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PubMed2002

Structural dependencies of interresidue scalar coupling (h3)J(NC') and donor (1)H chemical shifts in the hydrogen bonding regions of proteins.

A study is presented of the structural dependencies for scalar J-coupling and the amide donor (1)H chemical shifts in the hydrogen bonding regions of proteins. An analysis of the interactions between the donor hydrogen and acceptor oxygen orbitals in an N-H...O=C moiety suggests that there are three major structural factors for (15)N-(13)C coupling across hydrogen bonds: (1) the H...O' internuclear separation r(HO)('), (2) the H...O'=C' angle theta(2), and (3) indirect contributions involving the oxygen loan pair electrons should lead to a dependence on the H...O'=C'-N' dihedral angle rho. Density functional theory (DFT) and finite perturbation theory (FPT) were used to obtain the Fermi contact (FC) contributions to interresidue coupling in formamide dimers with systematic variation of these structural parameters. The computed (h3)J(NC)(') exhibit good correlations with cos(2) theta;(2) combined with an exponential dependence on r(HO)('). The correlation is further improved by including a dependence on the dihedral angle rho. For each of the 34 H-bonds having observable interresidue coupling in the immunoglobulin binding domain of streptococcal protein G, a formamide dimer was generated from the crystallographic structure with energy-optimized donor H-atom positions. The computed coupling constants are in reasonable agreement with the experimental, and there are excellent correlations with the simple equations involving theta;(2) and r(HO) if alpha-helix and beta-sheet regions are treated separately. This dichotomy is removed by introducing the dependence on the dihedral angle rho. Justification for the use of formamide dimers is provided by almost identical interresidue coupling constants for larger sequences extracted from the X-ray structure. The amide donor (1)H chemical shifts, which were based on DFT and GIAO (gauge including atomic orbital) methods, are in poorer agreement with the experimental data but exhibit excellent correlation with r(HO)('), theta(2), and rho.

Bacterial Proteins↗
PubMed1976

Hybridization of RNA to double-stranded DNA: formation of R-loops.

RNA can hybridize to double-stranded DNA in the presence of 70% formamide by displacing the identical DNA strand. The resulting structure, called an R-loop, is formed in formamide probably because of the greater thermodynamic stability of the RNA-DNA hybrid when it is near the denaturation temperature of duplex DNA. The rate of R-loop formation is maximal at the temperature at which half of the duplex DNA is irreversibly converted to single-stranded DNA (the strand separation temperature of tss) of the duplex DNA and falls precipitously a few degrees above or below that temperature. This maximal rate is similar to the rate of hybridization of RNA to single-stranded DNA under the same conditions. At temperatures above the tss the rate is proportional to the RNA concentration. However, at temperatures below tss the rate of R-loop formation is less dependent upon the RNA concentration. Once formed, the R-loops display considerable stability; the formamide can be removed and the DNA can be cleaved with restriction endonucleases without loss of R-loop structures.

Coliphages↗
PubMed1978

E.s.r. study of spin-trapped radicals formed during the photolysis of aqueous solutions of acid amides and H2O2.

Free radicals formed by the reactions of OH radicals with amides and their N-methylated derivatives in aqueous solutions have been studied. The OH radicals were produced by U.V.-photolysis of H2O2, and the short-lived amide radicals were converted to more stable nitroxide radicals by addition to a spin-trap, tert-nitrosobutane. The spin-trapped radicals were identified by e.s.r. spectroscopy. For acetamide, chloroacetamide, malonamide, succinamide and propionamide, the observed radicals were formed by H-abstraction from the carbon atoms attached to the carbonyl group. The H atom attached to the carbonyl group was abstracted in formamide. For N-methyl acetamide, N,N-dimethyl acetamide and the corresponding formamide derivatives, H-abstraction occurred only from the N-methyl group. The non-equivalency of the amide protons was observed in the spin-trapped radicals for acetamide, formamide, malonamide, succinamide and propionamide. The identification of the site of OH attack on N-methyl amides is helpful for the study of radical formation in peptides and proteins.

Acetamides↗
PubMed1978

A new method for excitation-contraction uncoupling in frog skeletal muscle.

The mechanical activity of frog sartorius muscle fibers can be uncoupled from the electrical activity of their surface membranes by immersing the preparation in Ringer solution containing either 1.5 or 2.0 M of formamide for 15--20 min. This uncoupling is not reversed when the muscle is transferred to normal frog Ringer solution. Formamide does not affect the electrical activity of the sciatic nerve branch, and both endplate potentials and miniature endplate potentials may be recorded from the uncoupled muscles. Prolonged exposure to formamide, beyond the time needed to paralyze, causes neuromuscular block.

Animals↗
PubMed2004

A simple and robust method for preparation of cDNA nylon microarrays.

DNA array technology has made remarkable progress in recent years and has become an indispensable tool in molecular biology research. However, preparing high-quality custom-made DNA arrays at a reasonable cost is still an important concern because we cannot abandon the use of DNA array systems designed for specific purposes. To address these problems, we here report the use of rolling circle amplification products of cDNA plasmids dissolved in 80% formamide as DNA probes immobilized on a nylon membrane. First, because formamide is practically non-volatile under ambient conditions and nucleic acids are easily dissolved in it, the use of formamide as a DNA solvent ensures that the DNA concentration of the solution will not change during arraying, which often takes several hours to a day depending on the number of DNA spots and arrays to produce. Secondly, the use of rolling circle amplification technology greatly reduced the labor needed to prepare the spotted DNA. The results in this study demonstrate that the introduction of these two modifications in preparation of nylon DNA array greatly improved its quality.

Cost Control↗
PubMed1990

The self-cleaving domain from the genomic RNA of hepatitis delta virus: sequence requirements and the effects of denaturant.

The sequence requirements for self-cleavage of hepatitis delta virus genomic RNA were examined using precursor RNAs which were labeled at either the 5' or 3' ends and progressively deleted from the unlabeled end. In the presence of 50% formamide, which enhances self-cleavage in 2 mM MgCl2 at 37 degrees C, 84 nucleotides (nt) 3' of the break site were required. In the absence of formamide the minimum was reduced to 82 nt. Under both sets of conditions, precursors with 1 nt 5' to the break site cleaved. These results allowed two condition-dependent minimal domains for self-cleavage to be defined. However, in the absence of formamide, sequences flanking the minimal domain inhibited cleavage, possibly through involvement in the formation of non-cleaving structures. These data are consistent with the idea that cleavage in vivo could be regulated by alternative RNA structures.

Base Sequence↗
PubMed1976

Stepwise dissociation of high molecular weight avian myeloblastosis virus RNA: 30-40S RNA subunits--the best natural template-primer for viral reverse transcriptase.

Controlled disruption of 60S AMV RNA with formamide was used to prepare 50-55S and 30-40S RNAS. When the activities of these RNAs as templates for AMV reverse transcriptase were compared it was found that 50-55S RNA was 1-5 times and 30-40S RNA 2 to 3 times more active than 60S RNA. The 30-40S RNA produced by heating, instead of formamide disruption, was inactive as a template but activity was restored by addition of oligo(dT). 40% of the 4S RNA initially associated with the 60S RNA remained associated with all the RNA species obtained by formamide treatment but was lost on heating. It is concluded that this RNA acts as resident primer whereas the other 60% of the 4S RNA is less firmly bound and appears to have little or no primer activity. Removal of the less firmly bound 4S RNA increases the template activity of the viral RNA.

Avian Leukosis Virus↗
PubMed1982

Partial purification and characterization of messenger RNA coding 14-3-2 protein from rat brain.

14-3-2 Protein (neuron-specific enolase) is a neuron-specific protein. Using a reticulocyte lysate cell-free system for translation of 14-3-2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))-Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14-3-2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8-fold. The size of 14-3-2 protein mRNA appears to be about 19-20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.

Animals↗
PubMed1970

Somatic antigens of Streptococcus group E. I. Comparison of extraction techniques.

Eleven Streptococcus group E strains, representing serotypes I, II, III, IV, V, and "untypable" isolates, were extracted by formamide, trichloroacetic acid, and hydrochloric acid under various conditions in an effort to determine the best method for recovering maximum amounts of group and type antigens. The group antigen was found to be relatively stable, and adequate amounts for identification purposes were recovered by a wide spectrum of conditions. Type-specific antigens were relatively labile, and were destroyed at low pH in acid hydrolysis or by prolonged heating in formamide hydrolysis. The best single procedure for recovering both type and group antigens from Streptococcus group E was formamide hydrolysis for 30 min at 180 C.

Animals↗
PubMed1999

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies.

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.)

Animals↗
PubMedDate not supplied

The molecular weight of rRNA precursor molecules and their processing in higher plant cells.

Actively dividing callus cells of higher plants (Petroselinum crispum, Daucus carota, Acer pseudoplatanus) were used to detect the primary gene product of rDNA in vivo. Parsley and carrot cells were labelled with [32P]orthophosphate. Under non-denaturing conditions, in both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels. Its molecular weight did not exceed 2.5 x 10(6) dalton. Under denaturing conditions, 2.0--2.1 x 10(6) dalton were determined on formamide gels. This rRNA precursor was already present after a labelling period of 5--10 min. In parsley cells labelled mature rRNA (25S and 18S) arrived in the cytoplasm 45 min after onset of incubation. In Acer pseudoplatanus incubated with [3H]uridine two rapidly labelled components did emerge from polyacrylamide gels without formamide; their molecular weights were 2.3 and 3.2--3.4 x 10(6) dalton. After electrophoresis in formamide, the larger component disappeared, thus indicating that it would be an intermolecular aggregate of different RNAs. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 x 10(6) dalton.

Cells, Cultured↗
PubMed1996

Evaluation of C-(beta-D-galactosyl) and C-(2-deoxy-D-lyxo-hex-1-enopyranosyl) (D-galactal type) derivatives as inhibitors of beta-D-galactosidase from Escherichia coli.

C-(2-Deoxy-D-lyxo-hex-1-enopyranosyl)formamide was prepared from acetylated C-(beta-D-galactopyranosyl)formamide by a radical-mediated bromination-zinc/N-methylimidazole-induced reductive elimination-Zemplén deacetylation reaction sequence. The preparation of acetylated 5-(2-deoxy-D-lyxo-hex-1-enopyranosyl)tetrazole was improved. Methyl C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formimidate was transformed by benzylamine into N-benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamidine and, after hydrolysis to methyl C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formate, into N-benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamide. A series of C-(beta-D-galactopyranosyl) and C-(2-deoxy-D-lyxo-hex-1-enopyranosyl) derivatives was comparatively investigated for E. coli beta-D-galactosidase inhibitory activity. N-Benzyl-C-(2-deoxy-D-lyxo-hex-1-enopyranosyl)formamidine was the best inhibitor and had K(i) = 6 microM (on the basis of its free base concentration, K(i) = 8.3 nM was obtained). Basicity and hydrophobicity of the aglycon proved to be more important factors for the inhibition than the conformation of the sugar ring.

Carbohydrate Conformation↗
PubMed2002

Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls.

BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells.

Animals↗
PubMed2005

Mass spectrometric detection of biotinylated peptides captured by avidin functional affinity electrophoresis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to detect biotinylated peptides captured by avidin functional affinity electrophoresis (AFAEP). Peptide samples loaded onto AFAEP were heated with sodium dodecyl sulfate to ensure that the peptides are negatively charged, and thus migrate electrophoretically toward the cathode through the embedded avidin zone in the middle of the gel. To detect the biotinylated peptides, the band containing the avidin-biotinylated peptide complexes was excised from a 7.5% w/v native polyacrylamide gel, and biotinylated peptides were extracted with aqueous 95% v/v formamide (pH 8.2), aqueous 6 M guanidine HCl (pH 1.5), or water, at temperatures from 4 to 95 degrees C for periods from 5 min to 24 h. It was observed that all three solvents are capable of extracting biotinylated peptides and avidin from the gel, but the best results were obtained with aqueous 95% v/v formamide (pH 8.2) at 65 degrees C for 20 min. However, some AFAEP-captured biotinylated peptides are not stable and are extensively modified by formamide during extraction at too high a temperature or too long an extraction time.

Avidin↗
PubMed1998

Asialoglycoprotein receptor-mediated gene transfer using novel galactosylated cationic liposomes.

We synthesized three novel galactosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((1-imino-c-beta-D-thiogalactosyl+ ++-ethyl)amino) butyl)formamide (Gal-C4-Chol) and its ethyl formamide and hexyl formamide analogues (Gal-C2-Chol, Gal-C6-Chol), to prepare liposomal gene carriers possessing the cationic charge necessary for plasmid DNA binding and galactose residues as a targetable ligand for liver parenchymal cells. Liposome/DNA complexes prepared with these lipids showed low cytotoxicity in human hepatoma HepG2 cells. Gal-C4-Chol/DC-Chol/DOPE(3:3:4) liposomes, consisting of 3:3:4 mixtures of Gal-C4-Chol, 3beta[N',N', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), and dioleoylphosphatidylethanolamine (DOPE), showed higher transfection activity and [32P]DNA uptake than DC-Chol/DOPE(6:4) liposomes. The presence of 20 mM galactose significantly inhibited both transfection efficiency and uptake of DNA of Gal-C4-Chol/DC-Chol/DOPE(3:3:4) and Gal-C4-Chol/DOPE(6:4) liposomes, but not those of DC-Chol/DOPE(6:4) liposomes. These results indicate that the liposome/DNA complexes prepared using novel galactosylated cholesterol derivatives are efficiently recognized by asialoglycoprotein receptors and internalized and lead to gene expression. In addition, we found that the galactosylated cholesterol derivative with a longer spacer showed higher transfection activity.

3T3 Cells↗
PubMed1990

Study of stringency conditions for human papillomavirus DNA detection on cell lines, frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes.

In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Sihà and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35 degrees C below the melting temperature of DNA (Tm-35 degrees C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35 degrees C; C-medium stringency, hybridization at Tm-35 degrees C and washings at Tm-12 degrees C; D-high stringency, hybridization at Tm-12 degrees C and washing without formamide; E-very high stringency, hybridization and washings at -12 degrees C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes. When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type. Stringent (Tm-12 degrees C) and non stringent (Tm-35 degrees C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopsy↗
PubMed1975

Ultraviolet-gas phase and -photocatalytic synthesis from CO and NH3.

The major photoproduct obtained on irradiation of gaseous NH3 and CO mixtures is ammonium cyanate; lesser amounts of urea, biurea, biuret semi-carbazide, formamide and cyanide were observed. The formation of the major gas phase photolysis product may be rationalized by the following reaction sequence: (see article). Urea is probably formed from NH4NCO in a thermal reaction while formamide may result from the disproportionation of NH2CO. Photocatalytic syntheses of 14C-urea, -formamide, and -formadehyde are effected by irradiation of 14CO and NH3 in the presence of Vycor, silica gel, or volcanic ash shale surfaces. These syntheses are catalyzed by ultraviolet wavelengths longer than those absorbed by the gaseous reactants. The syntheses are also effected when the surface material is first irradiated in the presence of CO followed by a dark incubation with NH3. Apparently, the initiating step is a light dependent formation of a reactive form of CO on the surface. A discussion is given on the possible contribution of these reactions to the abiotic synthesis of organic nitrogen compounds on Mars, on the primitive Earth and in interstellar space.

Ammonia↗
PubMed1975

Improvements of the membrane filter method for DNA:rRNA hybridization.

We describe and recommend the following improvements of DNA:rRNA membrane filter hybridization methods. One of our aims was to avoid DNA release from filter discs during hybridization. 1. Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr. 2. Duplexing is over in 8-10 hr. 3. Formamide has to be very pure (O.D. less than or equal to 0.2/cm light path at 270 nm). 4. RNAase treatment: 250 mug/5 ml 2 SSC/filter at 37 C for 1 hr. 5. Our conditions for stepwise thermal denaturation are: 5 degrees C steps from 50 C to 90 C in 1.5 SSC in 20% formamide. 6. Single-stranded DNA, fixed on membrane filters, and stored in vacuo at 4C can be used reliably for hybridization for up to 20 months. 7. Concentrated DNA in 0.1 SSC, quick-frozen at -50 C and stored at -90 C for up to 2 years can be used for hybridization without much change. 8. A CsCl gradient purification step yields much purer DNA, but increases the release of DNA from filters by about 20%. Filters with 20% more DAN is a compensation. 9. rRNA can be stored for 20 months in SSC or 2 SSC at -12 C without changing the hybridization results.

Arthrobacter↗