Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Formamides”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,261 records · Page 70Linked to original sources
PubMed2004

Enzymatic genosensor on streptavidin-modified screen-printed carbon electrodes.

Voltammetric enzyme genosensors on streptavidin-modified screen-printed carbon electrodes (SPCEs) for the detection of virulence nucleic acid determinants of pneumolysin and autolysin genes, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate were used as the enzymatic label and substrate, respectively. The oligonucleotide probes were immobilized on electrochemically pretreated SPCEs through the streptavidin/biotin reaction. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 degrees C on the surface of the SPCEs. After the hybridization reaction with FITC-labeled complementary targets, the enzyme is captured using an anti-FITC antibody conjugated to AP. In nonstringent experimental conditions, these genosensors can detect 0.49 fmol of 20-mer oligonucleotide target and discriminate between a complementary oligo and an oligo with a three-base mismatch. In the presence of 25% formamide in the hybridization buffer, a single-base mismatch on the oligonucleotide target can be detected.

Adsorption↗
PubMed2006

Capillary zone electrophoresis of some extremely weak bases in acetonitrile.

In water capillary zone electrophoresis cannot be used to investigate basic compounds, which are so weak that their pK(a,HB+) values are less than zero. In acetonitrile the basic strength of such compounds is increased by many orders of magnitude. Accordingly, several extremely weak bases are protonated at low pH in acetonitrile, thus, allowing their investigation by CZE. In this work the CZE separation of thioacetamide, acetamide, thiourea, benzamide, and 4-nitrobenzamide as well as that of N-methylformamide, N,N-dimethylformamide, formamide, and dimethyl sulfoxide is demonstrated in acetonitrile using 10 mmol/L perchloric acid as an electrolyte. The effect of BGE additives, like water and acetic acid, on the CZE performance is discussed. The problem of finding a suitable electroosmotic flow marker at low pH in acetonitrile is addressed, and nitromethane is shown to be a proper marker compound under such extreme conditions. This work demonstrates how organic solvents can enlarge the field of application of CZE.

Journal Article↗
PubMed1996

Chiral separations by nonaqueous capillary electrophoresis.

This paper reports successful chiral separations of pharmaceutical racemic amines by nonaqueous capillary electrophoresis (NACE) using beta- and gamma-cyclodextrins (CDs) and various derivatives of beta-CDs. The results in three organic solvents, formamide (FA), N-methylformamide (NMF), and N,N-dimethylformamide (DMF) were compared to those in pure water and in 6 M urea in water systems. The binding constants of trimipramine, mianserin, and thioridazine with beta-CD were determined in the following five solvent systems: water, 6 M urea in water, FA, NMF, and DMF. The binding constants decreased systematically from approximately 10(4) in water to approximately 10 in FA and approximately 10(-2) in DMF. As a result, the optimum CD concentration in the aqueous media is in the high micromolar range, while that in the FA is around 100 mM. In the aqueous media, the occurrence of the optimum at very low concentrations and the rapid changes in enantioselectivity with CD concentration would make it difficult to develop methods based on trial and error. Nevertheless, it is shown that, even under nonoptimum concentrations of the chiral selector, other experimental parameters such as ionic strength, addition of tetraalkylammonium (TAA+), and temperature can be adjusted to achieve acceptable resolutions. This, however, is often achieved at the expense of longer analysis times. In addition, the effects of apparent pH (pH*) and type of cyclodextrin on chiral separations in NACE are studied. The application of negatively charged beta-CD in FA is also reported. Chiral separation of trimipramine was achieved at lower concentration of anionic CD due to the additional Coulombic interactions.

Cyclodextrins↗
PubMed1998

Near-infrared heavy-atom-modified fluorescent dyes for base-calling in DNA-sequencing applications using temporal discrimination.

A series of near-IR fluorescent dyes were prepared which contained an intramolecular heavy atom for altering the fluorescence lifetimes to produce a set of probes appropriate for base-calling in a single-lane DNA sequencing format. The heavy-atom modification consisted of an intramolecular halogen situated on a remote section of the chromophore in order to minimize the perturbation on the lifetimes and fluorescence quantum yields. In addition, the dye series possessed an isothiocyanate functional group to allow facile attachment to sequencing primers. The unconjugated dyes showed similar absorption and emission maxima (lambda abs = 765-768 nm; lambda em = 794-798 nm) as well as fluorescence quantum yields that were invariant, within experimental error, with the heavy atom. However, the lifetimes of these dyes were found to vary with the identity of the halogen substitution (I, tau f = 947 ps; F, tau f = 843 ps, measured in methanol), with an average variation within the dye series of 35 ps. The spectroscopic properties of the free dyes and the dyes conjugated to sequencing primers on the 5'-end of the oligonucleotide were determined in a DNA-sequencing matrix (denaturing gels containing formamide). The results indicated slight differences in the fluorescence properties of the free dyes compared to those of the dye/ primer conjugates in this particular matrix. Inspection of the ground-state absorption spectra showed significant aggregation for the free dyes in this solution, but the conjugated dyes exhibited no sign of aggregation due to the highly anionic nature of the oligonucleotide. The fluorescence lifetimes of the dye/primer conjugates demonstrated lifetimes which ranged from 735 to 889 ps, with an average variation of 51 ps, an adequate difference to allow facile discrimination of these dyes in DNA-sequencing conditions. In addition, the free solution electrophoretic mobilities of the native heavy-atom-modified dyes were found to be very similar. When the dye/primer conjugates were electrophoresed in a cross-linked polyacrylamide gel electrophoresis capillary column, they comigrated, indicating that, in single-lane sequencing applications, when utilizing these dyes, no postrun corrections would be required to correct for dye-dependent mobility shifts.

DNA↗
PubMed2000

Modulations in the abundance of salt clusters in electrosprays

The abundance of tetraheptylammonium bromide clusters produced from formamide solutions by electrospray ionization (ESI) is investigated. It exhibits a wavy pattern when plotted as a function of cluster size, successive waves being ordered according to the initial cluster charge z. This structure confirms the ion evaporation mechanism of Iribarne and Thomson and simplifies drastically the determination of the distribution of cluster charge z and radius R, from which the kinetics of ion evaporation from charged drops can be inferred.

Journal Article↗
PubMed2001

Multiple isotope effects on the acyl group transfer reactions of amides and esters.

Acyl group transfer reactions, especially those to amides and esters, are important in biochemistry. Multiple kinetic isotope effects for the atoms at the reactive center of these molecules have provided the most detailed bonding picture of the transition state to date. These kinetic isotope effect studies are reviewed for several reactions of formamide, methyl benzoate, and methyl formate. In these cases all the evidence is consistent with a stepwise mechanism, involving tetrahedral intermediates. In the case of p-nitrophenyl acetate, the change to an excellent leaving group causes the tetrahedral intermediates to become kinetically unstable; the kinetic isotope effects are best fitted to a concerted mechanism.

Acylation↗
PubMed1995

Kinetic studies by fluorescence resonance energy transfer employing a double-labeled oligonucleotide: hybridization to the oligonucleotide complement and to single-stranded DNA.

A single 16-base oligodeoxyribonucleotide was labeled at the 3'-end with fluorescein and at the 5'-end with x-rhodamine (R*oligo*F); the chromophores served as a donor/acceptor pair, respectively, for Förster resonance energy transfer. We exploited the striking differences in the steady-state emission spectra of the R*oligo*F as a single strand and in a duplex structure to signal hybridization in solution and to determine the kinetics of duplex formation as the probe bound to its oligomer complement and to its target sequence in M13mp18(+) phage DNA. The binding followed second-order kinetics; in 0.18 M NaCl (pH 8) with 25% formamide, the rate constant for binding to the oligomer complement was 5.7 x 10(5) M-1 s-1, and that to M13mp18(+) was 5.7 x 10(4) M-1 s-1. The source of the 10-fold decrease in the rate of binding to M13mp18(+) was examined to differentiate between multiple nonproductive nucleation and rapid fluctuations in the structure around the target site. From simulations based on each model combined with associated experimental results, we concluded that the slower binding was due to rapid structural fluctuations around the target site, with an effective target concentration 0.1 of that of the total. Comparisons of total fluorescein emission derived from both steady-state and lifetime measurements suggest that the 5'-x-rhodamine induces a conformational change that affects the interaction at the 3'-end between the fluorescein and the polymer. The effects of salt on the fluorescence were complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

A quantum mechanical study of the active site of aspartic proteinases.

We have performed ab initio self-consistent field (SCF) and configuration interaction (CI) calculations on the active site of the aspartic proteinases pepsin and endothiapepsin. The active site, which carries a formal negative charge to effect hydrolysis, was modeled as a formic acid/formate anion moiety and a water molecule, and the nearest hydrogen bonding residues (Gly34, Ser35, Gly217, and Thr218, with respect to the residue numbering in endothiapepsin) were modeled as formamide and methanol molecules. Four possible binding modes for the active-site water molecule were considered. In contrast to previous theoretical studies, we predict that the most stable form has the water molecule forming a bifurcated hydrogen bond to the inner oxygens of Asp32 and -215, with Asp32 being ionized. The calculations suggest that the water molecule prefers to bind across the shortest OD32 ... OD215 diagonal of the active-site carboxyl groups and therefore the binding mode of the water molecule for all the native aspartic proteinases can be readily predicted by measuring these distances.

Amino Acid Sequence↗
PubMed1993

Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA.

The abasic site (apurinic/apyrimidinic site) is the most common lesion in DNA and is suggested to be an important intermediate in mutagenesis and carcinogenesis. We have recently reported a novel assay for the detection and quantitation of abasic sites in DNA [Kubo, K., Ide, H., Wallace, S. S., & Kow, Y. W. (1992) Biochemistry 31, 3703-3708]. In this assay, the aldehyde group in an abasic site is first modified by a probe bearing a biotin residue, called the Aldehyde Reactive Probe (ARP) and then the tagged biotin is quantified by an ELISA-like assay. However, in the previous study, ARP was prepared only in a crude form, and no solid chemical data concerning the structure and specificity of ARP were reported. In this study, an improved method for the preparative synthesis of ARP has been established, and its structure has been unambiguously characterized using spectroscopic means. In order to elucidate the specificity of ARP to DNA damages, ARP was incubated with a variety of damaged bases or nucleosides and the reaction mixtures were analyzed by HPLC. Of the 14 compounds tested for their reactivity to ARP, 2-deoxyribose (a model compound for an abasic site) and 5-formyluracil reacted with ARP. Interestingly, compounds bearing a formamide group such as formamidopyrimidine and deoxyribosylformamide did not react with ARP, indicating that ARP is specific to damages having an alkyl or allyl aldehyde group. Furthermore, the ability of ARP synthesized by the defined chemical route to detect abasic sites has been substantiated using natural DNA containing abasic sites. Potential applications and limitations of the ARP assay are discussed.

Animals↗
PubMed1984

Solvent effects on allosteric equilibria: stabilization of T and R conformations of Escherichia coli aspartate transcarbamylase by organic solvents.

The activity of Escherichia coli aspartate transcarbamylase (ATCase) is markedly influenced by the addition of organic solvents to the assay medium. The cosolvents tested, which include simple aliphatic alcohols, amides, and ureas, as well as acetone and dioxane, fall into two different classes: the most polar ones (formamide, acetamide, N-methylformamide, and urea) stimulate the enzyme activity for all concentrations tested. In contrast, solvents that are less polar than water inhibit the enzyme at low concentrations but stimulate it at higher concentrations. No comparable effects are observed in the case of the isolated catalytic subunits, a non-regulated form of ATCase. Extensive kinetic studies on ATCase and on two of its Michaelian derivatives, 2-thioU-ATCase and carbamylated ATCase, indicate that solvents modulate the same allosteric transition that is responsible for homotropic interactions between the catalytic sites. The stabilization of the R state of ATCase by comparatively high concentrations of cosolvents is reminiscent of similar findings made on hemoglobin and glycogen phosphorylase, suggesting a common underlying mechanism. Addition of organic cosolvents to water is known to reduce hydrophobic interactions, and we suggest that this effect may preferentially stabilize the more "relaxed" conformations of allosteric proteins, because they have a larger surface exposed to solvent [Chothia, C. (1974) Nature (London) 248, 338-339]. On the other hand, we suggest that the stabilization of the T state by low concentrations of all but the most polar cosolvents simply reflects stronger electrostatic interactions in this conformation.

Allosteric Regulation↗
PubMed1985

Solvent effects on the stability of A7U7p.

The thermodynamics of double-helix formation were measured spectrophotometrically for A7U7 in water at 1 M NaCl and for A7U7p in a variety of solvent mixtures and salt. Comparison of the A7U7 results with calorimetric measurements indicates duplex formation involves intermediate states. For A7U7p between 0.06 and 0.55 M Na+, dTm/d(log [Na+]) = 17.4 degrees C, similar to the value of 19.6 degrees C for poly-(A).poly(U) [Krakauer, H., & Sturtevant, J. M. (1968) Biopolymers 6, 491-512]. At 1 M NaCl, the A7U7p duplex is most stable in 100% water. For 10 mol % solutions, the order for A7U7p duplex stability is ethylene glycol greater than glycerol greater than ethanol greater than 2-propanol greater than dimethyl sulfoxide greater than 1-propanol greater than formamide greater than N,N-dimethylformamide greater than urea greater than dioxane. Comparison of changes in stability and thermodynamic parameters with literature results for proteins suggests proteins and A7U7p interact differently with solvent. The results suggest hydrophobic bonding is not a major contributor to the stability of the A7U7p duplex. Comparisons with bulk solvent surface tension suggest the energy of cavity formation is also not a major contributor to duplex stability.

Calorimetry↗
PubMed1981

Processing of the ribonucleic acid in the large ribosomal subunits of Urechis caupo.

Ribosomal subunits were isolated from eggs or embryos of Urechis caupo, and the ribonucleic acid (RNA) was characterized by electrophoresis under denaturing conditions. The small ribosomal subunit contains a single 17S RNA sequence with a molecular weight of 6.20 X 10(5). The large ribosomal subunit contains four polynucleotide sequences. The 5S RNA has a molecular weight of 4.09 X 10(4). The 26S RNA complex isolated under nondenaturing conditions dissociates in the presence of formamide to yield a 5.8S RNA, molecular weight 5.46 X 10(4), and two approximately 17S and 17.5S RNA sequences with molecular weights of 6.04 X 10(5) and 6.61 X 10(5). The 17S and 17.5S RNAs of the large ribosomal subunits are formed in vivo from a 26S RNA precursor after assembly of the large ribosomal subunit. Large ribosomal subunits are transferred from the nucleus to the cytoplasm with the 26S RNA precursor intact. The hidden break to form the 17S and 17.5S RNAs is introduced in the cytoplasm. No intact 26S RNA could be detected in polysomes; this indicates that the conversion of the 26S RNA to the 17S and 17.5S RNAs may be required to produce large ribosomal subunits capable of participating in protein synthesis.

Animals↗
PubMed1980

Laser temperature jump study of solvent effects of poly(adenylic acid) stacking.

The dynamics of the single-stranded, helix--coil transition of poly(adenylic acid) have been investigated by using the Raman laser temperature jump technique. The driving forces of this conformational transition have been probed by varying the cosolvent in mixed aqueous solutions. The rate of helix formation correlates well with the inverse of the solution viscosity. This correlation and the low activation barrier of approximately 4 kcal/mol for this process indicate a rotational diffusion controlled reaction. The rate of coil formation has a much higher activation barrier, presumably due to the strength of base stacking in the helix. This unstacking rate is virtually unaffected by alcohol cosolvents. A significant increase in this rate occurs when polar cosolvents such as urea, formamide, or acetonitrile are added. Absorbance changes with temperature suggest that urea specifically solvates the adenine base. The polar cosolvents appear to break up the stacked, helical conformation by interacting with the bases.

Cacodylic Acid↗
PubMed1980

Termination of transcription by Escherichia coli ribonucleic acid polymerase in vitro. Effect of altered reaction conditions and mutations in the enzyme protein on termination with T7 and T3 deoxyribonucleic acids.

Both bacteriophage T7 and the related bacteriophage T3 have strong termination sites for bacterial RNA polymerase located near 20% on the standard genome map. These termination sites are used with 90% efficiency in vivo, even in cells which contain a defective p protein. Under normal reaction conditions in vitro, Escherichia coli RNA polymerase terminates with 90% efficiency at the T7 terminator site but shows little or no termination at the corresponding T3 locus. Thus, the two templates form an ideal in vitro test system with which to study the parameters that govern transcriptional termination. Termination at these sites has been monitored by following the time course of RNA synthesis under conditions where only a single transcriptional cycle is carried out and by following the size distribution of RNA chains by gel electrophoresis. Termination of the T7 termination site is unaffected by a large variety of changes in reaction conditions, by quantitative cleavage of the nascent RNA during the reaction with a mixture of single- and double-stranded specific ribonucleases, or by a number of different mutations in the subunits of RNA polymerase, including sigma. Similarly, a large variety of changes in reaction conditions fail to enhance termination at the T3 terminator site, including changes in temperature, MgCl2 concentration, and glycerol concentration or the addition of dimethyl sulfoxide, ethanol, or spermidine to the reaction. However, in the presence of elevated salt concentrations, at low ribonucleoside triphosphate concentrations, and in the presence of formamide, efficient in vitro utilization of the T3 terminator is seen. Changes in the RNA polymerase protein can also enhance utilization of the T3 site. A class of rifampicin-resistant rpoB mutants has been identified which produce a rifampicin-resistant RNA polymerase which is able to utilize the T3 terminator site in vitro. Similarly, the normal Bacillus subtilits RNA polymerase utilizes the T3 terminator site in vitro with high efficiency.

DNA, Viral↗
PubMed1980

Single-stranded poly(deoxyguanylic acid) associates into double- and triple-stranded structures.

Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text). The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG). At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase. This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA. All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis. At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer.

Base Sequence↗
PubMed1979

Synthesis of poly(A)-containing RNA by isolated spinach chloroplasts.

Chloroplasts were isolated from spinach leaves and the intact chloroplasts separated by centrifugation on gradients of silica sol. Chloroplasts prepared in this way were almost completely free of cytoplasmic rRNA. The purified chloroplasts were incubated with 32PO4 in the light. The nucleic acids were then extracted and the RNA was fractionated into poly(A)-lacking RNA and poly(A)-containing RNA (poly(A)-RNA) via oligo(dT)-cellulose chromatography. The poly(A)-RNA had a mean size of approximately 18--20 S as determined by polyacrylamide gel electrophoresis. The poly(A)-RNA was digested with RNase A and RNase T1, and the resulting poly(A) segments were subjected to electrophoresis on a 10% w/v polyacrylamide gel 98% v/v formamide). Radioactivity was incorporated into both poly(A)-RNA and poly(A)-lacking RNA and into the poly(A) segments themselves. The poly(A) segments were between 10 and 45 residues long and alkaline hydrolysis of poly(A) segments followed by descending paper chromatography showed that they were composed primarily of adenine residues. There was no 32PO4 incorporation into acid-insoluble material in the dark. We conclude that isolated chloroplasts are capable of synthesizing poly(A)-RNA.

Chloroplasts↗
PubMed1979

Estrogen withdrawal in chick oviduct. Evidence for continued expression of active unique genes using an "expressed" DNA probe.

We have analyzed the effect of estrogen on the kinds of unique DNA sequences which are transcriptionally expressed in chick oviduct with an "expressed" DNA probe. Steady-state nRNA in estrogen-stimulated chick oviduct represents about 25% of the complexity of total chick unique DNA. To purify this expressed DNA fraction, chick unique DNA was isolated, nick-translated, and hybridized to chemically mercurated oviduct nRNA (Hg-nRNA); the resulting hybrids were bound to sulfhydryl-Sepharose, and DNA was selectively recovered by thermal elution in formamide buffer. To compare the sequence homology between nRNAs isolated from oviduct before or up to 6 days after estrogen withdrawal, trace amounts of expressed DNA derived from estrogen-stimulated oviduct were hybridized in RNA-excess reactions. All nRNAs hybridized with equal efficiency. Furthermore, hybridization of expressed DNA to nRNA mixtures showed that nRNA from nonwithdrawn and withdrawn oviduct contained a similar set of unique sequences. The data indicate that, at most, only a small percentage (0--5%) of transcriptionally active unique DNA sequences are shut down when estrogen is removed from the circulation.

Animals↗
PubMed1979

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.

Avian Leukosis Virus↗