatidylserine decarboxylase and base exchange enzyme measured in vitro probably reflect the minor contribution of these two pathways to phosphatidylethanolamine biosynthesis.
The ribosome of Physarum polycephalum was found to contain 69 different proteins, with molecular weights ranging from 10 900 to 50 000. The same result was obtained when ribosomes were prepared in presence of a protease inhibitor. A spot frequently observed in two-dimensional gels was identified as an oxidized form of S3, the major phosphoprotein.
The thermodynamic parameters for the protein–ligand binding have been obtained by microcalorimetry on three albumin samples (fatty acid free human serum albumin (I), fraction V human serum albumin (II), and fraction V bovine serum albumin (III)) bound with L-tryptophan (A) and three-ring tryptophan analogs (B and B*). The percentage, u, of binding molecules (equivalent to the number of sites) is found to be 1.0 for I and 0.65 for II (in good agreement with dialysis results on the same systems) and 1.0 for III.The large negative ΔH(bind) (−27.2 to −33.2 kJ∙mol−1) constitutes the main contribution to ΔG(bind) (−23.0 to −31.2 kJ·mol−1). The better binding of I–III towards B and B* compared with A is due to ΔS(bind) being less negative. This is interpreted as a lesser loss of entropy for the three-ring ligands than for the normal tryptophan when they are bound.Data obtained on the proteins (heat of dilution; Huggins' constant, k′) correlate well with u or Qmax. This indicates that these physicochemical data could be used to characterize and compare rapidly some albumins of different sources. The unexpected finding that the parameters of I and III are nearer to each other than they are from II is discussed.
Hepatocyte nuclei from young (3--5 week), mature (8--12 week), and aged (over 32 weeks) rats were isolated and characterized by flow cytometry. Nuclei were bulk separated into diploid (2C), tetraploid (4C), and octoploid (8C) enriched fractions on sucrose gradients. Total, 0.35 M NaCl soluble, and residual proteins were prepared from all nuclear stages and examined by one- and two-dimensional gel electrophoresis. Within limits of sensitivity of these techniques, the following general features emerged. (a) A majority of proteins visualized were common to and present in similar relative quantities in nuclei from all age and ploidy groups. (b) A relatively higher proportion of nonhistone proteins (NHP) were saline-soluble in 2C nuclei from young rats than at any subsequent stage of development. (c) Several age-related and to a lesser extent ploidy-related fluctuations in pattern among the NHP were evident. These reflected primarily differences in solubility rather than major quantitative changes among individual proteins. (d) Exceptions to the foregoing included a group of high molecular weight components (greater than 100 000), a major and a minor component between 45 000 and 50 000, and a heterogeneous group of proteins in 2C nuclei from very young animals. There were no obvious differences among the histones, although these proteins were not examined in detail. The complex pattern of changes observed are discussed in terms of known aspects of hepatocyte differentiation and are related to possible changes in nucleoplasmic, nuclear matrix and Hn-RNP associated proteins.
Some properties of rat liver nuclear membrane-bound UDPglucuronosyltransferase were compared with those of the endoplasmic reticulum bound enzyme. The activity of nuclear membrane-bound UDPglucuronosyltransferase was stimulated only about 1.5-fold by Lubrol WX. Under the same conditions microsomal UDPglucuronosyltransferase was, as usual, highly activated (up to 10-fold), when 4-nitrophenol was the acceptor of glucuronic acid. Specific activities of the detergent-activated enzyme were similar in microsomal and nuclear membrane preparations, when the following aglycone substrates were used: 4-methylumbelliferone, 4-nitrophenol, 1-naphthol, phenolphthalein, and testosterone. Apparent Km values for UDP-glucuronic acid ranged between 0.15-0.25 mM for glucuronidation of 4-nitrophenol and 1-naphthol, by either Lubrol WX activated or non-activated, nuclear membrane-bound UDPglucuronosyltransferase. These values were comparable to those found for detergent activated microsomal enzyme. The results show a similarity in behavior of detergent-activated UDPglucuronosyltransferase regardless of subcellular membrane source and, therefore, suggest the association of the same glucuronosyltransferase with nuclear membrane and endoplasmic reticulum. A possible significance of the presence of high activity of this enzyme in nuclear membrane is discussed.
Human transferrin consists of a single chain polypeptide which supports two N-glycosidically linked glycans at sequons a and b. Glycopeptides were released from human transferrin by proteolytic digestion, desialylated by mild acid hydrolysis, and then isolated by chromatographic methods. The structures of the glycans located on each sequon were determined by a combination of analytical techniques including Smith degradation, permethylation, and enzymic degradation. Approximately 79% of the total glycan from sequon a was of the biantennary type as previously described by Dorland and his colleagues (FEBS Lett. 77, 15-20 (1977)). The remaining 21% consisted of a mixture of triantennary and tetraantennary glycans, each amounting to approximately 10% of the total glycan for this sequon. The triantennary structure resembled that described for the N-glycosidic triantennary glycans of bovine fetuin by Nilsson and his colleagues (J. Biol. Chem. 254, 4545-4553 (1979)). Of the tetraantennary glycan, approximately half of the structures were incomplete, i.e., one antenna terminated by N-acetylglucosamine. On sequon b, 81% of the glycan was biantennary, identical to those biantennary glycans of sequon a, and the reminder was triantennary, also of the fetuin type. The glycan structures and their locations on the polypeptide are related to the known subpopulations of human transferrin.
The role of thyroid gland in cold-induced growth of brown adipose tissue and in cold-induced adaptive changes in brown adipose tissue mitochondria was investigated. Interscapular brown adipose tissue of thyroidectomized rats maintained at 28 °C was of normal size (protein, cytochrome oxidase content) and its mitochondria were normal, as judged from the level of GDP binding and the polypeptide composition. (GDP binds to a 32 000 dalton polypeptide component of the inner mitochondrial membrane. This component is part of the thermogenic proton conductance pathway. The level of GDP binding is a sensitive indicator of the thermogenic state of the tissue.) Brown adipose tissue mitochondria of thyroidectomized rats exposed to 4 °C for 15 h did not show the usual increase in GDP binding, an indication of a lack of a thermogenic response; the response was restored in thyroxine-treated thyroidectomized animals. In thyroidectomized rats treated with a low maintenance dose of thyroxine (25 μg/kg, s.c., three times per week) and acclimated to cold (4 °C) for 2 weeks, a normal growth of brown adipose (increase in protein and cytochrome oxidase) and normal changes in mitochondria (increase in GDP binding and an increase in the proportion of polypeptides of molecular weight 31 200 – 34 400) occurred. Treatment of intact rats with a large dose of thyroxine (1000 μg/kg, s. c., per day) resulted in a large increase in wet weight, mainly due to lipid accumulation since only small increases in protein and cytochrome oxidase and no change in DNA content occurred; mitochondrial GDP binding was decreased and polypeptide composition unchanged. It is concluded that thyroid hormone exerts a permissive effect on the cold-induced, noradrenaline-mediated, unmasking of GDP-binding sites in brown adipose tissue. The failure of the thyroidectomized rat to survive at 4 °C is probably primarily due to its inability to activate thermogenesis in its brown adipose tissue. Thyroid hormone does not appear to be involved, other than in a permissive way, in long-term cold-induced growth and mitochondrial changes in brown adipose tissue, since these occur normally in the presence of only small amounts of thyroxine in thyroidectomized rats and do not occur in intact rats treated with large amounts of thyroxine.
Conformational changes of amphotericin B in the presence of cholesterol as well as in the presence of bilayer vesicles of phosphatidylcholine with saturated fatty acid chains of various lengths (14 less than n less than 22) have been monitored by circular dichroism (CD). It has been shown that the observed species are not only dependent on such parameters as the cholesterol content of the vesicles, the vesicles' physical state, and the number of amphotericin B molecules per vesicle, but also on the time elapsed after mixing and the thermal treatment of the system, which may create irreversible changes. In particular, heating through the transition temperature (Tc) vesicles containing cholesterol and loaded with amphotericin below Tc leads to the expulsion into the aqueous medium of a cholesterol-amphotericin complex, a phenomenon which affords an explanation for some of the electron paramagnetic resonance and resonance Raman results. It has also been shown by gel filtration, ultracentrifugation, and Tc determination that interaction of amphotericin B with vesicles in the gel state induces fusion or aggregation of the vesicles, which is not the case (or at least weakly) when the vesicles are in the liquid crystalline state. This aggregation is the more rapid the nearer the temperature of the reaction is to Tc. This study confirms the great complexity of events which may occur during interaction of amphotericin B with model membranes and presents some results which complement those of studies performed with other spectroscopic methods.
Brown adipose tissue (BAT) of rats is known to grow in response to acclimation to cold. The growth is accompanied by changes in mitochondrial polypeptide composition (an increase in the relative proportion of a polypeptide of molecular weight 32 000, known to be associated with the thermogenic proton conductance pathway). The mediator of the change in mitochondrial polypeptide composition is unknown. The objective of these experiments was to find out whether any of the pituitary hormones might be the mediator. Treatment of rats with growth hormone failed to alter BAT size or mitochondrial polypeptide composition. BAT grew and the change in BAT mitochondrial polypeptide composition occurred in cold-acclimated hypophysectomized rats, maintained on thyroxine and corticosterone to ensure their survival in the cold. It is concluded that none of the pituitary hormones is the mediator for the cold-induced change in BAT mitochondrial polypeptide composition or is required to exert a direct effect on BAT for cold-induced BAT growth to occur. It also seems unlikely that more than a maintenance amount of glucocorticoids is required for normal cold-induced growth of BAT; these hormones are thus also unlikely to mediate the change in BAT mitochondrial polypeptide composition. The requirement for no more than a maintenance amount of thyroxine for BAT growth and for the cold-induced change in BAT mitochondrial polypeptide composition confirms previous conclusions drawn from studies on cold-acclimated thyroidectomized rats.
Cardiac tropomyosin from 20-day-old chick embryos is composed of three different polypeptides with the same molecular weight but different isoelectric points. These polypeptides, which are designated as alpha 1, alpha 2, and alpha 3, have identical peptide maps. In vitro, however, only polypeptide alpha 1 is synthesized in a reticulocyte lysate programmed with cardiac RNA. These results, together with the observations indicating that tropomyosin alpha 2 corresponds to a phosphorylated polypeptide, suggest that only tropomyosin alpha 1 corresponds to a primary translational product and that forms alpha 2 and alpha 3 are derived from alpha 1 as a consequence of posttranslational modifications.
Nuclei and chromatin isolated in the presence of calcium or magnesium from Rana catesbeiana liver tissue exhibit considerable endogenous deoxyribonuclease activity. This activity is present in liver nuclei isolated from froglets as well as in liver nuclei isolated from untreated and thyroid hormone treated premetamorphic tadpoles. Nuclei and chromatin isolated in the absence of divalent cations and in the presence of spermine exhibit no detectable expression of the endogenous deoxyribonuclease activity. The endogenous deoxyribonuclease present, but not expressed, in spermine-isolated tadpole liver nuclei or chromatin is salt extractable. Once dialyzed, the salt-extracted deoxyribonuclease is activated by calcium or magnesium. This deoxyribonuclease shows maximal enzyme activity in 15 mM calcium at pH 8.0 or in 15 mM magnesium at pH 7.4. After Ca2+ activation, deoxyribonuclease activity is maximally inhibited by amounts of spermine similar to that required to completely inhibit DNase I. Destruction of the salt-extracted deoxyribonuclease activity by treatment with proteinase K or heat suggests that it is of a proteinaceous nature. The localization and nature of this enzyme activity established that it is associated with the salt-soluble proteins affiliated with tadpole and froglet liver chromatin.
The ts A1S9 cell is a dnats mutant of mouse L cells which exhibits enhanced sensitivity to killing by the antibiotic novobiocin, as compared with the parental wild-type (WT-4) cell, another dnats mouse L cell (ts C1), and other mammalian cells. Such a response was not detected with other cytotoxic agents tested. Novobiocin inhibits DNA synthesis in ts A1S9 cells, at concentrations which have little or no effect on RNA or protein synthesis. Enhanced novobiocin sensitivity is not due to altered cellular permeability to the drug, since ts A1S9 cells do not take up more antibiotic than do WT-4 or ts+ AR mouse L cells. A ts+ revertant of ts A1S9 cells exhibits a wild-type pattern of novobiocin sensitivity, suggesting that novobiocin resistance may be encoded in the ts A1S9 locus. These findings led to a study of DNA conformation which revealed that the nuclear, chromatin-bound DNA of temperature-inactivated ts A1S9 cells sediments differently in ethidium bromide sucrose density gradients than does that of WT-4 cells grown at 38.5 °C. These observations suggest that expression of the ts A1S9 defect results in a more highly constrained conformation of the DNA, relative to that in control cells, and that in this configuration the DNA may be unable to act as template for replication.
The activation of brown adipose tissue adenylate cyclase by catecholamines was studied in genetically obese (ob/ob) and lean mice. In obese mice, the maximum activation of the enzyme by several beta-adrenergic agonists was only two-thirds that in lean mice and, as an activator, noradrenaline was only one-eighth as potent. The adenylate cyclase was also less responsive to guanine nucleotides. In these respects, the defect in catecholamine-stimulated adenylate cyclase was similar in both white and brown adipose tissue of the obese mouse. The enzyme in brown adipose tissue differed from that in white adipose tissue in its sensitivity to other beta-adrenergic agonists and in its requirement for Mg2+. It is suggested that this abnormal catecholamine-activated adenylate cyclase in brown adipose tissue may be relate to the thermoregulatory defect of the obese mouse and hence may contribute to the obesity syndrome.
Change in RNA during the juvenile hormone (JH) stimulated synthesis of vitellogenin (Vg) in the fat body of adult female Locusta migratoria have been studied. Total RNA from mature females, but not that from males or prereproductive females, shows a 6300 nucleotide component, which has been isolated by binding to oligo(dT)-cellulose and sucrose gradient centrifugation, and identified as Vg mRNA by translation in Xenopus oocytes. It has been assayed quantitatively by photometric scanning after electrophoresis. During a gonotrophic cycle, Vg mRNA increased rapidly from 0 up to about 1% of the total fat body RNA, or more than 10(6) copies per cell. After destruction of the corpora allata (the source of JH) by treatment with ethoxyprecocene, Vg synthesis was stimulated by injection of 150 micrograms of the JH analog, methoprene. In primary stimulation, Vg mRNA was first detected at 24 h and showed a marked lag in accumulation; in secondary stimulation by methoprene after decay of the primary effect, Vg mRNA was detected after only 12 h and accumulation was much more rapid. Both in the natural cycle and in experimental stimulation, Vg mRNA did not disappear in correlation with declining Vg synthesis, which suggests conservation of mRNA in untranslated form. Total (chiefly ribosomal) RNA showed a different pattern, accumulating markedly during primary and only slightly during secondary stimulation. The data indicate that JH acts selectively (though not necessarily directly) on transcription of the Vg genes.
