Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification

The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.     

An adaptation of the filter paper disk technique for the differential estimation of DNA and RNA of rat liver

A method is described for the differential estimation of DNA and RNA from rat liver on filter paper disks. To differentiate between DNA and RNA, samples are subjected to alkaline hydrolysis in microtitration plates prior to absorption on the disks. This procedure eliminates the loss of DNA which is observed if the disks are subjected to RNA hydrolysis after the sample has been absorbed on disks. The procedure is directly comparable to standard methods for the measurement of nucleic acids.     

The presence of 5-hydroxymethylcytosine in animal deoxyribonucleic acid

A method is given for small-scale preparation of DNA from 1.0-1.5g of adult rat tissues. The product from brain or liver is characterized by base ratios and phosphorus content which accord with reported values for rat tissue. It is reasonably free of RNA, protein and glycogen. It contains 5-hydroxymethylcytosine at a content of about 15% of the total cytosine bases present. 5-Hydroxymethylcytosine is also demonstrable in mouse and frog brain DNA and in the crude cytidylic acid fractions obtained from RNA hydrolysates of rat brain and liver. 5-Hydroxymethylcytosine is identified by paper chromatography, u.v. spectra in acid and alkaline solutions and by its conversion into 5-hydroxymethyluracil.     

A nonradioactive method for measuring DNA damage and its repair in nonproliferating tissues

The fluorometric assay of Kissane and Robins has been modified to monitor DNA in alkaline sucrose gradient fractions. Using this procedure the sedimentation analysis of DNA not only of liver, but also of brain, thymus, lung, pancreas, kidney, and skin was carried out. Like liver DNA, DNA released by the alkaline lysis of the above organs sedimented as heavy DNA (> 1 × 10⁹ daltons). A good correspondence was obtained for the sedimentation profiles of liver DNA whether DNA in the gradient fractions was determined by the fluorometric method or by measuring radioactivity.Using the fluorometric assay, the strand breaks not only of liver DNA but also of brain and kidney DNA have been demonstrated following the intravenous administration ofN-methylnitrosourea. Carcinogen-induced DNA damage and repair (as measured by sedimentation of DNA in alkaline sucrose gradients) in any organ including human biopsy specimens are potentially measurable by this procedure.     

Relationship between tissue levels of S-adenosylmethionine, S-adenylhomocysteine, and transmethylation reactions.

The concentrations of S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and various methyltransferases were determined in the cerebrum, cerebellum, and liver of rats during development and aging. The liver contained from 3 to 7 and from 10 to 15 nmol AdoHcy per gram in young and adult rats, respectively. The AdoMet concentration was 60 to 90 nmol/g liver from rats of the same age and sex. It did not vary significantly with age. In the brain the AdoMet concentration was 45 to 50 nmol/g at birth and decreased to 20 nmol/ g tissue with maturity of the organ. The level of AdoHcy in this organ was less than 1 nmol/g tissue throughout the life-span of the rat. Since the ratio of AdoMet to AdoHcy is relatively high, the rate of methylation of histones, DNA, or phosphatidylethanolamine in the liver or brain was not significantly influenced by AdoHcy. Under normal nutritional conditions, the tissue concentration of AdoMet is far above the Km values of histone and phosphatidylethanolamine methyltransferases. The levels of activity of these enzymes in liver and brain did not correlated with the cellular concentration of AdoHcy. Thi histone methyltransferase activity was elevated in rapidly proliferating tissues and declined markedly in the absence of histone biosynthesis. Phosphatidylethanolamine methyltransferase activity was elevated during development of the liver. The specific activity of the AdoHcy hydrolase remained relatively constant in the rat brain and liver. The activity of this enzyme was 10 times higher in liver than in brain, yet the concentration of AdoHcy was much lower in the latter organ. The tissue levels of this compound are evidently dependent on the rates of removal of homocysteine and adenosine. Adenosine deaminase was present in the liver and brain at relatively high concentrations, particularly during development.     

An improved method for identifying 8-O-acetyl-N-acetylneuraminic acid

The periodic acid/thionin-Schiff/potassium hydroxide/periodic acid/fuchsin-Schiff sequence developed by Culling et al. frequently causes damage to sections and gives inconsistent results because of insufficient primary oxidation and difficulties in making the thionin-Schiff reagent. These disadvantages have been largely eliminated by more thorough primary oxidation and by replacing the original thionin-Schiff with a new cold thionin-Schiff. The effect of alkaline hydrolysis on thionin-aldehyde complexes was also studied and the reduction of color caused by this treatment was restored by a second thionin-Schiff reaction. The new sequence gives consistent results and imparts greater color to the thionin-Schiff reaction.     

An Improved Method for Identifying 8-O-Acetyl-N-Acetylneuraminic Acid

The periodic acid/thionin-Schiff/potassium hydroxide/periodic acid/fuchsin-Schiff sequence developed by Culling et al. frequently causes damage to sections and gives inconsistent results because of insufficient primary oxidation and difficulties in making the thionin-Schiff reagent. These disadvantages have been largely eliminated by more thorough primary oxidation and by replacing the original thionin-Schiff with a new cold thionin-Schiff. The effect of alkaline hydrolysis on thionin-aldehyde complexes was also studied and the reduction of color caused by this treatment was restored by a second thionin-Schiff reaction. The new sequence gives consistent results and imparts greater color to the thionin-Schiff reaction.     

Coefficients of distribution of cyclic polyether macrotetrolide antibiotics between hydrophobic and hydrophilic solvents

Dependence of distribution of 14C-macrotetrolide antibiotics between water and chloroform on the presence of various additives in the aqueous phase was studied with the radioindicator procedure. It was shown that in comparison to distilled water aqueous solutions of chlorine salts of ammonium, potassium and sodium increased the content of macrotetrolides in chloroform as a result of forming strong hydrophobic complexes. This is especially applied to the ions of ammonium whose addition to the aqueous phase led to an increase of macrotetrolide level in chloroform up to 98.4 per cent. Addition of weak hydrochloric acid or alkaline agents resulted in marked transfer of the ionophores into the aqueous phase at the expense of hydrolysis of the antibiotic cyclic molecules. The highest hydrolysis levels were induced by potassium hydroxide, the content of the ionophores in the hydrophobic phase decreasing up to 90.6 per cent. The effect of picric acid on distribution of the macrotetrolides between water and chloroform was different and depended on its concentration.     

Pharmacologic properties of a phosphate ester of dopamine

The 3 or 4 phosphate ester of dopamine (PD) was hydrolyzed by homogenates of rat tissues to give inorganic phosphate (Pi) and dopamine. The rate of hydrolysis of PD by kidney homogenates was increased by exogenous MgCl2 but not CaCl2 or KCl. The activity of brain, heart or liver homogenates was insensitive to the added salts. Several lines of evidence indicate that alkaline phosphatase activity contributes to the high rate of PD hydrolysis by the kidney but not brain homogenate. The intravenous infusion of PD at 12 mumole/kg in one hr to anesthetized rats increased the dopamine content of the plasma, kidney and heart without altering brain or liver dopamine. The results suggest that PD may be more effective than dopamine for increasing dopamine levels of the kidney. In addition, the hydrolysis of PD by brain homogenates, which is independent of alkaline phosphatase activity, suggests that specific enzymes exist for the metabolism of PD.     

DNA-DNA dot hybridization technique used as DNA determination method in the alkaline elution analysis of DNA damage

A DNA-DNA (‘Southern’) dot hybridization technique was adapted for use as a quantitative DNA detection method during alkaline elution analysis of irradiated rat cell material. In comparison to standard microfluorometric methods, similar γ-ray-dose-response relationships were obtained with less than 1% of the cell material when the dot hybridization assay was used. When a highly repetitive, long interspersed DNA element of the rat genome is used as a hybridization probe, as few as 10⁴ cells of rat tissue or rat cell culture cells per sample with approx. 50 ng of DNA were sufficient to detect single-strand breaks and protein cross-links in the DNA of rat hepatocytes and cells of the nasal epithelium after in vitro γ-irradiation. Since highly repetative DNA elements are available from nearly all higher eukaryotes, this alternative approach of detecting DNA in alkaline elution analysis is generally proposed for tissues which yield only low amounts of cell material and/or which are difficult to label by radioactive DNA precursors.     

Isolation of a malondialdehyde-deoxyguanosine adduct from rat liver DNA.

In previous studies, an adduct of malondialdehyde (MDA) with guanine was identified in rat and human urine. Subsequent detection of an adduct with deoxyguanosine (dG) in urine prompted an investigation of its possible occurrence in DNA. Rat liver DNA was hydrolyzed using nuclease P1 and alkaline P-ase and subjected to deoxyribonucleoside analysis using reverse phase high-pressure liquid chromatography (HPLC) with fluorescence detection. A compound was isolated that could not be separated from a synthetic pyrimidinopurine adduct of MDA and dG (dG-MDA). Partial hydrolysis released guanine (Gua), Gua-MDA, and dG in amounts that, in aggregate, were the molar equivalent of the starting material calculated by fluorescence analysis as dG-MDA. Complete acid hydrolysis of the isolate yielded an equimolar amount of MDA. Analysis of liver DNA isolated from growing rats yielded a value for dG-MDA content of 9.0 +/- 1.6 pmol/100 micrograms DNA (mean +/- SEM, N = 5). This value is approximately 7 times those reported for the 8-hydroxy deoxyguanosine content of rat liver nuclear DNA. This study demonstrates that DNA is modified in vivo by reactions of its guanylate moiety with MDA, and indicates that, at least in the case of rat liver DNA, the prevalence of such modifications is greater than those caused by reactions with hydroxyl radicals.     

Characterization of the pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate hydrolase activity in rat liver. Identity with alkaline phosphatase.

The tissue content of pyridoxal 5'-phosphate is controlled principally by the protein binding of this coenzyme and its hydrolysis by a cellular phosphatase. The present study identifies this enzyme and its intracellular location in rat liver. Pyridoxal-P is not hydrolyzed by the acid phosphatase of intact lysosomes. At pH 7.4 and 9.0, the subcellular distribution of pyridoxal-P phosphatase activity is similar to the for p-nitrophenyl-P, and the major portion of both activities is found in the plasma membrane fraction. The ratio of specific activities for pyridoxal-P and p-nitrophenyl-P hydrolysis remains relatively constant during the isolation of plasma membranes. These activities also behave concordantly with respect to pH rate profile, pH-Km profile, and response to chelating agents, Zn2+, Mg2+, and inhibitors. Kinetic studies indicate that pyridoxal-P binds to same enzyme sites as beta-glycerophosphate and phosphorylcholine. The data strongly favor alkaline phosphatase as the enzyme which functions in the control of pyridoxal-P and pyridoxamine-P metabolism in rat liver. Alkaline phosphatase was solubilized from isolated plasma membranes. The kinetic properties of the enzyme are not markedly altered by its dissociation from the membrane matrix. However, there are significant differences in its behavior toward Mg2+ which suggest a structural role for Mg2+ in liver alkaline phosphatase.     

The Preparation of Biologically Active Messenger RNA from Human Postmortem Brain Tissue

Abstract: Messenger RNA (mRNA) was extracted from human postmortem brain tissue by alkaline phenol extraction of polysomes followed by oligo (dT)-cellulose chromatography. The mRNA preparations stimulated protein synthesis in a cell-free system containing wheat germ homogenate. The products of protein synthesis were analyzed by one- and two-dimensional gel electrophoresis. These analyses indicated that numerous polypeptides, including tubulin subunits and actin isomers, were synthesized by the human mRNA. The molecular weight range of polypeptides synthesized by human mRNA fractions from two brain specimens were identical, and analysis by two-dimensional gel electrophoresis indicated qualitatively similar products. The yield of mRNA extracted per gram of human tissue was less than the yield obtained with rat forebrains from animals sacrificed immediately before brain removal and mRNA purification. A decrease in the amount of polysomes isolated from human tissue relative to rat brain tissue was a major factor contributing to the low yield. The molecular weight distribution of polypeptides synthesized by human and rat brain mRNA fractions in wheat germ homogenate was similar; thus, there was no indication for selective breakdown or inactivation of high molecular weight mRNA species in the human tissue. Our studies indicate that it is possible to utilize postmortem tissue for molecular biological investigations of human brain mRNA.     

A rapid and simple method for the purification of rat liver RNase inhibitor

A rapid and simple method for the purification of rat liver alkaline RNase inhibitor from a 105,000 g supernatant is reported. It involves protein precipitations by (NH₄)₂SO₄ and chromatography on carboxymethyl cellulose-RNase column. The purification procedure gives a 1020-fold increase in specific activity with a yield of 32%. This purified inhibitor can be stored for 5 weeks without any loss in activity.     

REGIONAL LEVELS OF GABA IN THE BRAIN: RAPID SEMIAUTOMATED ASSAY AND PREVENTION OF POSTMORTEM INCREASE BY 3-MERCAPTO-PROPIONIC ACID

Abstract— A simple and rapid semiautomated assay for GABA in central nervous tissue is described. The method is based on a simple manual procedure of isolating GABA from tissue extracts on small CM Sepharose Cl 6B columns, followed by an automated fluorimetric detection (continuous flow system) with o -phthalaldehyde (OPA) and β-mercapto-ethanol (β-ME) at an alkaline pH. GABA is separated from other compounds that fluoresce in our detection system. By using low concentration of OPA and β-ME and allowing only a short reaction time with these reagents, the detection is specific towards GABA. The detection limit of the assay is 1 nmol.
A procedure is described for the prevention of postmortem GABA increase in rat and mouse brain by intravenous injection of 3-mercapto-propionic acid (1.2 nmol/kg) 2min before decapitating the animal. This treatment and microwave irradiation result in similar GABA levels in mouse brain and substantia nigra tissue from rat brain. We found a great conformity in regional GABA levels in the rat and the mouse brain.     

Optimization of alkaline pretreatment of coffee pulp for production of bioethanol

The use of lignocellulosic raw materials in bioethanol production has been intensively investigated in recent years. However, for efficient conversion to ethanol, many pretreatment steps are required prior to hydrolysis and fermentation. Coffee stands out as the most important agricultural product in Brazil and wastes such as pulp and coffee husk are generated during the wet and dry processing to obtain green grains, respectively. This work focused on the optimization of alkaline pretreatment of coffee pulp with the aim of making its use in the alcoholic fermentation. A central composite rotatable design was used with three independent variables: sodium hydroxide and calcium hydroxide concentrations and alkaline pretreatment time, totaling 17 experiments. After alkaline pretreatment the concentration of cellulose, hemicellulose, and lignin remaining in the material, the subsequent hydrolysis of the cellulose component and its fermentation of substrate were evaluated. The results indicated that pretreatment using 4% (w/v) sodium hydroxide solution, with no calcium hydroxide, and 25 min treatment time gave the best results (69.18% cellulose remaining, 44.15% hemicelluloses remaining, 25.19% lignin remaining, 38.13 g/L of reducing sugars, and 27.02 g/L of glucose) and produced 13.66 g/L of ethanol with a yield of 0.4 g ethanol/g glucose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:451–462, 2014     

Potential artifacts in the measurement of DNA deamination

Attack on DNA by some reactive nitrogen species results in deamination of adenine and guanine, leading to the formation of hypoxanthine and xanthine, respectively. Published levels of these products in cellular DNA have varied widely. Although these two deamination products are often measured by GC-MS analysis, the procedure of acid hydrolysis to release DNA bases for derivatization poses a risk of artifactual deamination of the DNA. In this study, we demonstrated the artifactual formation of these two deamination products during acid hydrolysis and hence developed a method for detecting and measuring 2'-deoxyinosine, the nucleoside of hypoxanthine. Our assay for 2'-deoxyinosine employs nuclease P1 and alkaline phosphatase to achieve release of the nucleosides from DNA, followed by HPLC prepurification with subsequent GC-MS analysis of the nucleosides. This assay detected an increase in the levels of 2'-deoxyinosine in DNA when commercial salmon testis DNA was treated with nitrous acid. We also used it to measure levels in various rat tissues of both normal and endotoxin-treated rats, but could not find increased 2'-deoxyinosine formation in tissues even though *NO production was substantially increased.     

A quantitative method for analyzing specific DNA sequences directly from whole cells

A quick, accurate assay for specific DNA sequences is described in which whole cells are treated with 0.4 M sodium hydroxide at 80 degrees C. DNA is relatively resistant to alkaline hydrolysis, whereas proteins and RNA are degraded rapidly. The DNA in NaOH is then transferred through a slot directly onto a nylon membrane and hybridized with a probe. Since the procedure is so simple, many samples can be analyzed in a short time. A single-copy gene can be detected in as few as 1000 cells and, since the DNA from 10(5) cells can be loaded through a single slot, the sensitivity is sufficient to detect one specific DNA sequence per 100 cells. Accurate quantitative analysis can be achieved by normalizing the amount of DNA available for hybridization in each slot, using a probe derived from total DNA.     

Percent satellite DNA as a function of tissue and age of mice.

A selective loss of satellite DNA was found to occur to different extents as a function of tissue and age of mice using several common DNA extraction and purification procedures. This result emphasizes a serious problem that may be encountered in comparative studies of DNA structure and composition if selective loss of specific DNA sequences occurs. We have developed a DNA extraction and purification procedure that is simple and reliable and gives a high percent DNA yield, which substantially reduces the selective loss of heterochromatin DNA sequences. The method features a centrifugation step of a proteolytic digest of chromatin in 2.4 M CsCl. Percent DNA yield of 82-98% are routinely obtained with no apparent loss of satellite DNA sequences from different tissues or ages of mice. Utilizing this method, percent satellite DNA was found to remain essentially constant at 11 +/- 1% for spleen, kidney, and brain tissues obtained from mice of 10-780 days of age. However, for liver, percent satellite DNA remained at about 7-8% from 10 to 300 days of age and then increased to about 12-13% from 300 to 600 days of age. During this latter time interval (300-600 days), an increase of DNA per nucleus of about 3-fold occurred, due to the formation of tetra- and octaploid cell types. A steady loss in the total number of nuclei per gram of liver as a function of age was also found. These two opposing effects resulted in a nearly constant amount of DNA per gram and per organ for liver throughout the lifespan of the mouse.