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PubMed1986

Effects of toxin II from the sea anemone Anemonia sulcata on contractile and electrical responses of frog skeletal muscle fibres.

The effects of Anemonia sulcata toxin II (ATX-II) were studied on mechanical and electrical activities of frog muscle fibres isolated from semitendinosus or tibialis anterior muscles of Rana temporaria (2.8-7.7 degrees C). In concentrations ranging between 7.7 and 100 microM, ATX-II greatly potentiated the isometric twitch of single muscle fibres in a time-dependent manner. Increase in twitch amplitude by ATX-II was associated with an increase in time to peak tension and time from peak tension to half relaxation. ATX-II caused no change in maximum force production during fused tetanus, but the tension was maintained for several seconds after the cessation of stimulation. Such long tetanic contractions were also obtained in low-Na Ringer solution, but their duration was somewhat shorter. No specific action of ATX-II was detected on relaxation kinetics during a tetanus. The twitch potentiating effect of ATX-II was markedly increased by 3,4-diaminopyridine. Action potentials recorded from single muscle fibres in the presence of ATX-II showed a delayed onset of repolarization with a reduced rate of fall. In addition, ATX-II caused repetitive spontaneous firing of action potentials after the cessation of tetanic stimulation. ATX-II (3.3 - 10 microM) also increased action potential duration by reducing the rate of repolarization in surface fibres of formamide-treated sartorius or cutaneous pectoris muscles (20 degrees C) stimulated indirectly or directly. The potentiation of twitch force and the prolongation of contractions caused by tetanic stimulation can be attributed to the membrane action of ATX-II, which leads to prolongation of action potentials, to repetitive muscle firing and to the appearance of plateau potentials.

4-Aminopyridine↗
PubMed1977

R loop mapping of the 18S and 28S sequences in the long and short repeating units of Drosophila melanogaster rDNA.

Cleavage of D. melanogaster rDNA with the Eco R1 restriction endonuclease reveals two major classes of repeating units: a long class of 17 kilobases (kb) and a short class of 11.4 kb. R loop mapping has been used to determine the topography of the sequences corresponding to the 18S and 28S rRNAs in both classes, including a cloned member (Dm103) of the long class. This mapping procedure derives from a novel reaction that we discovered between single-stranded RNA and homologous regions in duplex DNA molecules. In high formamide and at an elevated temperature, the RNA pairs with one of the two DNA strands in the region of homology to form an R loop in which one element is an RNA/DNA duplex and the other is single-stranded DNA (Figure 1). Mapping is accomplished by visualization of R loops in the electron microscope. The R loop map of Dm103 parallels that determined independently by Glover and Hogness (1977) from an analysis of its restriction fragments. Both maps indicate that the 28S rDNA in this cloned unit is divided into two blocks by a 5 kb insertion segment. R loop mapping of a population of long units obtained directly from the rDNA (that is, without cloning) has demonstrated that this interruption of the sequence coding for the 28S rRNA is a characteristic of the long class. By contrast, the 28S rDNA in the short units that we examined is not interrupted by an insertion segment. Otherwise, the R loop maps of the long and short units do not differ significantly. The two classes therefore correspond to repeating units that do (IN+) or do not (IN-) contain an insertion segment. Models for the transcription and function of these two classes of repeating units are discussed.

Animals↗
PubMed1988

Macromolecular crowding extends the range of conditions under which DNA polymerase is functional.

The nick-translation reaction of E. coli DNA polymerase I (Pol I) was used as a model system to demonstrate the ability of macromolecular crowding to alter the response of an enzyme to a number of basic parameters, such as pH, temperature or inhibitors. In the presence of high concentrations of non-specific polymers, nick translation occurred under a variety of otherwise strongly inhibitory conditions. The conditions tested included a range of pH values or temperatures or inhibitory concentrations of urea, formamide or ethidium bromide. These crowding effects are accentuated at higher ionic strengths, suggesting their origin in increased binding between the polymerase and its DNA template-primer under crowded conditions. Kinetic measurements were consistent with such a mechanism.

DNA↗
PubMed1995

Sucrose reduces the efficiency of protein denaturation by a chaotropic agent.

Sugars and polyols are used to stabilize proteins. The degree of stabilization conferred on a model protein by sucrose was calculated in terms of the free energy of folding. Phosphoglycerate kinase (PGK) was denatured by guanidine hydrochloride (GuHCl) in different sucrose concentrations. The linear extrapolation method [1,2] was used to calculate the free energy of folding in the absence of denaturant. Although sucrose increased the concentration of GuHCl required to unfold the protein, the free energy of folding in water was unchanged. In order to probe the nature of the stabilizing effect of sucrose, an FT-Raman spectroscopic study of denaturant-polyol systems was undertaken. Investigations of interactions between GuHCl, urea or formamide and polyhydric compounds, revealed no evidence for hydrogen bonding or dipole-dipole associations. Polyhydric compounds caused minor changes in denaturant spectra although the converse was not observed. The structure of deuterated water changed on addition of denaturants. For non-ionic denaturants, addition of polyhydric solutes countered this change in water structure. Thus polyhydric compounds oppose the effect of denaturants on water structure. The observed increase in GuHCl concentration required to unfold PGK in the presence of sucrose may be attributed to this property of sucrose.

Formamides↗
PubMed1984

Study on in vivo and in vitro metabolism of dimethylformamide in male and female rats.

The study of dimethylformamide (DMF) metabolism by rat tissues in vitro indicates that formaldehyde is not a metabolic product as previously reported [1]. Furthermore, no other monocarbon derivative (CO, CH3OH, HCOOH, CH4) was detected when DMF was incubated with a fortified liver preparation. One metabolic product is methylhydroxymethylformamide (DMF-OH) measured as N-methylformamide (NMF) due to the breakdown of the hydroxymethyl group during gas chromatography. It was usually believed that the main metabolite excreted in urine following administration of DMF to male and female rats was NMF. The results of this study indicate that DMF-OH constitutes the main metabolite in vivo. A quantitatively less important urinary metabolite, hydroxymethylformamide (NMF-OH), is determined as formamide (F) by gas chromatography. In male and female rats, partial hepatectomy reduces markedly the in vivo biotransformation of DMF. Following administration of DMF or NMF, the total amount of metabolites (DMF-OH and/or NMF-OH) excreted in urine is identical in both sexes, but female rats excrete more unchanged parent compound than male rats. The rate of NMF-OH excretion in urine following high doses of DMF supports the hypothesis that DMF may inhibit its own biotransformation.

Aminopyrine↗
PubMed1991

Reversed effect of caffeine on non-contractile and contractile Ca2+ mobilization operated by acetylcholine receptor in mouse diaphragm muscle.

Caffeine effects on contractile and acetylcholine receptor-related non-contractile Ca2+ mobilization were investigated in phrenic nerve-diaphragm muscles of mice with neostigmine. Caffeine enhanced at 0.25-5 mM, and decreased at 7-20 mM the total amount of contractile Ca(2+)-aequorin luminescence (Ca2+ transients), but only decreased at 2-10 mM non-contractile Ca2+ transients. Pretreatment with formamide (2 M for 30 min) abolished contractile Ca2+ transients, but did not affect non-contractile ones. These results suggest that non-contractile Ca2+ mobilization is not due to direct Ca2+ release from sarcoplasmic reticulum, but due to direct modulation by nicotinic acetylcholine receptor.

Animals↗
PubMed1994

Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases.

We describe conditions that improve the specificity of amplification of a G + C-rich (57% G + C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that accounts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal conditions for amplification of the segment of the G + C-rich satellite, we used two genetically engineered enzymes, AmpliTaq DNA polymerase and AmpliTaq DNA polymerase, Stoffel fragment (SF), and a number of denaturants or co-solvents. In the absence of denaturants or co-solvents, amplified products of both enzymes contained non-specific bands upon gel electrophoresis. Addition of certain denaturants or co-solvents to PCR mixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF was more specific and efficient. The products of AmpliTaq DNA polymerase included one or more extra bands, even in the presence of denaturants or co-solvents, except for glycerol or DMSO.

Base Sequence↗
PubMed1989

Polyethylene glycol increases specificity of hybridization typing of HPV specimens.

Different hybridization conditions for the typing of human papillomaviruses (HPV) in clinical biopsy specimens were tested. The stringency of the hybridization reaction was varied by altering the formamide concentration in the solution. Two polymers, dextran sulphate and polyethylene glycol (PEG), were compared as accelerators of the hybridization reaction. The PEG-containing hybridization solution was found to be suitable for typing clinical HPV specimens. Single-stranded RNA probes proved to be more specific than DNA probes in typing clinical specimens. Specimens that were positive both for HPV6 and HPV11 in spot hybridization were confirmed by Southern hybridization. In total, 467 biopsy specimens from genital, anal, oral, aural and nasal lesions were examined for HPV6, HPV11, HPV16 and HPV18 DNA by spot hybridization. Approximately one-third of the specimens were positive for these types.

DNA Probes↗
PubMed1996

Peroxyl radical formation in aqueous solutions of N,N-dimethylformamide, N-methylformamide, and dimethylsulfoxide by ultrasound: implications for sonosensitized cell killing.

Sonodynamic therapy, which refers to a synergistic effect of drugs and ultrasound, is a promising new modality for cancer treatment. The sonodynamic effect was found for a number of structurally unrelated compounds, and the underlying mechanisms are still unknown. Recently, Jeffers et al. (J. Acoust. Soc. Am. 97:669-676; 1995) have shown that the sonodynamic action of nontoxic concentrations of N,N-dimethylformamide (DMF), N-methyl formamide (MMF), and dimethylsulfoxide (DMSO) combined with ultrasound, on killing of cultured HL-60 human promyelocytic leukemia cells, and attributed this toxic effect to unknown short lived reactive species produced from these solutes by ultrasonic cavitation. Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in nitrogen-saturated aqueous solutions of DMF, MMF, or DMSO exposed to 50 kHz ultrasound, we detected formation of .CH3 and .CH2N(CH3)CHO radical adducts for DMF, mostly .CH2NHCHO adducts for MMF, and .CH3 adducts for DMSO. These radicals were formed either by reactions of the solutes with ultrasound-generated .H and .OH radicals (such as .CH2R-type radicals in DMF and MMF, and .CH3 radicals in DMSO), or by direct pyrolysis of the weak bonds in the solute molecules (e.g., .CH3 radicals from DMF). In air-saturated sonicated solutions these carbon centered radicals were converted to the corresponding peroxyl radicals and spin trapped with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); .OOCH2N(CH3)CHO radicals were identified in DMF, .OOCH2NHCHO radicals in MMF, and .OOCH3 radicals in DMSO solutions. We suggest that these radical species by virtue of their longer lifetimes and higher selectivity, compared to .OH radicals, which are also formed in sonicated solutions, are the species responsible for sonodynamic cell killing by the combined effect of ultrasound with DMF, MMF, or DMSO.

Benzenesulfonates↗
PubMed2005

Denaturing RNA electrophoresis in TAE agarose gels.

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.

Acetates↗
PubMed2004

Dextran sulfate provides a quantitative and quick microarray hybridization reaction.

Microarray technology is a powerful tool to speed up genomics study, yet many technical aspects need to be improved. The hybridization reaction of microarray experiments is carried out for 16h or overnight in order to obtain reasonably strong signals for analysis in the presence of high salt buffer, like SSC. However, the quantitative aspect of microarray hybridization has seldom been investigated. In this study, we showed that higher overall signals from hybridization were achieved in a buffer system containing dextran sulfate, which can accelerate the kinetics of reaction by increasing the local concentration of the reactants. The dextran sulfate containing hybridization solution increases the reaction 4-fold (median) for cDNA microarray and 29-fold for oligonucleotide microarray. More importantly, the solution also provides a quantitative hybridization reaction, where the hybridization signals are proportional to the abundance of transcript added. The enhancement in the kinetics of hybridization is due to both dextran sulfate and formamide present in the solution, but the effect is not due to the higher temperature used during the reaction. With a slightly longer reaction time the hybridization reaction with the solution allows the detection of hybridization signals from rare transcripts that is not possible with regular hybridization buffers. With appropriate washing, the enhancement of kinetics by the solution does not increase the background signals at all, allowing higher signal-to-noise ratios to be achieved.

Arabidopsis↗
PubMed2006

Differential distribution and regulation of mouse cardiac Na+/K+-ATPase alpha1 and alpha2 subunits in T-tubule and surface sarcolemmal membranes.

OBJECTIVES: Two Na+/K+-ATPase (NKA) alpha-subunit isoforms, alpha1 and alpha2, are expressed in the adult mouse heart. The subcellular distribution of these isoforms in T-tubule and surface sarcolemmal (SSL) membranes and their regulation by cAMP-dependent protein kinase (PKA) is unclear. METHODS: We used formamide-induced detubulation of mouse ventricular myocytes to investigate differential functional distribution and regulation by PKA of alpha1 and alpha2 in T-tubule versus SSL membranes by measuring NKA current (I(pump)) and NKA-mediated Na+ efflux (-d[Na](i)/dt). RESULTS: I(pump) is composed of 88% alpha(1)-mediated I(pump) (Ialpha1) and 12% alpha2-mediated I(pump) (Ialpha2). alpha1 and alpha2 subunits demonstrate distinct ouabain affinities (105+/-6 and 0.3+/-0.1 micromol/L respectively) but similar affinity for intracellular Na+ (K(1/2)Na+ of 16.6+/-0.8 and 16.7+/-2.6 mmol/L respectively). Detubulation reduced (i) I(pump) density (1.42+/-0.1 to 1.20+/-0.04 pA/pF), (ii) cell capacitance (181+/-12 to 127+/-17 pF), and (iii) Ialpha2 contribution (12 to 6%). Total I(pump) density was approximately 60% higher in T-tubule (1.94 pA/pF, derived) vs. SSL membranes. Although T-tubule membranes represent only 30% of total surface area, they generate approximately 70% of Ialpha2 and approximately 37% of Ialpha1. Ialpha1 density was substantially higher than Ialpha2 in SSL (Ialpha1:Ialpha2 = 16:1) but this was markedly reduced in T-tubules (4:1). In addition to differential localisation, isoprenaline (ISO, 1 micromol/L) significantly increased alpha1-mediated NKA Na+ affinity (from 16.6+/-0.8 to 13.3+/-1.4 mmol/L) and caused a small increase in maximal NKA Na+ efflux rate. ISO had no effect on alpha2-mediated NKA activity. CONCLUSION: These data suggest that NKA alpha1 and alpha2 subunits are differentially localised and regulated by PKA in T-tubule and SSL membranes and may have distinct regulatory roles in cardiac excitation-contraction coupling.

Adrenergic beta-Agonists↗
PubMed2005

Adsorption on hydrophobized surfaces: clusters and self-organization.

The arrangement of liquid molecules on surfaces bristling with alkyl chains is deduced from adsorption studies, X-ray powder diffraction data, and microcalorimetric measurements of swelling-type layered materials, especially clay minerals. Small polar molecules such as water, ethanol, formamide, dimethylsulfoxide, and aromatic compounds are clustered between the alkyl chains pointing away from the surface. The energetic contribution related to the movement of the alkyl chains from direct contact with the surface atoms into upright positions is decisive. The importance of the interactions between the liquid molecules on the structure of the adsorption layer is clearly indicated by the changes of the adsorption layer thickness by salt addition. Thermodynamic data are obtained from surface excess adsorption isotherms from binary liquid mixtures combined with microcalorimetric measurements. Long-chain adsorptives such as long-chain alcohols interact with the surface alkyl chains by forming stable bimolecular films. These films undergo a series of higher-order phase transitions into kink- and gauche-block structures as the consequence of rotational isomerization of the alkyl chains. Such transitions are considered elementary processes in self-assembling films (layer-by-layer deposition, fuzzy films, Langmuir-Blodgett technique), and lipid membranes.

Adsorption↗
PubMed2005

Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro.

Polysaccharide extracted from Ulva pertusa (Chlorophyta) is a group of sulfated heteropolysaccharide; for simplicity, the sulfated polysaccharide is referred to as ulvan in this paper. In this study, different sulfate content ulvans were prepared with sulfur trioxide/N,N-dimethylformamide (SO3-DMF) in formamide, and their antioxidant activities were investigated including scavenging activity of superoxide and hydroxyl radicals, reducing power and metal chelating ability. As expected, we obtained several satisfying results, as follows: firstly, high sulfate content ulvans had more effective scavenging activity on hydroxyl radical than natural ulvan. Secondly, comparing with natural ulvan, high sulfate content ulvans exhibited stronger reducing power. Thirdly, HU4 (sulfate content, 30.8%) and HU5 (sulfate content, 32.8%) showed more pronounce chelating ability on ferrous ion at high concentration than other samples.

Antioxidants↗
PubMed2005

Tandem mobility mass spectrometry study of electrosprayed tetraheptyl ammonium bromide clusters.

Multiply charged electrospray ions from concentrated solutions of Heptyl4N+Br- (designated A+B- hereafter) in formamide are analyzed mass spectrometrically (MS) following mobility selection in ambient air in a differential mobility analyzer (DMA). Most of the sharp mobility peaks seen are identified as (AB)(n)A+ clusters, with 0 < or = n < ot = 5. One anomalously abundant and mobile ion is identified as NH4+(AB)4. Six ions in the (AB)n(A+)2 series are also identified, completing and correcting earlier mobility data for singly and doubly charged ions up to masses of almost 9000 Da. The more mobile of two broad humps seen in the mobility spectrum includes m/z values approximately from 2500 up to 12,000 Da. It is formed primarily by multiply charged (AB)n(A+)z clusters with multiple ammonium bromide adducts. Because of overlapping of many peaks of different m/z and charge state z, only a few individual species can be identified by MS alone in this highly congested region. However, the spectral simplification brought about by mobility selection upstream of the MS reveals a series of broad modulations in m/z space, with all ions resolved in the second, third, ...sixth modulation being in charge states z = 2, 3, ...6, respectively. Extrapolation of this trend beyond the sixth wave fixes the ion charge state (in some cases up to z = 15) and mass (beyond m = 175,000 u). This wavy structure had been previously observed and explained in terms of ion evaporation kinetics from volatile drops, though without mass identification. All observations indicate that the clusters are formed as charged residues, but their charge state is fixed by the Iribarne-Thomson ion evaporation mechanism. Consequently, the measured curve of cluster diameter versus z yields the two parameters governing ion evaporation kinetics. Clusters with z > 1 and electrical mobility Z > 0.495 cm2/V/s are metastable and evaporate a singly charged cluster, probably (AB)2A+, between the DMA and the MS. Plotting the electrical mobilities Z of the clusters in the form (z/Z)1/2 versus m(1/3) (both proportional to cluster diameter) collapse the data for all cluster sizes and charge states into one single straight line for Z below 0.495 cm2/V/s. This linear relation reveals a uniform apparent cluster density of 0.935 g/cm3 and an effective hard-sphere diameter of the air molecules of 0.44 nm. An anomalous mobility increase is observed at diameters below 3 nm.

Bromides↗
PubMed2005

FORMAzol as an RNA storage medium: a cautionary note when performing RT-PCR.

The labile nature of RNA demands careful procedures for its extraction, purification, and storage. Generally, RNA is solubilized in aqueous buffers or organic solvents, or precipitated with alcohol and then kept at -20 degrees C or colder. A commercially available product for RNA storage is FORMAzol(R) (Molecular Research Center). We began using FORMAzol because the Application Notes from the Product Description sheet claims that reverse-transcription (RT) is not inhibited so long as FORMAzol does not exceed 5% (v/v) in the reaction mix. This is ostensibly more convenient than having to precipitate RNA, resolubilizing it in water or buffer, and then proceeding with RT-PCR. However, amplicon yields for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were poor when using RNA directly from FORMAzol, even though its final concentration was typically much less than 5%. By contrast, satisfactory RT-PCR products were obtained with RNA stored frozen in water or that had been resolubilized from alcohol precipitates. When RT-PCR was then performed on ethanol-precipitated and resolubilized RNA from FORMAzol, yields of GAPDH amplicons were acceptable. Although a revision to the FORMAzol Product Description sheet is now available at the manufacturer's website (http://www.mrcgene.com/formazol.htm), if users of the product implicitly follow the directions found in the package insert sheet-not being aware of the inhibitory effects of formamide (the denaturant in FORMAzol)-unsatisfactory results may be obtained from RT-PCR experiments. It is suggested that FORMAzol only be used for RNA storage and that RNA be precipitated with alcohol, washed, and resolubilized prior to use.

Chemical Precipitation↗
PubMed2006

Detection and quantification of Vibrio populations using denaturant gradient gel electrophoresis.

Bacteria affiliated with the genus Vibrio are endemic in marine and estuarine ecosystems and are also found in many freshwater environments. Vibrios can enter viable but non-culturable states and since many species are pathogenic, there is a great need for culture-independent methods that identify and quantify multiple Vibrio populations. We adopted Vibrio-specific 16S rRNA-directed primers and a competitive PCR protocol (QC-PCR; [Thompson, J.R., Randa, M.A., Marcelino, L.A., Tomita-Mitchell, A., Lim, E., Polz, M.F., 2004b. Diversity and dynamics of a North Atlantic coastal Vibrio community. Appl. Environ. Microbiol. 70, 4103-4110]) for separation and quantification of Vibrio populations using denaturant gradient gel electrophoresis (DGGE). Sixteen Vibrio isolates and eight environmental samples were used to assess the precision and resolution of the method. A 45-70% gradient of Urea and formamide enabled separation of Vibrio populations with single nucleotide differences in the amplified fragment. A titration curve for the QC-PCR-DGGE, verified by amending surface water bacterioplankton samples with up to 3 x 10(5)Vibrio cholerae cells, could be approximated by a linear regression of log-transformed values (R(2)=0.96). The limit of detection for single populations was 180 cells per extracted sample or about 4 cells per PCR reaction. Environmental samples from the southern Stockholm archipelago in the Baltic Sea and the more saline coastal waters of Skagerrak each carried between 2 and 6 Vibrio populations, and there were major differences between the locations. Notably, multiple Vibrio populations could be detected and quantified against a background of native bacterioplankton exceeding Vibrio population abundance by more than 6 orders of magnitude. Putative identification based on migration in the DGGE gel was verified by parallel cloning and sequencing of PCR products, and representative clones were also characterized by DGGE. This general approach could also be useful for targeting other phylogenetically constrained bacterial groups and assess their abundance and distribution in complex environmental settings.

DNA, Bacterial↗