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In the comparison of formamide and urea photocatalytic degradation, despite their similar structures, the final fate of bound nitrogen under illumination with TiO2 has shown a different behaviour; both the rate and the ratio of NH4+ and NO3- ion evolution seem not to be linked to the initial nitrogen oxidation state, but to the carbon oxidation state.
The heterodyned two-dimensional (2D) IR spectra and equilibrium dynamics of the N-H stretching motion of DCONHD in deuterated formamide, DCOND(2), were studied with 80 fs pulses at 3 microm. The time evolution of the heterodyned 2D IR spectra, pump-probe spectra, and photon echo peak shift demonstrate that interstate dynamics is occurring by relaxation of the original N-H excitation. The N-H vibrational frequency correlation function can be expressed as a sum of three exponentials with correlation times 0.24 ps, 0.8 ps, and 11 ps. The intermediate component is attributed to motions of the N-Hcdots, three dots, centeredO unit involving only slight angular variations of the N-H bond. The slow component is attributed to the structure breaking and making. The anisotropy decay confirmed that the significant angular N-H bond motion occurs on the 11 ps time scale. The fast component, which is the least well determined, might correspond to the modulation of the H-bond distance without angular motion. The correlation coefficient between the pumped and relaxed state distributions was +0.51, implying that the excited state phase memory is only slightly diminished by the relaxation of the N-H excitation. The relaxed modes are concluded to be local to the driven N-H mode.
By means of heterodyned two-dimensional IR photon echo experiments on liquid formamide and isotopomers the vibrational frequency dynamics of the N-H stretch mode, the C-D mode, and the C=O mode were obtained. In each case the vibrational frequency correlation function is fitted to three exponentials representing ultrafast (few femtoseconds), intermediate (hundreds of femtoseconds), and slow (many picoseconds) correlation times. In the case of N-H there is a significant underdamped contribution to the correlation decay that was not seen in previous experiments and is attributed to hydrogen-bond librational modes. This underdamped motion is not seen in the C-D or C=O correlation functions. The motions probed by the C-D bond are generally faster than those seen by N-H and C=O, indicating that the environment of C-D interchanges more rapidly, consistent with a weaker C-D...O=C bond. The correlation decays of N-H and C=O are similar, consistent with both being involved in strong H bonding.
We show in this report that DNA fragments smaller than 300 bp are separated with high resolution by electrophoresis in concentrated (up to 7%) agarose gels containing 50% formamide. The separated DNA fragments can subsequently be quantitatively transferred to DBM-paper [Alwine, J.C. et al., Proc. Natl. Acad. Sci. USA (1977) 74, 5350-54] using the Southern technique [Southern, E.M., J. Mol. Biol. (1975) 98, 503-517], while preserving the sharpness of the original gel pattern. Since thin (0.2-0.4 mm) and thick (up to 5 mm) agarose slab gels can be easily handled in vertical or horizontal apparatus, this method should prove to be a very useful extention of the Southern technique, applicable to a variety of analytical and preparative purposes.
The ribozymes derived from Hepatitis delta virus (HDV) RNA appear unique in their sequence requirements for self-cleavage. While truncating the 1679 nucleotide antigenomic HDV RNA, we have characterized the cleavage requirements of a number of ribozymes of intermediate length. Two of these, containing 186 and 106 HDV nucleotides respectively, cleaved to completion in the presence of 18 M formamide. The 186 nucleotide ribozyme also cleaved to completion in 10 M urea. Removal of an additional 10 nts from the 3' terminus of the 106 nt ribozyme resulted in a loss of the ability to cleave in high concentrations of the denaturants. The interaction of nucleotides near the cleavage site with a sequence within this 10 base region may confer unusual stability on these ribozymes.
The thermal stabilities of RNA:DNA hybrids are substantially greater than those of DNA:DNA duplexes in aqueous electrolyte solutions containing high concentrations of formamide. Association rates to form DNA:DNA duplexes and DNA:RNA hybrids have been measured in these solvents. There is a temperature range in which DNA:DNA rates are negligible and RNA:DNA rates close to optimal.
Acid-catalyzed hydrolysis of the formamide linkage of N-acetyl-N'-formyl-L-kynurenineamide in frozen dilute hydrochloric acid solution followed first-order kinetics, yielding N-acetyl-L-kynurenineamide as the sole reaction product. The maximum rate of reaction in the frozen solution was found at around -7.5 degrees and approximated that of the reaction in liquid solution at 40 degrees. By freezing the dilute acid solution at -8 degrees the reaction was accelerated by 60 times compared with that in super-cooled liquid solution at the same temperature.
Phylogenetic and epidemiological relatedness among transferable plasmids belonging to the IncC, IncM and IncH incompatibility groups has been studied by DNA-DNA filter hydridization. Hybridization was carried out on nitrocellulose microfilters, at low temperature, in formamide and under paraffin oil. The degree of hybridization among plasmids belonging to the IncC and IncM groups supported the conclusions drawn from genetic classification. Studies on relatedness among plasmids belonging to the IncH group allowed their classification into three phylogenetic sub-groups. Comparison of DNA sequences of three plasmids sharing the same genetic properties and isolated from different bacterial species suggested an epidemiological spread of the same plasmid.
The n.m.r. spectrum of N-methyl formamide oriented in a lyotropic liquid crystal solvent shows the presence of the cis as well as the trans forms. Analysis of the spectra due to both the species has been carried out. Possible non-planar distortions at the nitrogen atom for the trans species have been computed using the derived dipolar couplings and the available structural data.
A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA - RNA hybrids and double-stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45 degrees and 55 degrees C, respectively. In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 10(4) - 10(7). Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights the size of animal cell was reexamined. Precursor tRNA from HeLa cells migrates according to a molecular weight of 4.1 x 10(6). Nascent precursor mRNA has molecular weights of up to 5 x 10(6) in the case of duck erythroblasts and of up to 10(7) in HeLa cells. This seems to represent the largest size of non-viral animal-cell RNA molecules.
Rabbit skeletal muscle actin labeled with 125 iodine by an enzymic method is shown to be capable of polymerization and to bind to matrix-bound pancreatic DNAse I like unlabeled G-actin. It was used to demonstrate that actin can be released from DNAse-I-agarose by 35--40% formamide. Actin which was only shortly exposed to this solvent was able to bind again to DNAse I and to form filaments indicating that it has been recovered functionally intact from the affinity matrix.
The present study characterized the effects of TS-011 [N-(3-chloro-4-morpholin-4-yl) phenyl-N'-hydroxyimido formamide], a new selective inhibitor of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), on the metabolism of arachidonic acid by human and rat renal microsomes and the inhibitory effects of this compound on hepatic cytochrome P450 enzymes involved in drug metabolism. The effects of TS-011 on the fall in cerebral blood flow following subarachnoid hemorrhage (SAH) and in reducing infarct size in ischemic stroke models were also examined since 20-HETE may contribute to the development of cerebral vasospasm. TS-011 inhibited the synthesis of 20-HETE by human renal microsomes and recombinant CYP4A11 and 4F2, 4F3A, and 4F3B enzymes with IC50 values around 10 to 50 nM. It had no effect on the activities of CYP1A, 2C9, 2C19, 2D6, or 3A4 enzymes. TS-011 inhibited the synthesis of 20-HETE by rat renal microsomes with an IC50 of 9.19 nM, and it had no effect on epoxygenase activity at a concentration of 100 microM. TS-011 (0.01-1 mg/kg i.v.) reversed the fall in cerebral blood flow and the increase in 20-HETE levels in the cerebrospinal fluid of rats after SAH. TS-011 also reduced the infarct volume by 35% following transient ischemic stroke and in intracerebral hemorrhage in rats. Injection of 20-HETE (8 or 12 mg/kg) into the carotid artery produced an infarct similar to that seen in the ischemic stroke model. These studies indicate that blockade of the synthesis of 20-HETE with TS-011 opposes cerebral vasospasm following SAH and reduces infarct size in ischemic models of stroke.
5-Aminoimidazole-4-carboxamide ribonucleotide (ZMP) and N-(beta-D-ribofuranosyl)formamide 5'-phosphate (FAR-P) have been identified as products of the metabolism of ATP and 5-phospho-alpha-D-ribosyl diphosphate by Methanobacterium thermoautotrophicum delta(H), a member of the domain Archaea. Evidence indicates that the first three steps in the pathway to the formation of these compounds are the same as the first three steps of histidine biosynthesis and lead to the generation of pro-phosphoribosyl formimino-5-aminoimidazole-4-carboxamide ribonucleotide (5'-proFAR). The 5'-proFAR then undergoes hydrolysis to ZMP and FAR-P. The reaction was detected by an unexpected high concentration of ZMP in cell extracts of M. thermoautotrophicum delta(H).
Class a and class b 30 to 40S RNA subunits obtained by heat dissociation from the 60 to 70S RNA complex of avian tumor viruses were compared with several RNA standards by electrophoresis in formamide-polyacrylamide gels. Class a RNA was found to have a lower electrophoretic mobility and hence probably a higher molecular weight than class b RNA. The absolute molecular weight of class a and b RNA could not be determined with accuracy, because the relationship between logarithm of molecular weight and mobility of the RNA standards was not linear. The size of class a RNA fell into the range of 2.4 x 10(6) to 3.4 x 10(6) daltons and that of class b into the range of 2.2 x 10(6) to 2.9 x 10(6) daltons, depending on the standards used. The possible biological significance of this difference is discussed.
Single strand conformation polymorphism (SSCP) analysis using ethidium bromide can be improved by adding formamide as the denaturant. This gives higher resolution than previous SSCP methods; it had 100% sensitivity in the discrimination of 14 PCR samples from two different genes, even for a long fragment close to the upper limit of 250 base pairs. This modified procedure is a rapid, simple, safe, and yet highly sensitive method for detecting structural differences in DNA fragments.
Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 degrees C, kcat has the values 5870, 85, 0.55, and 0.075 s-1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato-enzyme intermediate.
Chronically infected Friend leukemia cells (FLC), grown in the presence of dimethyl sulfoxide (DMSO) (2%, v/v), dimethyl formamide (DMF) (1% v/v), or bromodeoxyuridine (BrdU) (3 or 20 mug/ml) for 4 or 7 days, were examined under the electron microscope. It was found that at the 4th day all three compounds induced comparable increases in the number of budding viruses (3 to 5 times that of the control). At the 7th day, the number had remained relatively constant in the BrdU-treated cells in contrast to the cells of the DMSO- or DMF-treated cultures, which showed a further increase of budding viruses. The greatest increase was seen when BrdU was added in combination with either DMSO or DMF, and this was reflected in the apparent increase in the number of extracellular viruses seen in cell pellets. Scanning electron microscopy on whole FLC mounts provided a rapid means of counting budding viruses and a good correlation was obtained between these counts and those made on thin sections by transmission electron microscopy. Attempts to quantitate the number of released viruses in controls and treated cultures after 4 days of growth revealed a 5- to 10-fold increase per cell in the samples treated with a combination of BrdU and either DMSO or DMF. Thymidine failed to prevent the increase of budding viruses induced by BrdU treatment. The number of budding viruses found after treatment with 3 mug/ml BrdU in the presence of 12 mug/ml thymidine was at a level comparable to that found after the individual BrdU treatment Finally, although FLC always contained varying amounts of intracisternal particles, their number, as compared to the paired controls, always decreased after BrdU treatment.
We investigated the optimum culture conditions for the production of a novel enzyme, N-substituted formamide deformylase, which acts mainly on N-benzylformamide, in Arthrobacter pascens F164. The highest enzyme activity was obtained when this strain F164 was cultivated in a synthetic medium with N-benzylformamide as sole nitrogen source. This deformylase was found to be an inducible enzyme depending on N-benzylformamide.