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PubMed1995

Biological monitoring of workers exposed to N-N-dimethylformamide. II. Dimethylformamide and its metabolites in urine of exposed workers.

N,N-Dimethylformamide (DMF) exposure was monitored in a synthetic leather factory; at the same time, urinary dimethylformamide and its metabolites were measured in urine samples collected before and at the end of workshifts. The study was run during two different periods. During the first phase ten workers were observed for 3 days (Monday, Tuesday and Wednesday) in the same week. In the second phase 16 workers were involved in the study on a Friday and on the following Monday. Urinary DMF, as well as hydroxymethyl-N-methylformamide and hydroxymethylformamide [measured as N-methylformamide (NMF) and formamide, respectively], were measured as a "physiological" product in subjects not exposed to dimethylformamide. Environmental exposure to DMF ranged between 10 and 25 mg/m3. The unmodified solvent found in urine collected at the end of the exposure was significantly related to the environmental concentrations of DMF; its urinary concentrations were found to range between 0.1 and 1 mg/l. Higher concentrations of NMF (mean 23.3 mg/l) and formamide (24.7 mg/l) were measured in urine samples collected at the end of workshifts. The same concentrations were related to individual exposures to DMF. N-Acetyl-S-(N-methylcarbamoyl)cysteine in the urine of workers exposed to DMF showed a mean concentration of 40.4 mg/l on Friday (before and after the workshift) and a mean concentration of 10.3 mg/l on Monday. Its slow kinetic profile favours its body accumulation during the working week.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗
PubMed2002

K(+) current distribution in rat sub-epicardial ventricular myocytes.

In the rat ventricle, the transient outward K(+) current ( I(TO)) is carried by heteromeric channels composed of Kv4.2 and Kv4.3. However its distribution in the cell membrane is unclear: immunohistochemical studies of Kv4.2 distribution in the cardiac ventricular cell membrane have given equivocal results, and there are no corresponding studies of Kv4.3. We therefore used detubulated cardiac cells to investigate the functional distribution of I(TO) between the t-tubules and surface membrane. I(TO), the delayed rectifier ( I(K)), the inward rectifier ( I(K1)) and steady-state ( I(SS)) K(+) currents were monitored using the patch-clamp technique in control and formamide-treated (detubulated) cells from rat left ventricular sub-epicardium. Formamide treatment decreased cell capacitance by 20%, did not significantly change the density of I(TO), I(K) or I(K1) but decreased the density of I(SS) and L-type Ca current ( I(Ca)). These data suggest that I(TO), I(K), and I(K1) are uniformly distributed between the surface and t-tubule membranes, but that I(SS) and I(Ca) are concentrated in the t-tubules.

Animals↗
PubMed1991

A single-reaction method for DNA sequence determination.

A chemical method for the determination of DNA sequence is presented. Heating of DNA, labeled at a single 3' extremity, in the presence of formamide results in efficient cleavage of phosphodiester bonds 3' of A, G, and C residues. The relative efficiency is A = G greater than C. The bias between A and G is solved by a simple pretreatment (photoreaction in methylene blue) followed by heating in formamide. The entire procedure does not require any intermediate purification step or handling of hazardous chemicals and allows determination of the sequence on only two electrophoretic lanes. Its simplicity and rapidity favor automatization.

Base Sequence↗
PubMed1991

Comparative study of chromatography on thin layers impregnated with organic stationary phases. Chromatographic separation of nitrophenols.

The chromatographic behaviour of nitrophenols on thin layers of silica gel and cellulose was compared, both without impregnation and impregnated with non-aqueous polar stationary phases (formamide, dimethylformamide) and less polar stationary phases (liquid paraffin, octan-1-ol, 1-bromonaphthalene). Cellulose is preferred when using formamide or dimethylformamide if a pure partition process is required. For each particular analyte a certain amount of the stationary phase is always required to suppress the adsorption activity of silica gel. Separation by reversed-phase thin-layer chromatography is strongly affected by the type of stationary phase (the possibility of forming charge-transfer complexes with 1-bromonaphthalene) and its support (the acidic properties of silica gel and its adsorption activity), the mobile phase (content of organic modifier, pH, presence of salts) and the properties of the solutes (polarity, ionizability).

Cellulose↗
PubMed2005

Relevance of Frank's solvent classification as typically aqueous and typically non-aqueous to activities of firefly luciferase, alcohol dehydrogenase, and alpha-chymotrypsin in aqueous binaries.

Effects of cosolvent concentration on activity of fire fly luciferase, alpha-chymotrypsin, and alcohol dehydrogenase from baker's yeast (Saccharomyces cerevisiae) have been studied for several solvents with varying hydrophobicities (logP from +1.0 to -1.65) and polarities (dielectric constant from 7.4 to 109). The inhibitory effect of the cosolvent is examined in light of Frank's classification of solvents into 'typically aqueous (TA)' and 'typically non-aqueous (TNA).' The solvent concentration at which the enzyme activity decreases to half, the C(50) values, for TA solvents such as 1-cyclohexyl-2-pyrrolidinone, 2-butoxyethanol, 1-methyl-2-pyrrolidinone, tetrahydrofuran, t-butanol, and ethanol correlate quite well with their critical hydrophobic interaction concentration, rather than logP, while those for TNA solvents such as acetonitrile, dimethyl formamide, formamide, and dimethyl sulfoxide correlate well with logP. The interactions of TA solvents with proteins appear to be governed mainly by hydrophobic interactions while both hydrophobic and hydrophilic interactions play important role in case of TNA solvents.

Acetonitriles↗
PubMed2006

A micellar model system for the role of zeaxanthin in the non-photochemical quenching process of photosynthesis--chlorophyll fluorescence quenching by the xanthophylls.

To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2-0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary-an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and beta-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and beta-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and beta-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non-photochemical quenching process in photosynthesis are discussed.

Carotenoids↗
PubMed2001

Enzyme-linked immunosorbent assay (ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening.

We have developed a solid-phase ELISA for the specific and sensitive detection of apoptotic cells. This method is based on the ability of a monoclonal antibody (MAb) against single-stranded DNA (ssDNA) to specifically identify apoptotic cells. The assay involves binding of cells to 96-well microtiter plates, treatment of the attached cells with formamide to denature DNA in apoptotic cells and one-step staining of the denatured DNA with a mixture of anti-ssDNA MAb and peroxidase-conjugated anti-mouse IgM. A near linear increase in signal was seen as the number of apoptotic cells increased from 500 to 5000. Untreated and necrotic cells or cells with single-stranded DNA breaks induced by H(2)O(2) did not produce signal above the background level. In leukemic cell cultures grown, treated with ID(50) concentration of etoposide, stained and analyzed in the same 96-well assay plate, intense ELISA signal was detected. The ratio of absorbance values from drug resistant and drug-sensitive cell lines treated with etoposide was in agreement with the degree of resistance determined by growth inhibition assays. These data show that this ELISA has sufficient sensitivity for use in drug screening protocols. In breast cancer cell cultures treated with cisplatin, ELISA absorbance increased only after treatment with drug concentrations 10-fold higher than concentrations inducing 95% growth inhibition. In cultures treated with staurosporine, there was a near linear relation between the ELISA absorbance values and cytotoxicity in the range of 15-92% growth inhibition. The absence of apoptotic signal in breast cancer cells treated with cytotoxic concentrations of cisplatin indicated that this drug kills cells by non-apoptotic mechanisms, whereas apoptosis was the dominant mechanism of cell death caused by staurosporine. The formamide-MAb apoptosis ELISA described here may provide a basis for high-throughput screening of drugs based on their ability to induce or suppress apoptosis.

Antibodies, Monoclonal↗
PubMed2001

Chiral separations of 1,3,4-thia- and 1,3,4-selenadiazine derivatives by use of non-aqueous capillary electrophoresis.

Our aim was to establish suitable conditions for the chiral separation of 12 1,3,4-thia- and 1,3,4-selenadiazine derivatives; some of them were identified in screening tests as potential antituberculotics. To overcome possible problems with the water insolubility of most analytes, we profited by the advantages of non-aqueous capillary electrophoresis. Methanol, formamide, and a mixture of formamide with acetonitrile (1:2, v/v) were used as separation media. Hydroxyethyl-, hydroxypropyl-, and methyl-beta-cyclodextrin were applied as chiral selectors in concentrations of 200 mM. Besides the effect of these different electrophoretic media and selectors, we also investigated the consequences of using different electrolytes (25 mM ammonium acetate/1 M acetic acid and 25 mM citric acid/12.5 mM TRIS). Distinct differences of the separation factors in the different separation media were observed. Depending on structure characteristics of the analytes, we established clear classifications to these cyclodextrins (CD), which were most appropriate for the separation of the enantiomers of the particular analytes.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1990

Self-cleavage of hepatitis delta virus genomic strand RNA is enhanced under partially denaturing conditions.

Self-cleavage of a polyribonucleotide containing an autocleaving sequence from the genomic strand of hepatitis delta virus was enhanced by conditions that destabilized RNA structure. Self-cleavage of the transcripts used in this study required Mg2+ (or another divalent cation), and in the absence of denaturants, maximum cleavage was observed at very low Mg2+ concentrations (0.05-0.1 mM). However, at 37 degrees C and in the presence of 2-10 mM Mg2+ the rate of cleavage was increased as much as 50-fold with the addition of urea to 5 M or formamide to 10 M. Cleavage was prevented by higher concentrations of the same reagents (9.5 M urea or 22.5 M formamide), presumably because a structure required for self-cleavage is disrupted by strongly denaturing conditions. In contrast to a previous report [Wu, H.-N., & Lai, M. M. C. (1989) Science 243, 652-654], we find that chelating Mg2+ with EDTA terminates the cleavage reaction without promoting measurable amounts of ligation of the cleavage products. The ability of denaturants to promote rapid self-cleavage in vitro raises the possibility that an unidentified factor could have a similar effect in vivo.

Base Sequence↗
PubMed2005

Distinct transport selectivity of two structural subclasses of the nodulin-like intrinsic protein family of plant aquaglyceroporin channels.

Major intrinsic proteins (MIPs) are a diverse class of integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray structures of MIPs indicate that a tetrad of residues (the ar/R region) form a narrow pore constriction that constitutes the selectivity filter. In comparison with mammalian and microbial species, plants have a greater number and diversity of MIPs with greater than 30 genes encoding four phylogenetic subfamilies with eight different classes of ar/R sequences. The nodulin 26-like intrinsic protein (NIP) subfamily in Arabidopsis can be subdivided into two ar/R subgroups: the NIP subgroup I, which resembles the archetype of the family, soybean nodulin 26, and the NIP subgroup II, which is represented by the Arabidopsis protein AtNIP6;1. These two NIPs differ principally by the substitution of a conserved alanine (NIP subgroup II) for a conserved tryptophan (NIP subgroup I) in the helix 2 position (H2) of the ar/R filter. A comparison of the water and solute tranport properties of the two proteins was performed by expression in Xenopus laevis oocytes. Nodulin 26 is an aquaglyceroporin with a modest osmotic water permeability (P(f)) and the ability to transport uncharged solutes such as glycerol and formamide. In constrast, AtNIP6;1 showed no measurable water permeability but transported glycerol, formamide, as well as larger solutes that were impermeable to nodulin 26. By site-directed mutagenesis, we show that the H2 position is the crucial determinant that confers these transport behaviors. A comparison of the NIPs and tonoplast-intrinsic proteins (TIP) shows that the H2 residue can predict the transport profile for water and glycerol with histidine found in TIP-like aquaporins, tryptophan found in aquaglyceroporins (NIP I), and alanine found in water-impermeable glyceroporins (AtNIP6;1).

Amino Acid Sequence↗
PubMed2006

Purification and functional characterization of aquaporin-8.

BACKGROUND INFORMATION: Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0-AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported. RESULTS: We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped-flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2-mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8-injected oocytes showed significantly higher [14C]methylammonium uptake than water-injected oocytes. CONCLUSIONS: In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid-base equilibrium.

Amino Acid Sequence↗
PubMed1969

Deoxyribonucleic acid-ribonucleic acid hybridization. Annealing and quantitative recovery of intact ribosomal ribonucleic acid molecules from hybrids.

A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide-0.33m-potassium chloride-10mm-tris-hydrochloric acid buffer, pH7.4, at 33 degrees for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.

Buffers↗
PubMed1972

Effect of compounds of the urea-guanidinium class on renaturation and thermal stability of acid-soluble collagen.

The effects of guanidinium salts in decreasing the renaturation rate and lowering the thermal stability of acid-soluble calf-skin collagen have been compared with those of formamide and urea. With the exception of guanidinium sulphate at higher concentrations, no qualitative differences were apparent in the effects of these perturbants, which thus differed only in molar activity. Activity variation in the guanidinium salts reflected a net effect resulting from additivity of cation and anion contributions. As observed in other protein systems, lyotropic activity increased in the series formamide<urea<guanidinium ion, and in the guanidinium salts in the anion order fluoride<sulphate<chloride<bromide<nitrate<iodide. Low activities of guanidinium fluoride and sulphate were attributable to counter-effects of the anions, which acted as structural stabilizers. Changes in renaturation kinetics induced by either temperature or added perturbants appeared to conform with the Flory-Weaver model for the collagen transition. Additivity and non-specificity of the observed effects are discussed with particular reference to a common mechanism involving weak, non-saturated binding of perturbants at protein peptide groups.

Animals↗
PubMed1980

Isoelectric-focusing properties and carbohydrate content of pea (Pisum sativum) legumin.

Legumin from pea (Pisum sativum) is a molecule made up of six pairs of subunits, each pair consisting of an ;acidic' subunit (mol.wt. about 40000) and a ;basic' subunit (mol.wt. about 20000) linked by one or more disulphide bonds. The heterogeneity of legumin has been investigated by isoelectric focusing; undissociated legumin could not be focused satisfactorily, but legumin subunits could be analysed under dissociating conditions. 8m-Urea was not found to be a satisfactory medium for isoelectric focusing of legumin, as the ;basic' subunits showed a shift in pI with time of incubation in urea. A new dissociating medium for isoelectric focusing, namely 50% (v/v) formamide, was used for analysis of legumin, which gave pI values of 5.0-5.3 for the ;acidic' subunits, and 8.3-8.7 for the ;basic' subunits. Both types of subunits were shown to be heterogeneous in charge and molecular weight by two-dimensional analysis employing isoelectric focusing in the first dimension and sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the second. The ;basic' and ;acidic' subunits of legumin were separated on the preparative scale by ion-exchange chromatography in 50% formamide. Carbohydrate attached to the protein was investigated as a possible cause of the heterogeneity of legumin subunits. However, both a fluorescent-labelling technique and a sensitive radioactive-labelling technique failed to show any carbohydrate bound to legumin subunits, and it was concluded that legumin is not a glycoprotein.

Carbohydrates↗
PubMed2004

Double proton transfer behavior and one-electron oxidation effect in double H-bonded glycinamide-formic acid complex.

The behavior of double proton transfer occurring in a representative glycinamide-formic acid complex has been investigated at the B3LYP/6-311 + + G( * *) level of theory. Thermodynamic and, especially, kinetic parameters, such as tautomeric energy, equilibrium constant, and barrier heights have been discussed, respectively. The relevant quantities involved in the double proton transfer process, such as geometrical changes, interaction energies, and intrinsic reaction coordinate calculations have also been studied. Computational results show that the participation of a formic acid molecule favors the proceeding of the proton transfer for glycinamide compared with that without mediate-assisted case. The double proton transfer process proceeds with a concerted mechanism rather than a stepwise one since no ion-pair complexes have been located during the proton transfer process. The calculated barrier heights are 11.48 and 0.85 kcal/mol for the forward and reverse directions, respectively. However, both of them have been reduced by 2.95 and 2.61 kcal/mol to 8.53 and -1.76 kcal/mol if further inclusion of zero-point vibrational energy corrections, where the negative barrier height implies that the reverse reaction should proceed with barrierless spontaneously, analogous to that occurring between glycinamide and formamide. Furthermore, solvent effects on the thermodynamic and kinetic processes have also been predicted qualitatively employing the isodensity surface polarized continuum model within the framework of the self-consistent reaction field theory. Additionally, the oxidation process for the double H-bonded glycinamide-formic acid complex has also been investigated. Contrary to that neutral form possessing a pair of two parallel intermolecular H bonds, only a single H bond with a comparable strength has been found in its ionized form. The vertical and adiabatic ionization potentials for the neutral complex have been determined to be about 9.40 and 8.69 eV, respectively, where ionization is mainly localized on the glycinamide fragment. Like that ionized glycinamide-formamide complex, the proton transfer in the ionized complex is characterized by a single-well potential, implying that the proton initially attached to amide N4 in the glycinamide fragment cannot be transferred to carbonyl O13 in the formic acid fragment at the geometry of the optimized complex.

Electrons↗
PubMed2001

Dimethyl sulfoxide affects the selection of splice sites.

Depending on the cell lines and cell types, dimethyl sulfoxide (Me2SO) can induce or block cell differentiation and apoptosis. Although Me2SO treatment alters many levels of gene expression, the molecular processes that are directly affected by Me2SO have not been clearly identified. Here, we report that Me2SO affects splice site selection on model pre-mRNAs incubated in a nuclear extract prepared from HeLa cells. A shift toward the proximal pair of splice sites was observed on pre-mRNAs carrying competing 5'-splice sites or competing 3'-splice sites. Because the activity of recombinant hnRNP A1 protein was similar when added to extracts containing or lacking Me2SO, the activity of endogenous A1 proteins is probably not affected by Me2SO. Notably, in a manner reminiscent of SR proteins, Me2SO activated splicing in a HeLa S100 extract. Moreover, the activity of recombinant SR proteins in splice site selection in vitro was improved by Me2SO. Polar solvents like DMF and formamide similarly modulated splice site selection in vitro but formamide did not activate a HeLa S100 extract. We propose that Me2SO improves ionic interactions between splicing factors that contain RS-domains. The direct impact of Me2SO on alternative splicing may explain, at least in part, the different and sometimes opposite effects of Me2SO on cell differentiation and apoptosis.

3' Untranslated Regions↗
PubMed1986

There exists a distinct stage during mammalian DNA synthesis immediately after joining of replication intermediates.

We describe an approach, using alkaline cell lysis and digestion with nuclease S1, which permits to distinguish between newly ligated DNA and the DNA of mature chromatin. When cells with steady-state labelled DNA (mature DNA) are analyzed, the results show labelled "nucleosomal-sized" DNA. However, when DNA of cells pulse-labelled with thymidine for 45 seconds is examined one can detect only large DNA. The newly ligated DNA is not reduced to "nucleosomal-sized" DNA by nuclease S1. When the large DNA is denatured in formamide one can detect 10 kb DNA fragments. Furthermore in pulse-chase experiments there appear, after formamide-treatment, increasing amounts of "nucleosomal-sized" DNA with a parallel decrease in the amount of 10 kb DNA fragments. Hence the newly ligated, large, DNA differs from mature DNA and represents a distinct stage during DNA replication.

Cells, Cultured↗
PubMed1988

Helix stability and the mechanism of cruciform extrusion in supercoiled DNA molecules.

The kinetic properties of cruciform extrusion in supercoiled DNA molecules fall into two main classes. C-type cruciforms extrude in the absence of added salt, at relatively low temperatures, with large activation energies, while S-type cruciforms exhibit no extrusion in the absence of salt, and maximal rates at 50 mM NaCl, with activation energies about one quarter those of the C-type. These diverse properties are believed to reflect two distinct pathways for the extrusion process, and are determined by the nature of the sequences which form the context of the inverted repeat. C-type kinetics are conferred by A + T rich sequences, implying a role of helix stability in the selection. In this study we have shown that: 1. Helix-destabilising solvents (dimethyl formamide and formamide) facilitate extrusion by normally S-type molecules at low temperatures in the absence of salt. 2. C-type extrusion is strongly suppressed by low concentrations (2-4 microM) distamycin, at which concentrations S-type extrusion is enhanced. 3. Some extrusion occurs in a C-type construct in the presence of 50 mM NaCl. This is increased by addition of 3 microM distamycin, under which conditions extrusion becomes effectively S-type. Thus S-type constructs can behave in a quasi-C-type manner in the presence of helix-destabilising solvents, and C-type extrusion is suppressed by binding a compound which stabilises A + T rich regions of DNA. Helix destabilisation leads to C-type behaviour, while helix stabilisation results in S-type properties. These studies demonstrate the influence of contextual helix stability on the selection of kinetic mechanism of cruciform extrusion.

Bacteriocin Plasmids↗