Comparison of the thermal denaturation behaviour of DNA-solutions and metaphase chromosome preparations in suspension.

Hyperchromicity measurements are well established to analyse the thermal denaturation behaviour of pure DNA sequences in solution. Here, we show that under appropriate experimental conditions this technique can also be applied to study thermally controlled conformation changes of higher order DNA-protein complexes as for instance metaphase chromosome preparations in suspension. A computer controlled sensitive, upright double beam photometer with a heatable cuvette was constructed. Measurements of the temperature dependent extinction of both, solutions and particle suspensions are possible, since sedimentation effects of particles can be neglected due to the vertical optical axis in the probe cuvette. Thermal denaturation of metaphase chromosome preparations of human and Chinese hamster cells was investigated and compared to melting profiles of DNA solutions for two excitation wavelengths, 256 and 313 nm. The influence of neutral and low pH was considered. The results indicate that metaphase chromosome preparations show a thermal denaturation behaviour different from pure DNA. Whereas DNA solutions showed one pH dependent melting peak at 256 nm only, the peak pattern of metaphase chromosome preparations showed a large variability both at 256 and 313 nm. At neutral pH, in two temperature regions (40-55 degrees C and 75-82 degrees C) peaks were found indicating chromosome typical conformation changes independently from the mammalian cell species (Chinese hamster, human). In contrast to pure DNA, no typical reduction in the temperatures of peak maxima with decreasing pH was found for metaphase chromosome preparations of both cell types. These results may be relevant for further systematic studies of efficient thermal probe/target denaturation procedures in non enzymatic DNA-chromosome in situ hybridisation.     

Optical studies of the interaction of 33258 Hoechst with DNA,chromatin, and metaphase chromosomes

The interaction of the bisbenzimidazole dye 33258 Hoechst with DNA and chromatin is characterized by changes in absorption, fluorescence, and circular dichroism measurements. At low dye/phosphate ratios, dye binding is accompanied by intense fluorescence and circular dichroism and exhibits little sensitivity to ionic strength. At higher dye/phosphate ratios, additional dye binding can be detected by further changes in absorptivity. This secondary binding is suppressed by increasing the ionic strength. A-T rich DNA sequences enhance both dye binding and fluorescence quantum yield, while chromosomal proteins apparently exclude the dye from approximately half of the sites available with DNA. Fluorescence of the free dye is sensitive to pH and, below pH 8, to quenching by iodide ion. Substitution of 5-bromodeoxyuridine (BrdU) for thymidine in synthetic polynucleotides, DNA, or unfixed chromatin quenches the fluorescence of bound dye. This suppression of dye fluorescence permits optical detection of BrdU incorporation associated with DNA synthesis in cytological chromosome preparations. Quenching of 33258 Hoechst fluorescence by BrdU can be abolished by appropriate alterations in solvent conditions, thereby revealing changes in dye fluorescence of microscopic specimens specifically due to BrdU incorporation.     

Simultaneous measurements of intracellular pH in the leech giant glial cell using 2'',7''-bis-(2-carboxyethyl)-5,6-carboxyfluorescein and ion-sensitive microelectrodes.

We have employed two independent techniques to measure the intracellular pH (pHi) in giant glial cells of the leech Hirudo medicinalis, using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and double-barreled neutral-carrier, pH-sensitive microelectrodes, which also record the membrane potential. We have compared two procedures for calibrating the ratio of the BCECF signal, excited at 440 nm and 495 nm: 1) the cell membrane was H(+)-permeabilized with nigericin in high-K+ saline at different external pH (pHo) values, and 2) the pHi of intact cells was perturbed in CO2/HCO3(-) -buffered saline of different pH, and the BCECF ratio was calibrated according to a simultaneous microelectrode pH reading. As indicated by the microelectrode measurements, the pHi did not fully equilibrate to the pHo values in nigericin-containing, high-K+ saline, but deviated by -0.12 +/- 0.02 (mean +/- SEM, n = 37) pH units. In intact cells, the microelectrode readings yielded up to 0.15 pH unit lower values than the calibrated BCECF signal. In addition, larger dye injections into the cells (> 100 microM) caused an irreversible membrane potential loss indicative of some damage to the cells. The amplitude and kinetics of slow pHi changes were equally followed by both sensors, and the dye ratio recorded slightly higher amplitudes during faster pHi shifts as induced by the addition and removal of NH4+.     

The influence of formamide on thermal denaturation profiles of DNA and metaphase chromosomes in suspension

Systematic photometric studies are presented to analyze the thermal denaturation behaviour with and without formamide of metaphase chromosome suspensions in comparison to DNA solutions. Temperature dependent hyperchromicity measurements at 256 nm and 313 nm were performed using an appropriately designed computer-controlled photometer device. Due to an upright optical axis, this allowed absorbance measurements with negligible sedimentation effects not only for solutions of pure DNA, but also for particle suspensions of isolated metaphase chromosomes. This device has a temperature resolution of +/- 0.5 degrees C and an optical sensitivity of 10(-3) to 10(-4) optical density. For calf thymus DNA the reduction of the melting point with the increase of formamide in the solution was measured at pH 7.0 and pH 3.2. The good correlation of the theoretical approximation to experimental data indicated the suitability of the apparatus to quantitatively describe DNA conformation changes induced by thermal denaturation. For metaphase chromosome preparations of Chinese hamster culture cells, absorbance changes were measured between 20 degrees C and 95 degrees C with a temperature gradient of 1 degrees C/min. These measurements were performed at pH 7.0 and at pH 3.2. The denaturation profiles (= first derivative of the absorbance curve) resulted in a highly variable peak pattern at 256 nm and 313 nm indicating complex conformation changes. A statistical evaluation of the temperature values of the peak maxima resulted in temperature ranges typical for chromosomal conformation changes during thermal treatment. Especially the range of highest temperature values was independent from pH modifications. For pH 3.2 the influence of formamide on the denaturation behaviour of metaphase chromosome preparations was analyzed. In contrast to pure DNA solutions, a reduction of the "melting point" (i.e. the maximum temperature at which a conformation change takes place) was not found. However, the denaturation behaviour depended on the duration of formamide treatment before the measurement.     

Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.     

On the various types of circular dichroism induced on acridine orange bound to poly(S-carboxymethyl-L-cysteine).

Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.     

Hydrodynamic radii and lipid transfer in prostasome self-fusion.

The characteristics of human prostasomal vesicles have been investigated by three methods, namely, dynamic light scattering, transfer of a lipophylic fluorescent dye (R18), and electron microscope appearance. The vesicle preparations were stable for a long time and their diameters were in the range of 200 nm. The exposure to acidic pH values (about 5) increased both particle radii and the transfer of R18. The microscopic appearance was also affected by the pH value. We infer that these changes are due to a self-fusion of prostasome vesicles; this fusion is protein-dependent since various methods used by us and able to affect the protein component of membranes (boiling, extraction of lipid and liposome preparation, treatment with pronase) all abolished the effect seen at pH 5 on intact particles.     

Revised procedures for the certification of carmine (C.I. 75470, Natural red 4) as a biological stain

Carmine is one of the original dyes certified by the Biological Stain Commission (BSC). Until now it has lacked both an assay procedure for dye content and a means to positively identify the dye. The methods for testing carmine in the laboratory of the BSC have been revised to include spectrophotometric examination at pH 12.5-12.6 to determine that the dye is carmine (λ_max=530-335 nm). The maximum absorbance of a solution containing 100 mg of dye per liter of water, adjusted to pH 12.5-12.6, which provides a relative measure of dye content, should lie in the range 1.2 to 1.8. If the dye is not carmine, spectrophotometry at pH 1.9-2.1 shows whether it is carminic acid (λ_max=490-500 nm) or 4-aminocarminic acid (λ_max=525-530 nm). The latter two dyes, which are also called carmine when sold as food colorants, have physical properties different from those of true carmine. The functional tests for carmine as a biological stain are Orth's lithium-carmine method for nuclei, Southgate's mucicarmine method for mucus, and Best's carmine method for glycogen.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus

First, the enzyme immobilized on cyanide bromide agarose beads (CNBr) (that did not involve all enzyme subunits in the immobilization) has been crosslinked with aldehyde-dextran. This preparation did not any longer release enzyme subunits and become fully stable at pH 4 and 25 °C.Then, the stabilities of many different enzyme preparations (enzyme immobilized on CNBr, that derivative further crosslinked with aldehyde-dextran, enzyme immobilized on highly activated amino-epoxy supports, GDH immobilized on supports having a few animo groups and many epoxy groups, GDH immobilized on glyoxyl-agarose beads at pH 7, and that preparation further incubated at pH 10, and finally the enzyme immobilized on this support directly at pH 10) were compared at pH 4 and high temperatures, conditions where both dissociation and distortion play a relevant role in the enzyme inactivation. The most stable preparation was that prepared at pH 7 and incubated at pH 10, followed by GDH immobilized on amino and epoxy supports and the third one was the enzyme immobilized on glyoxyl-agarose at pH 10.The incubation of all enzyme preparations in saturated guanidine solutions produced the full inactivation of all enzyme preparations. When not all enzyme subunits were immobilized, activity was not recovered at all. Among the other derivatives, only glyoxyl preparations (the most inert supports and those where a more intense multipoint covalent attachment were expected) gave significant reactivation when re-incubated in aqueous medium. After optimization of the reactivation conditions, the enzyme immobilized at pH 7 and later incubated at pH 10 recovered 100% of the enzyme activity.     

Species specificity in the extrinsic absorption changes exhibited by some invertebrates stained with voltage-sensitive dyes

Summary The absorption changes of several invertebrate neuronal preparations stained by the potentiometric dyes (WW 375, WW 433, WW 401 and RGA 84) in response to electrical nerve stimulation were examined. The dyes did not penetrate the connective sheath of insect preparations, but stained it. Only a decremental spreading of optical signals was seen onPeriplaneta americana, Gryllus bimaculatus and GitGryllus campestris ganglia and nerves. In contrast to insect preparations, pond snail and leech neurons were well stained by these dyes. The dye WW 375 behaved somewhat distinctly on insect and pond snail preparations than had been previously reported on other invertebrates. Like the signals from vertebrate neurons, they usually had triphasic action spectra. Therefore, this kind of action spectrum is not found only in membranes of vertebrate neurons. The main conclusion of this work is that the species-specific effects of the dye on different invertebrate preparations have a common feature: the existence of three peaks in the change of absorption (at 575, 675 and 750 nm) in both kinds of WW 375 action spectra (monophasic or triphasic). The wavelength dependence of the change in absorption was not affected by concentration, staining time, pH, osmolarity or ionic composition of physiological saline.     

Utilization of modified silk cotton hull waste as an adsorbent for the removal of textile dye (reactive blue MR) from aqueous solution

Carbon prepared from silk cotton hull was used to remove a textile dye (reactive blue MR) from aqueous solution by an adsorption technique under varying conditions of agitation time, dye concentration, adsorbent dose and pH. Adsorption depended on solution pH, dye concentration, carbon concentration and contact time. Equilibrium was attained with in 60 min. Adsorption followed both Langmuir and Freundlich isotherm models. The adsorption capacity was found to be 12.9 mg/g at an initial pH of 2+/-0.2 for the particle size of 125-250 microm at room temperature (30+/-2 degrees C).     

Binding of Hoechst 33258 to chromatin in situ

The binding of Hoechst 33258 to rat thymocytes, human lymphocytes, and NHIK 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The results indicated that there were two different modes of binding of Hoechst 33258 to chromatin in situ at physiological pH. Type 1 binding, which dominated at total dye/phosphate ratios below 0.1 (0.15, M), was characterized by a binding constant of the order 10(7) M-1 and fluorescence with high quantum yield. Further binding of the dye resulted in a reduced blue/green fluorescence ratio, indicating that secondary sites were occupied. Binding at secondary sites above a certain density (0.1 less than or equal to bound dye/phosphate less than or equal to 0.2) induced strong quenching of fluorescence and precipitation of chromatin. Precipitation was quantitated by measuring the large-angle (greater than or equal to 15 degrees) light scattering of the cells above 400 nm, i.e., outside the Hoechst 33258/DNA absorption spectrum, as a function of dye concentration. In contrast, the light scattering at 365 nm, i.e., within the absorption spectrum of Hoechst 33258/DNA, was independent of the total dye/phosphate ratio. The coefficient of variation of the light-scattering (greater than or equal to 400 nm) histograms decreased with Hoechst 33258 concentration. Type 2 binding to histone-depleted chromatin was cooperative (Hill-coefficient approximately 2) and the apparent binding constant was 2-3 X 10(5) M-1 as determined from quenching and precipitation data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of recombination of heavy and light chains of a human IgG1 myeloma protein.

The recombination of alkylated H and L chains of a human myeloma protein (Jo) was studied by means of circular dichroism (CD). Marked CD changes were observed at 295 and 235 nm when H and L chains recombined. The change in the CD maximum at 235 nm was followed with time after mixing preparations of H and L chains in the pH range between 4 and 6. The recombination reaction was slow and followed second order kinetics. The observed rate constants were markedly dependent on pH. The pH dependence of the rate constant was analyzed assuming that there are two forms of H chain which are in a pH-dependent equilibrium with each other.     

A Membrane Filter Mounting Method and Spore Stain for Saccharomyces

Sporulated yeast cells seeded on 5 × 10 mm rectangles of ultrathin Millipore membrane filter are stained through the membrane with Ziehl's carbol-fuchsins. This is followed by 0.1% aqueous ruthenium red which both destains and counterstains. The preparations are made permanent with a water mounting medium. By a modification of the procedure microcolonies can be grown on the membranes, stained, and permanently mounted.     

Native-like tertiary structure in the Mucor miehei lipase molten globule state obtained at low pH

Studies on the acid-induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy and binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS). Acid denaturation of the lipase showed loss of secondary structure and alterations in the tertiary structure in the pH range 4 to 2 and 7 to 2 respectively, suggesting that the lipase exists as an acid-unfolded state approximately pH 2.0. A further decrease in pH (from 2.0 to 1.0) resulted in a second transition, which corresponded to the formation of both secondary and tertiary structures. The acid unfolded state at around pH 2.0 has been characterized by significant loss of secondary structure and a small increase in fluorescence intensity with a blue shift of 2 nm, indicating shift of tryptophan residues to less polar environment. Interestingly, the lipase at pH 1.0 exhibits characteristics of molten globule, such as enhanced binding of hydrophobic dye (ANS), native-like secondary structure and slightly altered tryptophanyl environments. That the molten globule of the lipase at pH 1.0 also possesses native-like tertiary structure is an interesting observation made for this lipase.     

Purification of maize streak virus and its relationship to viruses associated with streak diseases of sugar cane and Panicum maximum

Maize streak virus (MSV) was purified by homogenising infected leaf tissue in 0·01 m pH 3·9 phosphate buffer and clarifying the extract with n-butanol (7 ml/100 ml extract). Purified preparations contained particles 20 nm in diameter, some occurring singly, but most occurring in pairs, forming structures of 30 × 20 nm. The sedimentation coefficients of single and paired particles were 54 and 76 S respectively. When centrifuged in sucrose density gradients preparations made by extracting leaves at pH 3·9 gave a single intense light-scattering zone containing paired particles. Preparations made at pH 5·9 or 7·9 gave one or two additional upper zones containing single particles and fragmented material. Preparations treated with 0·05 or 0·1 m ethylene diamine tetra-acetic acid, disodium salt, (EDTA) contained no paired particles, few single particles and much fragmented material. In immunoelectrophoresis, the major component in preparations without EDTA migrated to the cathode whereas that in EDTA-treated preparations migrated to the anode. Virus isolates from streak-diseased sugarcane and guinea grass (Panicum maximum) were serologically related to MSV and had similar particles with identical sedimentation coefficients. No such particles were seen in purified preparations of healthy maize, sugarcane, or guinea grass. The viruses from sugarcane and guinea grass are probably host-adapted and are referred to correctly as the sugarcane and guinea grass strains of MSV. MSV probably contains single-stranded RNA, and the cryptogram is (R)/1:*/*:S/S:S/Au.     

Experimental Cyanine Red: A New Stain for Nucleic Acids and Acid Mucopolysaccharides

A new cationic dye, experimental cyanine red (du Pont), with an absorption maximum of 536 mμ and a pH of 2.9 in 0.5% aqueous solution, is shown to be suitable for staining nucleic acids and tissue materials presumed to contain acid mucopolysaccharides. Mammalian tissues fixed in 10% neutral buffered formalin or Bouin's fluid are dehydrated, embedded in paraffin, sectioned, mounted, deparaffinized, passed through ethanols to water, and stained for 3-30 min in 0.5% experimental cyanine red in water. Differentiation and dehydration in 3 changes (about 1 min each) of n-butanol is followed by clearing in xylene and mounting in resin.     

Species-specific effects on the optical signals of voltage-sensitive dyes

Summary The absorption changes of two merocyanine dyes in response to membrane potential changes were measured on several nueronal preparations to see whether the dyes would be useful in recording from these cells.We were able to record large signals without averaging from barnacle and leech neurons. The greatest signal with WW375 was seen at 750 nm. Much smaller increases in transmitted light intensity were seen at all other wavelengths between 500 and 780 nm. In contrast, vertebrate neuronal preparations produced much smaller signals with an entirely different action spectrum. Essentially the same spectrum was seen in cells of the sympathetic ganglion of the bullfrog,Rana catesbiana, dissociated chick spinal cord neurons, or dissociated rat superior cervical ganglion neurons. In each case an action potential was accompanied by increases in transmitted light intensity between 500 and 600 nm and 730 and 780 nm, and decreases in intensity between 600 and 730 nm with the dye WW375, the best dye tested. Similar results were obtained with dye NK2367 on both vertebrate and invertebrate preparations, except that the spectral properties were shifted 30 nm towards the blue. Both dyes caused some photodynamic damage to the cultured neurons after a few minute's exposure to the illuminating light. Several analogues of these dyes were also tried, but did not produce larger signals.     

Aerobic biodegradation of Azo dye by Bacillus cohnii MTCC 3616; an obligately alkaliphilic bacterium and toxicity evaluation of metabolites by different bioassay systems

An obligate alkaliphilic bacterium Bacillus cohnii MTCC 3616 aerobically decolorized a textile azo dye Direct Red-22 (5,000 mg?l^?1) with 95 % efficiency at 37 °C and pH?9 in 4 h under static conditions. The decolorization of Direct Red-22 (DR-22) was possible through a broad pH (7–11), temperature (10–45 °C), salinity (1–7 %), and dye concentration (5–10 g?l^?1) range. Decolorization of dye was assessed by UV–vis spectrophotometer with reduction of peak intensity at 549 nm (λ _max). Biodegradation of dye was analyzed by Fourier transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). The FTIR spectrum revealed that B. cohnii specifically targeted azo bond (N=N) at 1,614.42 cm^?1 to break down Direct Red-22. Formation of metabolites with different retention times in HPLC analysis further confirmed the degradation of dye. The phytotoxicity test with 5,000 mg?l^?1 of untreated dye showed 80 % germination inhibition in Vigna mungo, 70 % in Sorghum bicolor and 80 % in Vigna radiata. No germination inhibition was noticed in all three plants by DR-22 metabolites at 5,000 mg?l^?1. Biotoxicity test with Artemia salina proved the lethality of the azo dye at LC₅₀ of 4 and 8 % for degraded metabolites by causing death of its nauplii compared to its less toxic-degraded metabolites. Bioaccumulation of dye was observed in the mid-gut of A. salina. The cytogenotoxicity assay on the meristematic root tip cells of Allium cepa further confirmed the cytotoxic nature of azo dye (DR-22) with decrease in mitotic index (0.5 % at 500 ppm) and increase in aberrant index (4.56 %) over 4-h exposure period. Genotoxic damages (lagging chromosome, metaphase cluster, chromosome bridges, and dye accumulation in cytoplasm) were noticed at different stages of cell cycle. The degraded metabolites had negligible cytotoxic and genotoxic effects.