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[Food conversion in the digestive system of juvenile bulls. 2. Digestion of carbohydrates].

Each of ten rations containing 60% feed concentrate were tested on two bulls which were a fistulated at the duodenum. The carbohydrate contents varied considerably. Carbohydrate digestion in the forestomach was estimated by the detergent and Anthron methods and by quantitative gas chromatographic analysis for carbohydrate monomers. Apparent starch digestibility for starch-rich rations varied between 97.8 and 99%. Between 60 and 144 g of starch was found at the duodenum/animal.d in the case of the cereal-containing rations, the highest values being found for the one containing maize. The apparent digestibility of sugar was about 99%. The amount of sugar found at the duodenum varied between 16 and 67 g animal.d. Analysis by gas chromatography revealed that the feeds yielded less hemicelluloses than calculated from the difference NDF-ADF. The only other sugars found in appreciable amounts at the duodenum were arabinose and xylose.

Animal Feed↗

The lysosome periphery: biochemical and electrokinetic properties of the tritosome surface.

Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO(3), pH 7.2 at 25 degrees C the tritosomes had an electrophoretic mobility of -1.77 +/- 0.02 microm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm(2), and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10(-7) m. Treatment of the tritosomes with 50 microg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 +/- 0.02 microm/s/V/cm under the same conditions and caused the release of 2.01 microg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 microg (11.4 nmol)/mg tritosome protein. A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 +/- 3; glucosamine, 24 +/- 3; galactosamine, 10 +/- 2; glucose, 21 +/- 2; galactose, 26 +/- 2; mannose, 31 +/- 5; fucose, 7 +/- 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.

Animals↗

The chemistry of insect hemolymph; organic components of the hemolymph of the silkworm, Bombyx mori, and two other species.

1. Hemolymph was collected for analysis from the silkworm, Bombyx mori, in a series of developmental stages ranging from the second molt to the late pupa. The mean pH of larval hemolymph after collection was found to be 6.45, that of pupal hemolymph, 6.57; in vivo values may be slightly lower. Total dry solids ranged from 5.4 to 10.6 per cent. Total protein ranged from 1.2 to 5.3 per cent, increasing rapidly during the fifth instar. 2. Free amino acids were separated chromatographically and estimated. Of 19 amino acids identified, amounting collectively to 823 to 1497 mg. per 100 ml., glutamine, histidine, and lysine generally occurred in greatest amount. Tryptophan was not detected, and cystine (or cysteine) was found in only one sample. The total free amino acids account for 35 to 55 per cent of the non-protein nitrogen of the plasma. 3. Free sugars, estimated semiquantitatively on chromatograms, comprise glucose, fructose, and sucrose in total amount ranging from about 5 to 40 mg. per 100 ml. Total acid-soluble, ultrafiltrable carbohydrate, estimated as glucose by the anthrone reaction, ranged from 166 to 635 mg. per 100 ml., indicating the presence of low molecular weight sugar derivatives. 4. Inorganic phosphate amounted to 5 to 15 mg. per 100 ml., and acid-soluble organic phosphate to 100 to 200 mg. per 100 ml. The latter fraction includes several substances, of which one was tentatively identified as glucose-6-phosphate and the remainder are as yet unidentified. 5. Single samples of hemolymph were also taken from larvae of the wax moth, Galleria mellonella, and the spruce sawfly, Diprion hercyniae. These contained even higher concentrations of solutes than the silkworm samples, but with a generally similar distribution. The proportions of the free amino acids were different in each species.

Amino Acids↗

The chemistry of insect hemolymph. II. Trehalose and other carbohydrates.

alpha,alpha-Trehalose, a sugar previously regarded as a product characteristic of certain lower plants, has been identified as a major blood sugar of insects. Trehalose has been isolated in pure form from the blood of pupae of the silk moth, Telea polyphemus, and has been recognized chromatographically in all the insects examined, which comprise 10 species belonging to 5 different orders. Trehalose has been determined quantitatively with anthrone after either chromatographic separation or chemical degradation of other sugars. In larvae and pupae of 4 species of Lepidoptera it ranges from 0.2 to 1.5 gm. per 100 ml. of blood and makes up over 90 per cent of the blood sugar; in larvae of a sawfly, about 80 per cent of the blood sugar is trehalose. In Bombyx mori and Platysamia cecropia, the pupal blood trehalose level is about half that in the mature larva, suggesting utilization of trehalose for glycogen synthesis during pupation. Small amounts of glucose and apparent glycogen are also present in the plasma of these insects. In Bombyx larval plasma there is also 0.04 to 0.12 gm. per 100 ml. of glucose-6-phosphate and smaller amounts of an apparent ketose phosphate.

Animals↗

Quantitative fecal carbohydrate excretion in premature infants.

Despite relative lactase deficiency and pancreatic insufficiency, premature infants are fed formulas containing 50% lactose plus 50% glucose polymers. We measured total fecal carbohydrate excretion in six healthy 32-wk gestation premature infants who had been fed two 0.784-kcal/g formulas that were similar except for the carbohydrate source (100% lactose vs 50% lactose plus 50% glucose polymers). Using a cross-over design with the first formula randomly assigned, two 72-h balance studies were performed with carmine red, an intermittent external marker, and polyethylene glycol (PEG), a continuous internal marker. Formula and stools were analyzed for total carbohydrate (anthrone method) and PEG. There were no significant differences between the two formula periods for carbohydrate intake, mean daily stool output, or fecal carbohydrate excretion. Mean fecal carbohydrate excretion was less than 0.2 g/d, or less than 1% of carbohydrate intake. Thus, older (32-wk gestation) premature infants fed either 100% lactose or 50% lactose plus 50% glucose polymers have minimal fecal losses of intact carbohydrate.

Carbohydrates↗

Enhanced induction of epidermal ornithine decarboxylase activity in C57BL/6 compared to DBA/2 mice by protein kinase C-activating skin tumor promoters: relevance to genetically mediated differences in promotion susceptibility.

Previous work from our laboratory demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) or a synthetic diacylglycerol induced significantly higher epidermal ornithine decarboxylase (ODC) activity in C57BL/6 than in DBA/2 mice. To understand further the genetic basis for this strain difference, two tumor promoters were evaluated for their effects on epidermal ODC activity: teleocidin, which activates protein kinase C (PKC); and 1,8-dihydroxyl-3-methyl-9-anthrone (chrysarobin), which does not. In addition, the ODC induction response in B6D2F1 offspring and BXD recombinant inbred (RI) strains was examined following multiple treatments with TPA. A single topical application of teleocidin to mouse dorsal skin led to the hyperinduction of epidermal ODC activity in C57BL/6 mice. In contrast, while chrysarobin induced epidermal ODC activity, no significant differences in the magnitude of this response were observed in SENCAR, DBA/2 or C57BL/6 mice. Consistent with our previous findings, the magnitude of ODC induction by teleocidin in these three mouse lines (C57BL/6 greater than SENCAR greater than DBA/2) did not correlate with their susceptibility to tumor promotion by TPA (SENCAR greater than DBA/2 greater than C57BL/6). ODC activity induced by multiple application of TPA in B6DF1 mice, whose susceptibility to phorbol ester tumor promotion is inherited as an incomplete dominant trait, was comparable to that induced in C57BL/6 mice at all the doses examined. Cluster analysis of TPA-induced ODC activity in BXD RI strains allowed us tentatively to group them into four or five phenotypes and to estimate a minimum of two genetic loci controlling TPA-induced ODC activity. Furthermore, in BXD RI strains, there was no apparent relationship between the magnitude of ODC induction and responsiveness to tumor promotion or sustained hyperplasia. Collectively, these results suggest that hyperinducibility of ODC in response to PKC-activating tumor promoters is inherited as an autosomal dominant trait, and that genetic determinants for ODC induction, at least in C57BL/6 and DBA/2 mice, appear completely independent of those controlling tumor promotion susceptibility.

Animals↗

Regression and progression characteristics of papillomas induced by chrysarobin in SENCAR mice.

The present study was designed to test the effects of a free radical generating tumor promoter, chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone), on the growth and progression of papillomas generated in the skin of SENCAR mice. In the first set of experiments, papillomas were generated by initiation with 6.4 microg of 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with once-weekly applications of 52.8 microg chrysarobin for 10 weeks. The fate of individual papillomas was then monitored for a 20 week interval following cessation of promoter treatment. Five weeks after the cessation of chrysarobin treatment, the papilloma response reached a maximum of 13.2 papillomas/mouse. By the end of the 20 week interval 19% and 18% of the papillomas had regressed or coalesced respectively. A three-stage treatment protocol was also utilized to test the ability of chrysarobin to enhance the progression of pre-existing papillomas to squamous cell carcinomas (SCCs). In stage I, mice were initiated with 0.5 microg of DMBA. In stage II, mice were promoted with twice-weekly applications of 1 or 2 microg of 12-0-tetradecanoylphorbol-13-acetate (TPA) for 15 weeks. Then, in stage III, mice were treated with acetone, TPA (1 or 2 microg), chrysarobin (52.8 microg) or benzoyl peroxide (BzPo; 20 mg) for the next 45 weeks. The mean number of papillomas per mouse at plateau was very similar for all groups. The carcinoma incidence was also similar for all groups regardless of the treatment protocol used, as was the mean number of carcinomas per mouse. The ratio of papillomas that converted to SCCs in mice treated with chrysarobin during stage III was not significantly different from the acetone controls or any of the other treatment groups (P > 0.05, Kruskal-Wallis analysis). In addition, BzPo did not enhance the progression of papillomas to SCCs under the current experimental conditions. Collectively, the results indicate that papillomas promoted by chrysarobin have growth properties similar to those promoted by TPA under similar experimental conditions. Furthermore, despite its ability to generate free radical intermediates, chrysarobin does not enhance the malignant progression of pre-existing papillomas induced by TPA treatment.

9,10-Dimethyl-1,2-benzanthracene↗

Comparison of the histological changes in the skin of DBA/2 and C57BL/6 mice following exposure to various promoting agents.

The effects of multiple applications of 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 6.8 nmol), teleocidin (6.8 nmol), 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin, 220 nmol), mezerein (6.8 nmol), 4-O-Methyl-TPA (4-O-Me-TPA, 150 micrograms) and benzoyl peroxide (BzP, 20 mg) on the skin of DBA/2 and C57BL/6 mice were studied histologically. After four applications of TPA given over a 2-week period, the epidermis of DBA/2 mice showed a marked epidermal hyperplasia and the presence of a much greater number of dark basal keratinocytes (DCs) 48 h after the last treatment compared with C57BL/6 mice treated with a similar dose and protocol. A marked dermal infiltration of polymorphonuclear leukocytes (PMNs) was observed in DBA/2 mice 48 h after the last application of TPA, whereas little PMN infiltration was observed in skin of C57BL/6 mice. At 96 h after the last application of TPA, DBA/2 mice still showed a much greater degree of epidermal hyperplasia than C57BL/6 mice. PMNs were virtually absent in the dermis of both DBA/2 and C57BL/6 mice by 96 h after the last TPA treatment. Interestingly, treatment of both strains of mice with multiple applications of teleocidin induced a marked epidermal hyperplasia, a high percentage of DCs and a high labeling index (LI), similar to that observed in DBA/2 mice 48 h after the last treatment. Chrysarobin (given once-weekly for 4 weeks) induced a moderate sustained hyperplasia and DC response 48 h after the last treatment in both DBA/2 and C57BL/6 mice; however, C57BL/6 mice showed a greater epidermal hyperplasia than DBA/2 mice. Chrysarobin induced a significant infiltration of PMNs into the dermis of DBA/2 mice whereas in C57BL/6 mice there was only a slight dermal infiltration of PMNs. Mezerein (given twice-weekly for 2 weeks) induced a moderate epidermal hyperplasia, DC response and LI of similar magnitude in both DBA/2 and C57BL/6 mice, but did not induce PMN infiltration in either strain. BzP and 4-O-Me-TPA (given twice-weekly for 2 weeks) induced only a weak sustained epidermal hyperplasia, DC response and LI of similar magnitude in both strains of mice, and there was little, if any, dermal infiltration of PMNs either 48 or 96 h after the last treatment. Examination of the relationship between the extent of induced hyperplasia and the DC response showed an excellent linear correlation whereas the extent of PMN infiltration into the dermis was not well correlated with either parameter.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Female Aedes aegypti (Diptera: Culicidae) in Thailand rarely feed on sugar.

Female Aedes aegypti (L.) of two different body sizes and provided with different diets (20% sucrose, water only, or 20% sucrose + human blood) were marked and released together in a rural Thai village. Recaptured marked and unmarked (wild) adults were tested for fructose by the cold-anthrone reaction. Both released and wild females showed a low frequency of sugar feeding in nature; both small-bodied and large-bodied individuals failed to sugar feed significantly. Marked females released with sugar in their crop utilized this energy source over the following 2-3 d but failed to replenish it. In contrast, about one-third of wild, resting males showed evidence of recent sugar feeding. This indicated that the absence of plant sugar in females was not caused by a shortage of nectar sources in this rural domestic environment. Presumably, the nonutilization of plant sugar as an energy source increases the frequency of blood feeding and, therefore, the vectorial capacity of Ae. aegypti.

Aedes↗

Sugar feeding by Culicoides mississippiensis (Diptera: Ceratopogonidae) on the yaupon holly, Ilex vomitoria.

Adult Culicoides mississippiensis Hoffman were collected from 5 flowering yaupon holly plants at sunrise, late morning, early afternoon, and sunset from 5 flowering yaupon holly plants during the entire flowering season (16 March-15 April 1995). Individual insects were tested for fructose by using the cold anthrone test. Prevalence of fructose in C. mississippiensis was 55.6% (427 of 768), with positivity for gravid females greater than for males. Fructose positive rates decreased in gravid females from morning to evening, whereas male rates were constant until evening, when they decreased. Both sugar feeding by gravid females and host-seeking by parous females were highest at sunset, followed by sunrise.

Animals↗

A determinant of resistance of Neisseria gonorrhoeae to killing by human phagocytes: an outer membrane lipoprotein of about 20 kDa with a high content of glutamic acid.

A protein of about 20 kDa was extracted by sodium cholate (1%, w/v) from outer membranes of a strain of Neisseria gonorrhoeae, BS4 (agar), which is resistant to killing by human phagocytes. When the protein was purified by repeated fractionation on Sephadex G75, contamination with other outer-membrane proteins and lipopolysaccharide was negligible. The protein contained a full complement of amino acids, with high levels of glutamic acid. Carbohydrate, detected by the anthrone method and by sugar and hexosamine analysis, was present, but at very low levels. There was a significant content of fatty acids (about 5.7% of the protein), indicating a lipoprotein. The 20 kDa lipoprotein: (1) neutralized the ability of antiserum against whole organisms of BS4 (agar) to reduce the resistance of this strain to phagocyte killing; (2) evoked in mice an antiserum which reduced this resistance and immunoblotted only with 20 kDa lipoprotein in the cholate extract of outer membranes; and (3) promoted resistance to intracellular killing of an otherwise phagocyte susceptible gonococcal strain (BSSH). This is strong evidence that it is a determinant of gonococcal resistance to phagocyte killing.

Bacterial Outer Membrane Proteins↗

Purification and characterization of the extracellular aspartyl proteinase of Candida albicans: removal of extraneous proteins and cell wall mannoprotein and evidence for lack of glycosylation.

Aspartyl proteinase (AP) is an extracellular enzyme of Candida albicans implicated as a pathogenic factor. Previous reports on the purification and characterization of AP suggested that a single DEAE-Sephadex chromatographic step was sufficient for the removal of extraneous proteins and that the final product was glycosylated. We purified AP using a chromatographic series consisting of DEAE-Sephadex A25, Sephadex G75 and rechromatography on DEAE-Sephadex A25. Use of DEAE-Sephadex alone did not remove extraneous proteins and removed little contaminating mannoprotein (MP). The addition of a Sephadex G75 column to the purification scheme removed the majority of contaminating MP and proteins. The final DEAE-Sephadex A25 chromatographic step resulted in (a) removal of detectable extraneous proteins, (b) removal of immunologically detectable MP by dot blot and Western blot enzyme immunoassay, (c) loss of periodic acid-silver stain positivity, and (d) a high AP yield (1295 U l-1) and specific activity (1749 U mg-1). We conclude that a single DEAE-Sephadex A25 purification step is insufficient to remove extraneous proteins and MP, which could interfere with the production of AP-specific antibodies and the dissection of moieties responsible for immune reactivity. Reports of periodic acid-Schiff or anthrone positivity of AP preparations may reflect the presence of extraneous MP, which can be removed by the chromatographic series we describe.

Aspartic Acid Endopeptidases↗

Esters of indole-3-acetic Acid from Avena seeds.

The present studies showed that about 80% of the indole-3-acetic acid extractable from Avena kernels by aqueous acetone was esterified to polymers precipitable by ammonium sulfate and ethanol or acetone. The polymers were positively charged, being adsorbed to cation exchange columns at a pH of 3, or below, and eluted at a pH greater than 4. The polymers were heterogeneous with respect to size, about 5,000 to 20,000 daltons, and charge, exhibiting apparent pK(a) values of 4.2 and 4.7. The polymer fractions contained esterified IAA, anthrone-reactive material that liberated glucose upon acid hydrolysis, phenolic compounds, and peptidic material with a high proportion of hydrophobic amino acids. Since the esterified IAA was unstable, establishing polymer purity was not possible, and the designation IAA-glucoprotein fraction was adopted.Dehusked Avena kernels contained 8 mg/kg total IAA of which 5.5% was free and 94.5% esterified. IAA bound through a peptidic linkage was present, but in only trace amounts.

Journal Article↗

A technique for collection of exudate from pea seedlings.

Ethylenediaminetetraacetic acid (EDTA), at concentrations higher than 1.0 millimolar, is phytotoxic to etiolated seedlings of Pisum sativum. Substantial vascular exudation from pea epicotyls could be obtained without tissue damage at 0.5 millimolar EDTA if the solution was buffered at pH 7.5 with sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. Treated seedlings exuded 950 micrograms (leucine equivalents) of ninhydrin-positive material per day and 870 micrograms (glucose equivalents) of anthrone-positive material per day. Amino acid analysis showed the exudate to have glutamine as the major amido nitrogen containing compound and sucrose was shown to be the major sugar. Radiolabeled tryptophan and sucrose applied to cotyledons were transferred through the epicotyl and into the collection medium. The pH profile for exudation shows half maximal exudation at pH 7.2, indicating the promotion of exudation by EDTA is probably not due simply to Ca2+ chelation.

Amino Acids↗

Hydrogen-bonded dimers of 1,8,10-trihydroxy-10-(prop-2-enyl)anthracen-9(10H)-one: S(6), R(2)(1)(10) and R(2)(2)(14) motifs.

The central ring of the anthrone system in the title compound, C(17)H(14)O(4), has a shallow envelope conformation, and each of the two outer rings is inclined at an angle of 17.41 (3) degrees. In the solid state, the molecules exist as centrosymmetrically related O-H.O hydrogen-bonded dimers. Two intramolecular O-H.O hydrogen bonds, involving the central carbonyl O atom and having a graph-set motif of S(6), are observed. These intramolecular interactions lead co-operatively to an O-H.O.H-O pattern that has a binary graph-set motif of R(2)(1)(10).

Journal Article↗

A diiodo-substituted 9-anthracenone.

The central ring of the anthrone system in 1,8-diiodo-10-hydroxy-10-(3-methylbut-3-en-1-ynyl)anthrac en-9(10H)-one, C19H12I2O2, has a boat conformation and the two outer rings form a dihedral angle of 41.1 (2) degrees. The I-C bond lengths are 2.094 (4) and 2.083 (4) A. The hydroxyl and carbonyl groups form an intermolecular hydrogen bond having an O...O distance of 2.853 (4) A.

Anthracenes↗

Purification and some characteristics of the human epidermal SH-protease inhibitor.

An inhibitor of papain and other SH-proteases was purified 520-fold from human epidermis extracts by acetone fractionation, heat treatment, papain-Sepharose affinity chromatography, and Sephadex G-50 chromatography. The purified inhibitor had a molecular weight of 12,600 and contained no hexose, as tested by the anthrone reaction. The inhibitor survived in a boiling water bath, in 5% trichloroacetic acid, 20 mM Na3PO4 (pH 12.1) and 4 M NH4OH (pH 11.9). By isoelectric focusing 2 major activity peaks with pI's of 4.6 and 4.8, and a minor peak with a pI of 4.9 was fractioned, and 3 corresponding protein bands were seen after analytical isoelectric focusing. Immunization of rabbits with the purified inhibitor yielded a highly specific anti-inhibitor serum. The purified inhibitor inhibited papain, ficin, human cathepsins B and C, and slightly inhibited bromelain. No inhibition of serine proteases (bovine trypsin and chymotrypsin A, porcine elastase) or an acid protease (human cathepsin D) was observed. Evidence was obtained that the inhibitor formed a complex with both dithiothreitol-activated papain and enzymatically inactive mercuripapain.

Epidermis↗

Investigations of anthralin free radicals in model systems and in skin of hairless mice.

The antipsoriatic compound anthralin (1.8-dihydroxy-9-anthrone) is converted in skin into several oxidized products, including persistent free radicals that are not well characterized. Anthralin oxidation was investigated by electron spin resonance spectroscopy in a biological system and in a chemical system. Free radical formation in the skin of hairless mice is reduced by the antioxidant tocopherol. The data indicate that tocopherol acts by interfering with free radical formation rather than by scavenging persistent anthralin radicals directly. The skin radicals do not correspond to 10-anthranyl, the initial paramagnetic anthralin oxidation product. Similar radicals obtained in skin are formed by anthralin exposed to ultraviolet light or alkaline solution and by mitochondria and microsomes. The pertinent skin radical is attributed to products derived from anthralin dark structures, the final oxidation products of anthralin. It is suggested that resonance stabilized, paramagnetic polycyclic hydrocarbons are the compounds detected. Their stability and low reactivity indicate a low potential for cutaneous irritation and tumor promotion. Reactive oxygen species, which have been reported to be formed concomitantly during oxidation of anthralin, and the initially formed highly reactive 10-anthranyl radical are more potent candidates for mediating tumor promotion and inflammation.

Animals↗