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At least 1,153 records · Page 64Linked to original sources
PubMed2006

Comparison between denaturing gradient gel electrophoresis and phylogenetic analysis for characterization of A/H3N2 influenza samples detected during the 1999-2004 epidemics in Brazil.

In a preliminary study, a denaturing gradient gel electrophoresis method (DGGE) was described for influenza virus variants screening [Motta, F.C., Rosado, A.S., Couceiro, J.N.S.S., 2002. Standardization of denaturing gradient gel electrophoresis for mutant screening of influenza A (H3N2) virus samples. J. Virol. Meth. 105, 105-115]. Such a protocol has confirmed its usefulness, discriminating closely related samples by the evaluation of the HA1 portion of haemagglutinin coding RNA segment. In this study, the HA1 sequence/phylogenetic analysis was compared with DGGE results to evaluate the degree of agreement between these methods. Forty-one influenza clinical samples characterized as the A/H3 subtype by a multiplex-PCR throughout 1999-2004 epidemics were chosen at random. The 569 bp DGGE amplicons were generated by nested-PCR using the first round multiplex-PCR product as template. The amplicons were analyzed on a 6% polyacrylamide gel with a urea-formamide gradient (25-35%) at 60 degrees C/150 V/5 h, being differentiated by their melting profiles. Even with the multiple melting domains characteristic of the region used in this study, the 41 samples could be grouped in 7 distinct clusters by DGGE. Five of the clusters reproduced exactly the phylogenetic tree topology, including the most external branches. Although the other two clusters demonstrated a poorer match, the internal genetic correlations were conserved, and just four samples were grouped incorrectly in comparison with the phylogenetic results. The results demonstrated the usefulness of this method for screening of variant samples throughout or in subsequent epidemics, thus improving the detection of influenza variants.

Brazil↗
PubMed2006

PCR-based detection is unable to consistently distinguish HIV 1LTR circles.

Quantitative PCR methods are routinely used to measure multiple HIV cDNA forms, including linear cDNA, early and late reverse transcripts, 2LTR circles, and integrated provirus. PCR-based methods for the detection of 1LTR circles have been proposed, but are complicated by the inherent homology of the LTR sequence present in all cDNA forms. Amplicons with variable lengths of homology showed that it is difficult to discriminate 1LTR circles faithfully from other cDNA forms. Addition of formamide, DMSO, or glycerol did not eliminate amplification of spurious products. Thus, detection of 1LTR circles by PCR is not reliable.

DNA, Circular↗
PubMed2006

Detection of Fusarium culmorum in wheat by a surface plasmon resonance-based DNA sensor.

A surface plasmon resonance (SPR) sensor based on DNA hybridization has been developed for the detection of Fusarium culmorum, a fungal pathogen of cereals. A 0.57 kbp DNA fragment of F. culmorum was amplified by specific primers and a 25-mer oligonucleotide probe was selected within the sequence of the PCR amplicon. After biotinilation, the probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The effect of denaturating agents (formamide and urea) and ionic strength (NaCl) on hybridization efficiency of double-stranded PCR products with the immobilized probe and the specificity of the probe were investigated. The SPR biosensor was successfully used for the detection of F. culmorum in culture material of different strains and in naturally infected wheat samples. Tested on fungal cultures, it showed a good selectivity for F. culmorum against other species of either Fusarium or other fungal genera. A background signal was observed in wheat samples strictly depending on the DNA amount of the testing matrix. Testing 30 ng of durum wheat DNA the detection limit was 0.06 pg of F. culmorum DNA. The developed PCR-SPR assay allowed to detect F. culmorum with sensitivity and specificity higher than gel-electrophoresis analysis.

Biosensing Techniques↗
PubMed2006

Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter.

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.

Animals↗
PubMed2006

Photoinduced intermolecular electron transfer from dimethyl aniline to 7-amino coumarin dyes in the surface of beta-cyclodextrin.

The photoinduced electron-transfer reaction between Coumarin dyes and N,N-dimethylaniline has been investigated using steady-state and Time-resolved fluorescence spectroscopy in the surface of beta-cyclodextrin in dimethyl formamide (DMF) solvent. The electron-transfer rate constant has been correlated with the free-energy change during the process. We have observed a diffusion-controlled ET process in pure DMF by showing a normal Marcus region. However, the picture is different in presence of cyclodextrin. Here the ET is retarded at higher free-energy region. This unusual feature in bimolecular electron-transfer reaction is assumed to be arising from the different binding possibility of the Coumarin molecules to the cyclodextrin moity.

Aniline Compounds↗
PubMed2006

Vibrational spectroscopic studies of an organic non-linear optical crystal 8-hydroxyquinolinium picrate.

8-hydroxyquinolinium picrate (8-HQP) was synthesized by the addition of equimolar quantities of 8-hydroxyquinoline (8-HQ) and picric acid (PA). Single crystals were grown from N,N dimethyl formamide (DMF) by restricted evaporation method at room temperature. The solubility of 8-HQP was determined in different solvents at various temperatures. The structural characterization of the grown crystals was carried out by X-ray diffraction. Vibrational modes were classified on the basis of group theoretical analysis and the spectral bands were compared with those of parent compounds in order to propose a tentative assignment by recording FT-IR, FT-Raman and polarized Raman spectra in different crystal orientations. The crystal possess lower cut-off at 230 nm and good transparency as confirmed by optical transmittance studies.

Crystallography, X-Ray↗
PubMed2006

Spectroscopic studies of 2-(2-bromo-ethyl)-6-nitro-benzo[de]isoquinolene-1,3-dione in water/alkanol mixed solvents and nonionic micelle of Igepal CO series.

Spectroscopic studies of newly synthesized bioactive compound 2-(2-bromo-ethyl)-6-nitro-benzo[de]isoquinolene-1,3-dione (BNBIO) have been carried out in polar aprotic solvent, viz. acetonitrile, tetrahydrofuran, 1,4-dioxan, ethylene glycol, dimethyl formamide, and polar protic solvent, viz. methanol, ethanol, propanol, water. Variation in absorbance of BNBIO in water-methanol, water-ethanol and water-propanol mixtures at their different compositions have been observed. Absorption behaviour of the dye has been studied in poly(oxyethylene) nonylphenol surfactants Igepal CO 630, Igepal CO 720 and Igepal CO 890 containing same hydrophobic tail and different numbers of poly(oxyethylene) groups. Experimental results of the BNBIO nonionic micelles have been explained in terms of 1:1 electron donor-acceptor (EDA) complexation and the complexation equilibrium becomes suppressed with increasing number of poly(oxyethylene) residue on the Igepal surfactant. Variation in binding constant of dye-micelle complexation has been rationalized considering a competitive equilibrium process between the BNBIO-water interactions.

Alcohols↗
PubMed2005

Moving single bubble sonoluminescence in phosphoric acid and sulphuric acid solutions.

The phenomenon of sonoluminescence still presents some unsolved aspects. Recently [Y.T. Didenko, K. Suslick, Molecular Emission during Single Bubble Sonoluminescence, Nature 407 (2000) 877-879.], it was found that a single cavitating air bubble in polar aprotic liquids (including formamide and adiponitrile) can produce very strong sonoluminescence while undergoing macroscopic translation movements in the resonator, a condition known as moving single bubble sonoluminescing (MSBSL). Here we describe some experiments conducted in aqueous solutions of phosphoric and sulphuric acid. In these liquid media, it is possible to reproduce MSBSL and luminescence is emitted even if a trapped bubble is subjected to a strong shape instability, named in the literature "jittering phase". When a moving and luminescing bubble was present and the acoustic pressure gradually increased, we observed the generation of a discrete lattice of trapped bubbles. The bubbles in the lattice emit very intense light flashes and can change their position while maintaining the overall spatial distribution in time. Some preliminary results, obtained from Mie-scattering and measurements of relative light intensity, are reported.

Journal Article↗
PubMed2001

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages.

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.

Animals↗
PubMed1992

Novel variant transthyretin gene (Ser50 to Ile) in familial cardiac amyloidosis.

We detected a point mutation in the transthyretin (TTR) gene in a patient with familial cardiac amyloidosis by using PCR-DCP (DNA conformation polymorphism) analysis that is based on the diversity in electrophoretic mobility of single-stranded DNAs and/or heteroduplex DNAs in PCR products. The PCR products of the transthyretin gene were denatured in the presence of formamide and electrophoresed in a non-denaturing polyacrylamide gel to detect an electrophoretic change due to a sequence variation. An unusual DNA fragment was visualized by silver staining in the PCR products of the exon 3 from the patient. Subsequent sequencing analysis revealed a T to A transversion and led to a replacement of Ser by Ile at codon 50 of the TTR gene.

Amyloidosis↗
PubMed1988

Quantum mechanical modeling of aspartic proteinase interactions: difference in binding of diastereomeric statine models.

Quantum mechanical calculations were carried out for the interaction of two diastereomeric model inhibitors of aspartic proteinases with a model for the active site, based on crystallographic coordinates of endothiapepsin. The model inhibitor is formamide N-(2-hydroxy 3-methyl propane) and the active site is represented by the full backbone and most of the side chains of the two partial sequences D32-T33-G34-S35 and D215-T216-G217-T218. Those calculations demonstrate that the best binding mode for this short inhibitor is consistent with the X-ray experiments and somewhat stronger with the inhibitor in a 2(S) configuration, compared to 2(R). Another binding mode is possible for this model inhibitor only in the 2(S)-configuration, and is weaker than the first.

Amino Acids↗
PubMed2002

Enhanced activity of human N-myristoyltransferase by dimethyl sulfoxide and related solvents in the presence of serine/threonine-containing peptide substrates.

Human N-myristoyltransferase (hNMT) activity was found to be stimulated several-fold by DMSO and its analogues in the presence of serine-containing peptide substrates. DMSO caused a concentration-dependent 10-fold stimulation of hNMT activity in the presence of a pp60(src)-derived peptide substrate (Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys-Arg). However, the stimulation of hNMT activity was not observed by DMSO when a cyclic AMP (cAMP)-dependent protein kinase-derived Ser-free peptide substrate (Gly-Asn-Ala-Ala-Ala-Ala-Lys-Lys-Arg-Arg) was used. These findings suggested that the effect of DMSO is on the substrate rather than on the enzyme. When a MARCKS (myristoylated alanine-rich C-kinase substrate)-derived peptide substrate (Gly-Ala-Gln-Phe-Ser-Lys-Thr-Ala-Arg-Arg) and the M2 gene segment of the reovirus type 3 peptide substrate (Gly-Asn-Ala-Ser-Ser-Ile-Lys-Lys-Lys) were used, hNMT activity was increased by approximately 8.5- and 7-fold, respectively. Dimethyl sulfone (20%) increased hNMT activity between 2.5- and 3.5-fold in the presence of pp60(src), MARCKS, and M2 gene segment peptides. Dimethyl formamide (20%) increased the hNMT activity by 8.5-, 8.5-, 5.5- and 3.5-fold when pp60(src), MARCKS, M2, and cAMP-dependent protein kinase-derived peptide substrates were used, respectively. Acetone (20%) also increased the hNMT activity by 20-fold in the presence of the pp60(src) peptide substrate. Dimethyl ammonium chloride (20%) caused about 6.5- and 2.5-fold increases in the hNMT activity in the presence of the pp60(src) and cAMP-dependent protein kinase-derived peptide substrates, respectively. Infrared spectroscopy showed a decreased intensity in the band at 3500-3600cm(-1) when the infrared spectrum of the pp60(src)-derived peptide was determined in the presence of DMSO. These results suggest the involvement of hydrogen bonding between the heteroatoms of the organic molecules and the hydrogen atoms of the free hydroxyl groups of the serine/threonine-containing peptide substrates. Such interactions appear to enhance the activity of hNMT towards its serine-containing substrates.

Acyltransferases↗
PubMed2003

Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes.

Formamide-induced detubulation of rat ventricular myocytes was used to investigate the functional distribution of the Na/Ca exchanger (NCX) and Na/K-ATPase between the t-tubules and external sarcolemma. Detubulation resulted in a 32% decrease in cell capacitance, whereas cell volume was unchanged. Thus, the surface-to-volume ratio was used to assess the success of detubulation. NCX current (I(NCX)) and Na/K pump current (I(pump)) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I(NCX) density was significantly reduced by approximately 40% in detubulated cells. I(NCX) density at 0 mV decreased from 0.19 +/- 0.03 to 0.10 +/- 0.03 pA/pF upon detubulation. I(pump) density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na](i)) investigated (7-22 mM). At [Na](i) = 10 mM and -20 mV, I(pump) density was reduced by 39% in detubulated myocytes (0.28 +/- 0.02 vs. 0.17 +/- 0.03 pA/pF), but the apparent K(m) for [Na](i) was unchanged (16.9 +/- 0.4 vs. 17.0 +/- 0.3 mM). These results indicate that although thet-tubules represent only approximately 32% of the total sarcolemma, they contribute approximately 60% to the total I(NCX) and I(pump). Thus, the functional density of NCX and Na/K pump in the t-tubules is 3-3.5-fold higher than in the external sarcolemma.

Animals↗
PubMed1988

Use of weak acids to determine the bulk diffusion limitation of H+ ion conductance through the gramicidin channel.

The addition of 2 M formic acid at pH 3.75 increased the single channel H+ ion conductance of gramicidin channels 12-fold at 200 mV. Other weak acids (acetic, lactic, oxalic) produce a similar, but smaller increase. Formic acid (and other weak acids) also blocks the K+ conductance at pH 3.75, but not at pH 6.0 when the anion form predominates. This increased H+ conductance and K+ block can be explained by formic acid (HF) binding to the mouth of the gramicidin channel (Km = 1 M) and providing a source of H+ ions. A kinetic model is derived, based on the equilibrium binding of formic acid to the channel mouth, that quantitatively predicts the conductance for different mixtures of H+, K+, and formic acid. The binding of the neutral formic acid to the mouth of the gramicidin channel is directly supported by the observation that a neutral molecule with a similar structure, formamide (and malonamide and acrylamide), blocks the K+ conductance at pH 6.0. The H+ conductance in the presence of formic acid provides a lower bound for the intrinsic conductance of the gramicidin channel when there is no diffusion limitation at the channel mouth. The 12-fold increase in conductance produced by formic acid suggests that greater than 90% of the total resistance to H+ results from diffusion limitation in the bulk solution.

Amides↗
PubMed1992

Voltage-sensitive and solvent-sensitive processes in ion channel gating. Kinetic effects of hyperosmolar media on activation and deactivation of sodium channels.

Kinetic effects of osmotic stress on sodium ionic and gating currents have been studied in crayfish giant axons after removal of fast inactivation with chloramine-T. Internal perfusion with media made hyperosmolar by addition of formamide or sucrose, reduces peak sodium current (before and after removal of fast inactivation with chloramine-T), increases the half-time for activation, but has no effect on tail current deactivation rate(s). Kinetics of ON and OFF gating currents are not affected by osmotic stress. These results confirm (and extend to sodium channels) the separation of channel gating mechanisms into voltage-sensitive and solvent-sensitive processes recently proposed by Zimmerberg J., F. Bezanilla, and V. A. Parsegian. (1990. Biophys. J. 57:1049-1064) for potassium delayed rectifier channels. Additionally, the kinetic effects produced by hyperosmolar media seem qualitatively similar to the kinetic effects of heavy water substitution in crayfish axons (Alicata, D. A., M. D. Rayner, and J. G. Starkus. 1990. Biophys. J. 57:745-758). However, our observations are incompatible with models in which voltage-sensitive and solvent-sensitive gating processes are presumed to be either (a) strictly sequential or, (b) parallel and independent. We introduce a variant of the parallel model which includes explicit coupling between voltage-sensitive and solvent-sensitive processes. Simulations of this model, in which the total coupling energy is as small as 1/10th of kT, demonstrate the characteristic kinetic changes noted in our data.

Animals↗
PubMed1997

Solvent hydrogen-bond network in protein self-assembly: solvation of collagen triple helices in nonaqueous solvents.

Forces between type I collagen triple helices are studied in solvents of varying hydrogen-bonding ability. The swelling of collagen fibers in reconstituted films is controlled by the concentration of soluble polymers that are excluded from the fibers and that compete osmotically with collagen for available solvent. The interaxial spacing between the triple helices as a function of the polymer concentration is measured by x-ray diffraction. Exponential-like changes in the spacing with increasing osmotic stress, qualitatively similar to the forces previously found in aqueous solution, are also seen in formamide and ethylene glycol. These are solvents that, like water, are capable of forming three-dimensional hydrogen-bond networks. In solvents that either cannot form a network or have a greatly impaired ability to form a hydrogen-bonded network, strikingly different behavior is observed. A hard-wall repulsion is seen with collagen solvated by ethanol, 2-propanol, and N,N-dimethylformamide. The spacing between helices hardly changes with increasing polymer concentration until the stress exceeds some threshold where removal of the solvent becomes energetically favorable. No solvation of collagen is observed in dimethoxyethane. In solvents with an intermediate ability to form hydrogen-bonded networks, methanol, 2-methoxyethanol, or N-methylformamide, the change in spacing with polymer concentration is intermediate between exponential-like and hard-wall. These results provide direct evidence that the exponential repulsion observed between collagen helices at 0-8-A surface separations in water is due to the energetic cost associated with perturbing the hydrogen-bonded network of solvent molecules between the collagen surfaces.

Animals↗
PubMed1975

Photoamidation of unsaturated carbohydrates. Synthesis of 1-(3-C-carbamoyl-3-deoxy-beta-D-glucopyranosyl)-thymine and -cytosine.

The acetone-initiated photochemical addition of formamide to 1,2,4,6-tetra-O-acetyl-3-deoxy-alpha-D-erythro-hex-2-enopyranose (1) afforded 1,2,4,6-tetra-O-acetyl-3-C-carbamoyl-3-deoxy-alpha-D-glucopyransoe (2), 1,2,4,6-tetra-O-acetyl-3-C-carbamoyl-3-deoxy-alpha-D-allopyranose (3), 1,2,4,6-tetra-O-acetyl-3-C-carbamoyl-3-deoxy-alpha-D-altropyranose (4), and 1,2,4,6-tetra-O-acetyl-3-deoxy-3-C-(1-hydroxy-1-methylethyl)-alpha-D-mannopyranose (5), in 46, 13, 1, and 7% yields, respectively. Condensation of 2,4,6-tri-O-acetyl-3-C-carbamoyl-3-deoxy-alpha-D-glucopyranosyl bromide (produced from 2 with hydrogen bromide) with 2,4-bis(trimethylsilyl)thymine afforded 1-(2,4,6-tri-O-acetyl-3-C-carbamoyl-3-deoxy-beta-D-glucopyranosyl)thymine (6) in 40% yield. Deacetylation of 6 with sodium methoxide in methanol yielded the pure unprotected nucleoside 7. Similar condensation of the bromide from 2 with 2,4-bis(trimethylsilyl)-N4-acetylcytosine afforded the protected cytosine nucleoside 8 in 55% yield, which was converted into 1-(3-C-carbamoyl-3-deoxy-beta-D-glucopyranosyl)cytosine (9).

Anti-Bacterial Agents↗
PubMed2001

BD monomer and elastomer production processes.

The monomer 1,3 butadiene (BD) is a product of the petrochemical industry. It is used to make several elastomers including the very high volume styrene butadiene rubber (SBR) that comprises the bulk of automobile tires. It is also used to make polybutadiene rubber that is used in parts of tires, coatings, composites and other products. The monomer can be converted to chlorobutadiene (chloroprene) and used to make polychloroprene (neoprene). BD is one of the several olefins created by cracking hydrocarbons in the presence of steam. A mixed C4 stream from the steam cracker is then sent to a BD monomer extraction unit. Modern units typically use dimethyl formamide as the extraction solvent. SBR is commonly made by the copolymerization of BD and styrene, along with various additives to control the reaction, in a water emulsion. The reaction proceeds in a continuous chain of reactors until it is 'shortstopped' by a strong reducing agent. After removing unreacted monomers from the stabilized latex, it is blended, coagulated and dewatered. The resulting dry rubber crumb is bailed, film wrapped and stored in crates. The polymerization of BD to make polybutadiene rubber can be conducted as a water suspension type polymerization similar to SBR or in a solvent system followed by solvent recovery and transfer into water suspension.

Butadienes↗