Optimal conditions for the fluorometric determination of DNA

The Kissane and Robbins method of estimating DNA by the fluorescence of the product of its reaction with 3,5-diaminobenzoic acid dihydrochloride has been examined to determine the conditions which maximize the contribution of the reaction product to the observed fluorescence and minimize the level of the fluorescence in the assay blanks.     

Rapid filter method for the microfluorometric analysis of DNA.

A rapid filter method for the microfluorometric analysis of DNA is reported in this communication. Cells collected on cellulose filters are subject to an assay sequence developed from a fluorometric method initially described by J. M. Kissane and E. Robins ((1958) J. Biol. Chem.233, 184–188) for DNA quantitation. The assay is one of high specificity requiring no separation of DNA from other major cellular components. Samples containing as little as 0.2–0.3 μg of DNA have been analyzed by this filter method with a coefficient of variation for replicate standard samples of 3.3%. The DNA content of a number of cell types having different physiological characteristics has been determined by the use of this filter technique. The data obtained for Chlorella, Chlamydomonas, Ochromonas, and Stronglyocentrotus eggs were well within the values reported for these cells by other investigators using classical macromethods. We have taken advantage of the high specificity, sensitivity, and accuracy of this filter assay to determine DNA content during the synchronous growth cycle of the wall-less alga Olisthodiscus luteus using a single small volume culture as a sample source.     

DNA measurement by mithramycin fluorescence in chromatin solubilized by heparin

The mithramycin fluorescence procedure described by B. T. Hill and S. Whatley (1975, FEBS Lett., 56, 20–23) for DNA measurement tends to underestimate DNA concentrations in biological samples as compared to the results obtained by the diphenylamine reaction. This discrepancy disappears when DNA is first solubilized, by buffer containing heparin, from either cell homogenates or nuclear preparations. The optimal conditions for maximal fluorescence are 8 mm Mg²⁺, 10 μg/ml mithramycin, and heparin to DNA ratios ≥0.15 ($\text{w}\text{w}$). Background fluorescence is reduced 90% by dextran-coated charcoal adsorption of unbound mithramycin. The limit of sensitivity of the assay is 0.3 μg/ml and fluorescence is linear up to 30 μg DNA/ml.     

Improvement of DNA quantitation following proteolytic extraction

A reliable and sensitive method for quantitation of deoxyribonucleic acid (DNA) is described herein. It is based on a proteinase K digestion step followed by a fluorometric assay with 3,5-diaminobenzoic acid(Kissane and Robins, 1958). The method proved to be applicable to fresh liver and to hepatocytes either in suspension or in culture when attached to a collagen substratum. A linear relationship between biological sample size and fluorometric response was observed only if the assay was preceded by proteolytic extraction. Moreover, the method resulted in DNA values per fresh liver weight that were about 100% higher than values reported earlier. The data show that proteolysis results in a more exhaustive extraction of DNA from tissues and a better separation of extracted DNA from other tissue constituents interfering with the assay.     

Cotranslational cleavage of immunoglobulin light chain precursors by plasmacytoma microsomes.

Murine plasmacytoma endoplasmic reticulum which has been freed of ribosomes by EDTA treatment is capable of the cotranslational proteolytic processing of representative λ₁,λ₂, and k immunoglobulin light chain precursors. Messenger RNA fractions from the MOPC-104E, MOPC-315, and MOPC-46B tumor lines were used to direct the synthesis of the light chain precursors in a cell-free system derived from Krebs II ascites cells. The precursor cleavage activity of the plasmacytoma membranes is comparable in activity and in characteristics to that of two well-defined membrane preparations: Krebs II ascites intracellular membranes (E. Szczesna and I. Boime, 1976, Proc. Nat. Acad. Sci. USA73, 1179–1183) and EDTA-treated rough endoplasmic reticulum from canine pancreas (34., 35., J. Cell Biol.67, 852–862). The efficiency of the cleavage reaction appears to be dependent upon the precursor being utilized as a substrate. An assay suitable for a preliminary characterization of the plasmacytoma membrane preparations is described.     

A modified fluorometric method for the determination of microgram quantities of DNA from cell or tissue cultures.

Incubation of up to 8 μg of DNA with 30 mg DABA at 60°C for 30 min are the optimal conditions for development of fluorescence with DNA. At lower levels of DABA the time of incubation became crucial, and linearity at low DNA concentrations was lost. Application of the method to the estimation of DNA extracted with either 1 n KOH or 1 n PCA from monolayer cell cultures showed that fluorescence could still be developed satisfactorily in the presence of the alkali or acid without neutralizing or drying the sample, thus saving considerable time with no loss of accuracy or reproducibility of the assay, but with some loss of sensitivity.     

Design,synthesis and photoinduced DNA cleavage studies of [1,2,4]-triazolo[4,3-a]quinoxalin-4(5H)-ones

An expedient and eco-friendly synthesis of 1-aryl/heteroaryl-[1,2,4]-triazolo[4,3-a]quinoxalin-4(5H)-ones (4) has been accomplished via iodobenzene diacetate mediated oxidative intramolecular cyclization of 3-(2-(aryl/heteroarylidene)hydrazinyl)-quinoxalin-2(1H)-ones (3). Ten synthesized compounds 3 and 4 (10–40 μg) on irradiation with UV light at λ_max 312 nm could lead to cleavage of supercoiled pMaxGFP DNA (Form I) into the relaxed DNA (Form II) without any additive. Further, DNA cleaving ability of triazoles was quantitatively evaluated and was found to be dependent on its structure, concentration, and strictly on photoirradiation time. Mechanistic investigations using several additives as potential inhibitors/activator revealed that the DNA photocleavage reaction involves Type-I pathway leading to formation of superoxide anion radicals (O₂⁻) as the major reactive oxygen species responsible for photocleavage process.     

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of totalgenomic DNA from 15 red algal species. It resulted in 0.1 g high quality DNAfrom 1 mg fresh algal material, with an A₂₆₀/A₂₈₀ratio of 1.68–1.90.Using this rapidly isolated DNA, the 18S ribosomal RNA genes (rDNA) and the nuclearribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. Thetested DNA was suitable for restriction endonuclease digestion, genetic markeranalysis and polymerase chain reaction (PCR) amplification, and may be valid forother genetic manipulation.     

Synthesis of novel chalcones through palladium-catalyzed CO cross-coupling reaction of bromo-chalcones with ethyl acetohydroxamate and their antiplasmodial evaluation against Plasmodium falcipuram in vitro

An efficient method for palladium-catalyzed CO cross-coupling of ethyl acetohydroxamate (EAcHO) with 4-bromo-chalcones has been developed to synthesize novel chalcones. The two supporting ligands, namely tBuXPhos (L7), and cataCXium®PIntB (L16) were found to be effective ligands towards the Pd-catalyzed CO cross-coupling reaction to afford the desired product in moderate to excellent yields (50–99%). The coupled products were screened for in vitro blood stage antiplasmodial activity against Plasmodium falciparum (3D7) using the [³H] hypoxanthine incorporation inhibition assay. Of the twenty two compounds screened, eleven showed good antiplasmodial activity with IC₅₀ values ranging from 6–16 μg/mL. The selected active molecules 11, 16, 22, (IC₅₀ 12 μg/mL) and 19 (IC₅₀ 6 μg/mL) were studied for their cytotoxic effect against HepG2 Cells (human hepatocellular liver carcinoma cell lines), showing the selectivity index (SI) values are greater than 4 except chalcone 22. Our result demonstrates a methodology for synthesizing novel chalcones as a new class of antiplasmodial agent.     

A simplification of the protein assay method of Lowry et al. which is more generally applicable

Some recent modifications of the protein assay by the method of Lowry, Rosebrough, Farr, and Randall (1951, J. Biol. Chem.193, 265–275) have been reexamined and altered to provide a consolidated method which is simple, rapid, objective, and more generally applicable. A DOC-TCA protein precipitation technique provides for rapid quantitative recovery of soluble and membrane proteins from interfering substances even in very dilute solutions (< 1 μg/ml of protein). SDS is added to alleviate possible nonionic and cationic detergent and lipid interferences, and to provide mild conditions for rapid denaturation of membrane and proteolipid proteins. A simple method based on a linear log-log protein standard curve is presented to permit rapid and totally objective protein analysis using small programmable calculators. The new modification compared favorably with the original method of Lowry et al.     

L-arginine assay with the use of arginase I

A highly selective and sensitive method for the quantitative determination of L-arginine (Arg) with a fluorescent detection of the reaction product has been developed. The method is based on the use of human liver arginase I isolated from a recombinant producer strain, yeast Hansenula polymorpha, and 2,3-butanedione monoxime, which is used to detect carbamide—the product of enzymatic reactions. The linear concentration range for determining Arg in the final reaction mixture varies from 0.2 to 250 μM, and the detection limit is 0.16 μM. Tests of the new method using commercial Arg-containing pharmaceutical preparations showed a high correlation (R = 1.0) of the results with the manufacturer’s data and the results of other methods for Arg detection.     

Quantitative assay for submicrogram amounts of protein

An ultrasensitive protein assay, linear between 0.01 and 0.2 μg protein, is described. In this assay, copper is complexed to protein, excess copper is removed by adsorption to a small Sephadex column, and the copper-protein complex is destroyed by digestion with hydroperoxide. Phenol and chloramine-T are added to the reaction mixture and the copper catalyzes the production of a color-producing reaction between the two compounds. The assay is not affected by low levels of phosphate, Tris, metals, or a tenfold excess of nucleic acid over protein. Reducing agents, sucrose, certain anions, high salt concentrations, and EDTA seriously interfere. The method is about 500 times as sensitive as that of Lowry et al. [Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem.193, 265]. Like the biuret procedure, the assay measures copper complexed to the peptide bonds but is probably influenced by other factors, since equivalent amounts of different proteins give similar but not identical amounts of color.     

Cytochemical hybridisation with fluorochrome-labelled RNA

Summary A new method to localise specific DNA sequences in microscopic preparations by hybridocytochemistry using fluorochrome labelled complementary RNA has been described recently (Bauman et al. 1981). The present paper describes a procedure to increase the sensitivity of this method. RNA complementary to kinetoplasts DNA of Crithidia luciliae was labelled with fluorescein and hybridised with Sephadex beads to which kinetoplast DNA or heterologous DNA had been covalently bound as well as to Crithidia luciliae preparations. The fluorescein-labelled RNA was found to hybridize specifically with homologous DNA both on the beads and in the cells. The sensitivity of the hybrid detection could be increased by applying an indirect immunofluorescence reaction using rabbit antiserum raised against the hapten fluorescein as has been described for the amplification of a direct immunofluorescence reaction by Schmitz and Kampa (1979). The complete procedure resulted in an amplification of the original specific fluorescence both on the beads and in the cells. The increase was quantified by microfluorimetry. Several aspects of the immunocytochemical amplifying reaction were quantitatively investigated using Sephadex beads to which poly(A) or DNA was coupled and FITC-labelled poly(U) or cRNA was hybridised. A 5- to 10-fold amplification was obtained both in the beads and on the cell preparations. When the amplifying steps were repeated a proportional increase in background fluorescence was observed.This work was supported by the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.)     

Optimization of Folin-Ciocalteu reagent concentration in an automated Lowry protein assay

The Lowry method (G. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951, J. Biol. Chem.193, 265–275) for protein concentration measurement has been automated to permit assay of samples with concentrations from 1 to 400 μg/ml. Calibration with solutions of bovine serum albumin resulted in a nonlinear (quadratic) curve. The quantity of color developed in the assay was found to be strongly dependent on the concentration of the Folin-Ciocalteu phenol reagent. Color yield peaked sharply at a reagent concentration 40% lower than that used in the Lowry procedure. Optimization of the reagent concentration is necessary to obtain maximum sensitivity from the Lowry assay.     

A sensitive continuous and discontinuous photometric determination of oxygen, carbon dioxide, and carbon monoxide in gases and fluids.

The paper describes a sensitive, rapid, and precise photometric method for the continuous and discontinuous determination of O₂, CO₂, and CO. The method is based on highly specific color reactions: O₂ is determined by its reaction with alkaline catechol + Fe²⁺ yielding intensively colored products, CO₂ is determined by its color reaction with a solution of fuchsin + hydrazine; and CO is determined by its reaction with hemoglobin. The basic experimental equipment is that of the AutoAnalyzer (cf.Wolf, Zander, and Lang, 1976, Anal. Biochem.74, 585), with an additional chamber for the injection of small gas samples in the case of the discontinuous analysis. Continuously analyzing in a standardized gas flow of 1 ml · min^?1 (STPD), the lower limits of the sensitivities are 50 ppm for O₂, 100 ppm for CO₂, and 50 ppm for CO. The discontinuous analysis of the three gases requires the basic experimental equipment plus an airtight chamber. The lower limits of the amounts are 0.1 μl (STPD) for O₂, 0.2 μl for CO₂, and 0.1 μl for CO.     

Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay

The heads of mouse spermatozoa obtained 5 min after release from the excised caudae epididymides showed a characteristic fluorescence pattern in the presence of the fluorophore chlortetracycline (CTC). There was uniform fluorescence over the entire head with about half the sperm population showing a brighter line of fluorescence across the equatorial segment; this fluorescence pattern was designated “F.” After 90-min incubation in culture medium (CM) containing 2% (w/v) bovine serum albumin, most of the sperm heads showed a dark band of nonfluorescence over the equatorial and postequatorial segment, while the anterior portion of the head showed bright fluorescence. This fluorescence pattern was designated “B.” The time course for the disappearance of pattern F matched the time course of the appearance of pattern B, with a half-time of 30 min. The transformation was complete in 90 min. At longer times of incubation in CM, the percentage of spermatozoa showing pattern B declined; fluorescence over the entire head was lost, characteristic of the pattern for acrosome-reacted sperm (P. M. Saling and B. T. Storey (1979). J. Cell Biol.83, 544–555). Mouse sperm showing pattern B were able to undergo the acrosome reaction, either spontaneously or by induction with acid-solubilized zonae pellucidae from mouse eggs (H. M. Florman and B. T. Storey (1982). Dev. Biol.91, 121–130). The latter reaction was blocked by its specific inhibitor 3-quinuclidinyl benzilate (QNB). Mouse sperm showing pattern F could not be induced to undergo the acrosome reaction by exposure to solubilized zonae. This implies that the change from fluorescence pattern F to fluorescence pattern B corresponds with changes in the sperm which make them susceptible to undergo the acrosome reaction. This change occurs during the time interval previously determined to be needed for capacitation of mouse sperm in vitro in CM (M. Inoue and D. P. Wolf (1975). Biol. Reprod.13, 340–346). These results imply that spermatozoa showing CTC fluorescence pattern B can be considered to be capacitated and that a functional definition for capacitation is the acquired ability to undergo the acrosome reaction rapidly when treated with acid-solubilized zonae pellucidae. The CTC fluorescence assay provides for the first time a means to monitor the time course of epididymal mouse sperm capacitation in vitro.     

A new continuous, monodimensional electrophoretic system for the separation and quantitation of individual glycosaminoglycans

A rapid and sensitive method for agarose gel electrophoresis is described. By simply miniaturizing a conventional gel electrophoresis apparatus, we have decreased the time necessary for the separation of nucleic acid molecules by a factor of 10. The ability to detect DNA molecules by ethidium bromide fluorescence has simultaneously been increased fivefold. Transfer of DNA from these “minigels” onto nitrocellulose filters followed by hybridization using the procedure of C. M. Southern (1975, J. Mol. Biol.98, 503–517) was found to be efficient and rapid. This technique is sufficiently sensitive to detect radioactive quantities of [³²P]phosphate-labeled DNA or RNA microinjected into 500 chick embryo fibroblasts.