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PubMed1986

Extraction of adenine nucleotides from cultured endothelial cells.

The goal of this work was to find a method suitable for the extraction of adenine nucleotides from cultured vascular endothelial cells. Extraction of cell monolayers with 80% methanol in water yielded extracts with a higher content of ATP than did extraction of cells with perchloric acid, trichloroacetic acid, or boiling water. The optimal extraction solution was 80% methanol with 0.5 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or EDTA, heated to 70 degrees C immediately before use. Extraction of nucleotides by this solution was rapid and the recovery of exogenous ATP added during the extraction process was generally greater than 90%. An aqueous methanol or ethanol solution may be applicable for the extraction of nucleotides and other metabolites from cultured animal cells, dispersed cells, and frozen, powdered tissues.

Adenine Nucleotides↗
PubMed1989

18,21-Anhydroaldosterone and derivatives.

18,21-Anhydroaldosterone 8, 18,21-anhydro-19-noraldosterone 9, and 3 alpha, 5 beta-tetrahydro-18,21-anhydro-19-noraldosterone 13, which may be present in acid-processed urine, were prepared by cleaving their 20-ketal derivatives 2, 3, and 12 with hot mineral acid. Compounds 8 and 9 were also made by direct dehydration of aldosterone 5 and 19-noraldosterone 10 in good yield. The reverse ring opening of 8 to 5 could be carried out in moderate yield with an acetic acid-acetic anhydride-perchloric acid mixture, while an analogous ring opening of 9 gave only a poor yield of 10.

Aldosterone↗
PubMed2001

Capillary zone electrophoresis of basic analytes in methanol as non-aqueous solvent mobility and ionisation constant.

The electrophoretically relevant properties of monoacidic 21 bases (including common drugs) containing aliphatic or aromatic amino groups were determined in methanol as solvent. These properties are the actual mobilities (that of the fully ionised weak bases), and their pKa values. Actual mobilities were measured in acidic methanolic solutions containing perchloric acid. The ionisation constants of the amines were derived from the dependence of the ionic mobilities on the pH of the background electrolyte solution. The pH scale in methanol was established from acids with known conventional pK*a values in this solvent used as buffers, avoiding thus further adjustment with a pH sensitive electrode that might bias the scale. Actual mobilities in methanol were found larger than in water, and do not correlate well with the solvent's viscosity. The pK*a values of the cation acids, HB-, the corresponding form of the base, B, are higher in methanol, whereas a less pronounced shift was found than for neutral acids of type HA. The mean increase (compared to pure aqueous solution) for aliphatic ammonium type analytes is 1.8, for substituted anilinium 1.1, and for aromatic ammonium from pyridinium type 0.5 units. The interpretation of this shift was undertaken with the concept of the medium effect on the particles involved in the acid-base equilibrium: the proton, the molecular base, B, and the cation HB+.

Electrophoresis, Capillary↗
PubMed2001

Study of nuclear quadrupole interactions in different environments of decaying atoms of 75Se by sum peak method.

The sum peak intensity I400 (279 + 121 keV) relative to its singles peak intensity I279 or I121 has been determined in various environments of 75Se by gamma-gamma summing technique in a single HPGe detector. The change in intensity has been used to determine nuclear quadrupole interaction frequencies and electric field gradient components in different environments, e.g. Se-glycerol, Se-hydrochloric acid (HCl), Se-perchloric acid (HClO4), Se-blood samples with and without anti-coagulant, Se-alanine and Se-cysteine at different pH values. The electric field gradient is found to vary with chemical complex formation.

Journal Article↗
PubMed2002

Label-free determination of picogram quantities of DNA by stripping voltammetry with solid copper amalgam or mercury electrodes in the presence of copper.

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of the DNA sensors. We show that subnanomolar concentrations (related to monomer content) of unlabeled DNA can be determined using copper solid amalgam electrodes or hanging mercury drop electrodes in the presence of copper. DNA is first treated with acid (e.g., 0.5 M perchloric acid), and the acid-released purine bases are directly determined by the cathodic stripping voltammetry. Volumes of 5-3 microL of acid-treated DNA can easily be analyzed, thus making possible the determination of picogram and subpicogram amounts of DNA corresponding to attomole and subattomole quantities of 1000-base pair DNA. Application of this determination in DNA hybridization detection is demonstrated using surface H for the hybridization (superparamagnetic beads with covalently attached DNA probe) and the mercury electrodes only for the determination of DNA selectively captured at surface H.

Copper↗
PubMed2003

Redetermination of hydronium perchlorate at 193 and 293 K.

A sample of hydronium perchlorate, H(3)O(+) x ClO(4)(-), crystallized from ethanol at ambient temperature, was found to be orthorhombic (space group Pnma) at both 193 and 293 K, with no phase transition observed in this temperature range. This contrasts with the earlier observation [Nordman (1962). Acta Cryst. 15, 18-23] of a monoclinic phase (space group P2(1)/n) at 193 K for crystals grown at that temperature from perchloric acid. The hydronium and perchlorate ions lie across a mirror plane but it is not possible to define at either temperature a simple description of the H-atom positions due to the three-dimensional tumbling of the hydronium cation.

Journal Article↗
PubMed1991

F Nuclear Magnetic Resonance Analysis of 5-Fluorouracil Metabolism in Four Differently Pigmented Strains of Nectria haematococca.

F nuclear magnetic resonance spectroscopy was used to study the metabolism of 5-fluorouracil in four strains of Nectria haematococca which displayed similar sensitivities to growth inhibition by this compound but differed in their pigmentation. The major metabolites, 5-fluorouridine and alpha-fluoro-beta-alanine, were excreted into the medium by all four strains. The classical ribofluoronucleotides (5-fluorouridine-5'-monophosphate, -diphosphate, and -triphosphate) and alpha-fluoro-beta-alanine were identified in the acid-soluble fraction of perchloric acid extracts of mycelia. Two hydrolysis products of 5-fluorouracil incorporated into RNA were found in the acid-insoluble pool. They were unambiguously assigned to 5-fluorouridine-2'-monophosphate and 3'-monophosphate with specific hydrolysis reactions on isolated RNA. The lack of fluorodeoxyribonucleotides and the fact that the four strains incorporated similar amounts of fluororibonucleotides into their RNAs strongly suggest an RNA-directed mechanism of cytotoxicity for 5-fluorouracil. The heavily pigmented wild type differed from the three low-pigmented strains in its low uptake of 5-fluorouracil and, consequently, in its reduced biosynthesis of 5-fluorouridine and alpha-fluoro-beta-alanine. At present, it is not clear whether this change in 5-fluorouracil metabolism is a side effect of pigment production or results from another event.

Journal Article↗
PubMed2000

Simple and sensitive binding assay for measurement of adenosine using reduced S-adenosylhomocysteine hydrolase.

BACKGROUND: Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [(3)H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form. METHODS: SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid). RESULTS: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 +/- 2 nmol/L. Displacement of adenosine binding by the adenine 5'-nucleotides, adenine and hypoxanthine, required >1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC. CONCLUSIONS: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.

Adenosine↗
PubMed1987

[Selenium concentration in the cerebrospinal fluid of children].

Following a wet digestion of 0.5-2.0 ml cerebrospinal fluid in an open system using 2.0 ml nitric acid and 1.0 ml perchloric acid (240 degrees C) and a reduction step with 1.0 ml hydrochloric acid, Selenium can be determined polarographically after adding 100 micrograms Copper(II)-ions to the analyte (15 ml; water/perchloric acid). Selenium concentrations in cerebrospinal fluid of children younger than one year (2.49 +/- 1.67 ng/ml) are significantly higher (p = 0.0074) than those of older children (1.28 +/- 0.97 ng/ml). Independent of the childrens age and diseases the Selenium concentrations correlate distinctly with cell numbers and protein contents. A correlation between Selenium content and cell numbers alone could not be proved. The non-significant differences between the Selenium concentrations in cerebrospinal fluids of children with hydrocephalus, leukemia (with or without involvement of the central nervous system), and other diseases, respectively, may be interpreted by considering the protein content of the cerebrospinal fluid and the age of the children.

Adolescent↗
PubMed1987

[Selenium concentration in the cerebrospinal fluid of children].

Following a wet digestion of 0.5-2.0 ml cerebrospinal fluid in an open system using 2.0 ml nitric acid and 1.0 ml perchloric acid (240 degrees C) and a reduction step with 1.0 ml hydrochloric acid, Selenium can be determined polarographically after adding 100 micrograms Copper(II)-ions to the analyte (15 ml; water/perchloric acid). Selenium concentrations in cerebrospinal fluid of children younger than one year (2.49 +/- 1.67 ng/ml) are significantly higher (p = 0.0074) than those of older children (1.28 +/- 0.97 ng/ml). Independent of the children age and diseases the Selenium concentrations correlate distinctly with cell numbers and protein contents. A correlation between Selenium content and cell numbers alone could not be proved. The nonsignificant differences between the Selenium concentrations in cerebrospinal fluids of children with hydrocephalus, leukemia (with or without involvement of the central nervous system), and other diseases, respectively, may be interpreted by considering the protein content of the cerebrospinal fluid and the age of the children.

Adolescent↗
PubMed1979

A gas chromatographic method for determination of acetate levels in body fluids.

A simple, sensitive and accurate gas chromatographic method for the determination of acetate in plasma and urine is described. Following precipitation of proteins by perchloric acid and removal of potassium perchlorate by precipitation with KOH, acetic acid is quantitated by injection of the acidified sample on a highly polar column. A flame ionization detector is used. Propionic acid is added to the sample initially as internal standard. The sample volume required for the analysis is 0.5 ml. The standard curve is linear in the range from 0.01 to 10 mmol/l. Recovery of added acetate is complete, and the coefficient of variation is less than 4% even at low concentrations. The concentration of free acetate in plasma from 10, and urine from 4, healthy humans ranged from 0.04 to 0.07 and 0.09 to 0.24 mmol/l, respectively.

Acetates↗
PubMed1979

Plant histone 2 from wheat germ, a family of histone H2a variants. Partial amino acid sequences.

1. The 0.5 M perchloric acid extract prepared from chromatin of wheat germ, Triticum aestivum, contains a group of histones formerly called plant histones. These can be resolved by gel filtration on Bio-Gel P-60 with subsequent CM-cellulose ion-exchange chromatography into five histone fractions containing families of histones H2A and H2B. 2. The partial amino acid sequences of histone H2A variants H2A(1)Triticum, H2A(2)Triticum and H2A(3)Triticum are presented. Extensive sequence homology exists between calf thymus histone H2A and wheat embryo H2A histones. Differences are largely due to conservative amino acid substitutions and in two of the variants, viz. H2A(2) and H2A(3) to N-terminal extensions of the polypeptide chains.

Amino Acid Sequence↗
PubMed1986

Effect of matrix modification by strong mineral acids on the positive fast atom bombardment mass spectra of peptides.

We report an investigation of the effect of the addition of strong mineral acids to the most commonly used matrices for fast atom bombardment mass spectrometry of peptides. Among the acids tested we found that perchloric acid has a beneficial effect on: the sensitivity, the stability of quasi-molecular ion signals. Furthermore, in the presence of basic residues, ions related to perchlorate adducts are present. These further signals may be useful in differentiating peptides containing lysine from those containing the isobaric glutamine.

Acids↗
PubMed1978

A comparison of methods for blocking staining of nucleic acids.

Using rat spinal cord as test material 4 methods for blocking staining of nucleic acids with simple basic dyes have been compared. Two of these methods--a perchloric acid procedure for tissue blocks and a Ba(OH)2 technic for sections--block staining of RNA. The other two-immersion of sections in solutions of dichlorodiammine Pt II or in 10% zirconyl chloride-blocked staining of both RNA and DNA. None of these methods hydrolyze DNA sufficiently to produce a positive Feulgen and no loss of protein could be demonstrated. With the three metal containing procedures some metal could be demonstrated in the sections. This deposition was primarily in nucleic acid containing structures and was greatest and most widespread with the zirconyl chloride. The latter method altered the color of all hematoxylin technics to a deep red. The separation of nuclear stains on the basis of their bonding characteristics into three groups (Lillie et al. 1976) was confirmed.

Animals↗
PubMed1987

Measurement of ascorbic acid and dehydroascorbic acid in gastric juice by HPLC.

New methods are presented for measuring total vitamin C and the ascorbic acid/dehydroascorbic acid ratio in gastric juice. Extracts are prepared from a gastric juice which are suitable for direct injection onto a Waters Nova-pak C18 Radial-pak cartridge for high performance liquid chromatography (HPLC) using ultraviolet absorbance at 270 nm for detection. Both enable removal of interfering mucus and mucopolysaccharide breakdown products in a novel way. The first uses mini-columns of Sephadex G-50, run in acidic conditions to remove large molecular weight material while maintaining the ascorbic acid/dehydroascorbic acid ratio as it was in the fresh sample. Addition of dithiothreitol converts the dehydroascorbic acid quantitatively to ascorbic acid, thus enabling measurement of both components. The second method converts all the dehydroascorbic acid to ascorbic acid at the outset. A perchloric acid extract is neutralized and passed through a Sep-Pak C18. A new internal standard, reductic acid, is introduced for ascorbic acid analysis which behaves identically on Sep-Pak C18. Samples are analysed by ion-pair chromatography using 0.02 M NH4H2PO4 buffer (pH 7.1): methanol (80:20 v/v) containing 0.62 g/L tetrapentylammonium bromide. The detection limit was 1 ng ascorbic acid, and chromatography was completed in 5 min. The values obtained by the two independent HPLC methods were in good agreement with each other and with those obtained by the 2,4-dinitrophenylhydrazine colorimetric method.

Ascorbic Acid↗
PubMed1997

Hrp12, a novel heat-responsive, tissue-specific, phosphorylated protein isolated from mouse liver.

Previous studies from this laboratory identified a 28-kd nonreducible protein, liver-derived immunoinhibitory factor (LDIF) from the mouse liver. Isolation of this protein resulted in the co-purification of another unique protein called heat responsive protein 12 kd (Hrp12). In contrast to LDIF, Hrp12 was totally reducible to a protein of 12 kd suggesting a dimer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) purification, followed by sequencing of an in situ cyanogen bromide digest of membrane bound Hrp12, yielded an internal 20-amino acid polypeptide. Degenerate oligonucleotides made from this peptide were used to screen a murine liver complementary DNA (cDNA) library. A 1240-bp cDNA clone was obtained with an internal 521-bp open reading frame (ORF). Sequence analysis of the 173-amino acid ORF of mouse Hrp12 showed a high degree of homology with a 99 amino acid rat liver-kidney perchloric acid-soluble protein (LKPS) and a 136-amino acid perchloric acid soluble rat protein (PSP). Transcripts for Hrp12 were mainly restricted to the liver and kidney in mouse and man. The protein was estimated to be approximately 0.8% of the total liver-soluble cytosolic protein. A zoo-blot probed at moderate stringency with labeled cDNA revealed a strong conservation of the gene in all of the mammalian species tested. Analysis of the protein structure of Hrp12 revealed motifs predicted to be targets for protein kinase C (PKC). More importantly, purified mouse Hrp12 could be phosphorylated in vitro with PKC. The protein had significant similarity to DnaK heat shock protein (Hsp)70 and contained a 54-amino acid stretch with sequence similarity to Hsp90. This prompted us to investigate the heat shock response of Hrp12. Isolated hepatocytes and hepatoma cells were exposed to different heat shock temperatures (39.5 degrees C, 42.5 degrees C, and 44.5 degrees C); and then total RNA was extracted and Northern analysis carried out. The message for this novel protein responded atypically to heat shock. Although the steady-state level of the message increased after heat shock, a marked oscillatory pattern was superimposed on it. In contrast, the steady-state levels of Hsp90 and Hsp70 messenger RNA (mRNA) were found to respond to heat shock in the expected manner. Finally, the amount of Hrp12 protein was also found to increase after heat shock in a manner that was consistent with heat-responsive proteins.

Amino Acid Sequence↗