Emission characteristics of fluorescent labels with respect to temperature changes and subsequent effects on DNA microchip studies.
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The effects of temperature, salt concentration, and formamide concentration on the emission characteristics of commonly used fluorescent labels were evaluated on DNA microchips. The emission intensities of different fluorophores without hybridization were observed to vary, each to a different extent, to mainly temperature changes. Rhodamine red, TAMRA (tetramethylrhodamine), and dyes from the carbocyanide group exhibited the largest variations, and Texas Red and Oregon Green exhibited the smallest variations. This temperature dependency was shown to affect results obtained during melting curve analysis in DNA microarray studies. To minimize the bias associated with the temperature-dependent emission of different fluorescent labels, a normalization step was proposed. (+info)
Utilization of cyanide as nitrogenous substrate by Pseudomonas fluorescens NCIMB 11764: evidence for multiple pathways of metabolic conversion.
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The growth of Pseudomonas fluorescens NCIMB 11764 on cyanide as the sole nitrogen source was accomplished by use of a modified fed-batch cultivation procedure. Previous studies showing that cyanide metabolism in this organism is both an oxygen-dependent and an inducible process, with CO2 and ammonia representing conversion products, were confirmed. However, washed cells (40 mg ml-1 [dry weight]) metabolized cyanide at concentrations far exceeding those previously described; 85% of 50 mM KCN was degraded in 6 h. In addition, two other C1 metabolites were detected in incubation mixtures; their identities were confirmed as formamide and formate by 13C nuclear magnetic resonance spectrocopy, high-pressure liquid chromatography, radioisotopic trapping experiments, and other analytical means. The relative yields of all four metabolites (CO2, formamide, formate, and ammonia) were shown to be dependent on the KCN concentration and availability of oxygen; at 0.5 to 10 mM substrate, CO2 was the major C1 product, whereas at 20 and 50 mM substrate, formamide and formate were principally formed. The latter two metabolites also accumulated during prolonged anaerobic incubation, suggesting that P. fluorescens NCIMB 11764 can elaborate several pathways of cyanide conversion. One is formally similar to that proposed previously (R. E. Harris and C. J. Knowles, FEMS Microbiol. Lett. 20:337-341, 1983), involving the oxygen-dependent conversion of cyanide to CO2 and ammonia. The other two, occurring in the presence or absence of oxygen, involve separate reactions to yield, respectively, formate plus ammonia or formamide.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Origin of informational polymers: differential stability of phosphoester bonds in ribomonomers and ribooligomers.
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We have measured the stabilities of the bonds that are critical for determining the half-life of ribonucleotides and the beta-glycosidic and 3'- and 5'-phosphoester bonds. Stabilities were measured under a wide range of temperatures and water/formamide ratios. The stability of phosphodiester bonds in oligoribonucleotides was determined in the same environments. The comparison of bond stabilities in the monomer versus the polymer forms of the ribo compounds revealed that physico-chemical conditions exist in which polymerization is thermodynamically favored. These conditions were compared with those determining a similar behavior for 2'-deoxyribonucleosides, deoxyribonucleotides, and deoxyribooligonucleotides and were shown to profoundly differ. The implications of these facts on the origin of informational polymers are discussed. (+info)
Photocatalysis and the origin of life: synthesis of nucleoside bases from formamide on TiO2(001) single surfaces.
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We report the conversion of a large fraction of formamide (NH(2)CHO) to high-molecular-weight compounds attributed to nucleoside bases on the surface of a TiO(2) (001) single crystal in ultra-high vacuum conditions. If true, we present previously unreported evidence for making biologically relevant molecules from a C1 compound on any single crystal surface in high vacuum and in dry conditions. An UV light of 3.2 eV was necessary to make the reaction. This UV light excites the semiconductor surface but not directly the adsorbed formamide molecules or the reaction products. There thus is no need to use high energy in the form of photons or electrical discharge to make the carbon-carbon and carbon-nitrogen bonds necessary for life. Consequently, the reaction products may accumulate with time and may not be subject to decomposition by the excitation source. The formation of these molecules, by surface reaction of formamide, is proof that some minerals in the form of oxide semiconductors are active materials for making high-molecular-weight organic molecules that may have acted as precursors for biological compounds required for life in the universe. (+info)
Molecular dosimetry of ethylene oxide: formation and persistence of N-(2-hydroxyethyl)valine in hemoglobin following repeated exposures of rats and mice.
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The formation of N-(2-hydroxyethyl)valine (HEVal) in hemoglobin was investigated in male F344 rats (10/group) and B6C3F1 mice (20/group) exposed to 0, 3, 10, 33, 100, or 300 (rats only) ppm ethylene oxide (ETO) by inhalation for 6 h/day for 4 weeks (5 days/week) or exposed to 100 (mice) or 300 ppm (rats) ETO for 1 or 3 days, or 1, 2, or 4 weeks (5 days/week). The persistence of HEVal was studied in animals killed up to 10 days after cessation of the 4-week time-course studies. HEVal was determined by a modified Edman degradation and quantitation of the resulting pentafluorophenylthiohydantoin derivative, using gas chromatography-mass spectrometry. The resulting experimental data were compared to simulations derived with a model for the formation and removal of hemoglobin adducts (T.R. Fennell, S.C.J. Sumner, and V.E. Walker, Cancer Epidemiol., Biomarkers & Prev., 1: 213-219, 1992). Repeated exposures of rats and mice for 4 weeks to 300 and 100 ppm ETO, respectively, led to an accumulation of HEVal that was 14 (rats) and 15 (mice) times greater than that found after 1 day of exposure [28 +/- 2 (SE) and 9.4 +/- 0.4 (SE) pmol HEVal/mg globin in rats and mice, respectively]. After cessation of exposures, HEVal was lost faster than predicted by the normal erythrocyte life span alone. An initial phase of rapid decline in HEVal concentrations was consistent with the removal of older, more heavily alkylated populations of RBCs, accompanied by a burst of erythropoiesis. The dose-response curves for HEVal were linear between 3 and 33 ppm ETO, with 3.5 +/- 0.2 and 3.4 +/- 0.3 pmol adduct/mg globin formed in rats and mice, respectively, after 4 weeks of exposure to 3 ppm ETO. Above 33 ppm ETO, the slope of the dose-response curves increased. Comparison of the dose response for HEVal in rats exposed to ETO for 4 weeks to the dose-response for N tau-(2-hydroxyethyl)histidine in rats exposed to the same concentrations of ETO for 2 years (S. Osterman-Golkar et al., Teratog. Carcinog. Mutagen., 3: 395-405, 1983) suggested that exposures to ETO can reduce the life span of erythrocytes in a concentration- and time-dependent manner. Correlation of the experimental data and simulations for the formation and removal of HEVal demonstrated that perturbations in erythropoiesis and RBC life span complicate the estimation of exposures to ETO when estimates are based upon hemoglobin adduct measurements in heavily exposed individuals.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)
Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.
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The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process. (+info)
Thermal expansion of blood vessels in low cryogenic temperatures, Part II: Vitrification with VS55, DP6, and 7.05 M DMSO.
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As part of the ongoing effort to study the mechanical behavior of biological materials in cryopreservation processes, the current study focuses on thermal expansion during vitrification (vitreous in Latin means glassy). A new device is utilized in this study, which has been described in detail in the companion paper (Part I). The current study (Part II) focuses on measurements of vitrified blood vessels permeated with the cryoprotectants VS55, DP6, and DMSO. Data analysis in this study includes polynomial approximation of experimental results in the lower part of the cryogenic temperature range, where the material behaves as solid over a practical time scale. The study further includes a unified thermal expansion analysis throughout the entire cryogenic temperature range by compiling the current results with previously reported data. Finally, analysis of the glass transition temperature, based on thermal strain data is presented. (+info)
Induction of erythroid differentiation in murine virus infected eythroleukemia cells by highly polar compounds.
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Murine-virus-infected erythroleukemia cells cultured in a medium with dimethylsulfoxide or N,N-dimethylformamide are induced to differentiate to erythroid cells. A number of highly polar compounds have a similar effect in inducing erythroid differentiation of the virus-infected cells, as assayed by the appearance of hemoglobin. These compounds are 1-methyl-1-2-piperidone, N,N-dimethylacetamide, N-methylpyrrolidinone, N-methylacetamide, 2-pyrrolidinone, propionamide, pyridine-N-oxide, piperidone, N-methylformamide, acetamide, and triethylene glycol. It has been previously reported that dimethylsulfoxide must be present during DNA synthesis and, possibly, shortly therafter, to induce differentiation. These findings are consistent with the hypothesis that dimethylsulfoxide and related polar compounds act by changing the conformation of DNA or a DNA-protein complex, causing an alteration in transcription that leads to the expression of the program of erythroid differentiation. (+info)