Bioavailability of rifampin in experimental murine tuberculosis.
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Animals↗
Biomedical subjects
A Guy
Publications and source records attributed to A Guy.
[Total chemical synthesis of natural transfer RNA].
New improvements in the chemical synthesis of oligoribonucleotides are reported and they are applied to the first total chemical synthesis of a natural RNA. This E. coli K12 alanine tRNA contains in its sequence dihydrouridine, ribothymidine and pseudo-uridine. The synthetic tRNA was fully sequenced and showed a 42% aminoacyl acceptance activity. When tRNA was used as a template, reverse transcriptase directed the incorporation of adenine opposite dihydrouridine, ribothymidine and pseudouridine.
Base Sequence↗
Incorporation by chemical synthesis and characterization of deoxyribosylformylamine into DNA.
2-deoxyribosylformylamine is a major oxidative DNA damage type which occurs upon the action of ionizing radiation on DNA. The protected 2-deoxyribosylformylamine phosphoramidite was synthesized and used in conjunction with previously reported alkali labile base protected phosphoramidites ('PAC phosphoramidites') for the preparation of oligodeoxyribonucleotides containing this lesion. Final deprotection of the oligonucleotides was performed under mild alkaline conditions to preserve the integrity of the fragile defect. The presence of formylamino deoxyribosyl residue was confirmed by FAB mass spectrometry sequencing. Oligonucleotides bearing deoxyribosyl formylamine were used as templates for studying in vitro replication. They direct the insertion of guanine or induce a deletion opposite the lesion.
Base Sequence↗
The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.
We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.
Base Sequence↗
Structure and in vitro replication of DNA templates containing 7,8-dihydro-8-oxoadenine.
Adenine residues in DNA are oxidized under the action of ionizing radiation at the C-8 position to give 7,8-dihydro-8-oxoadenine. The formation of this lesion can be considered a cause of mutations and carcinogenesis. Oligodeoxyribonucleotides 39 and 47 bases long containing a single 7,8-dihydro-8-oxoadenine (8-hydroxyadenine) residue were synthesized by using nucleoside phosphoramidites. They were used as templates to study the copies obtained in vitro by the Klenow fragment and the thermostable Taq DNA polymerase. 7,8-Dihydro-8-oxoadenine does not block the replication and thymine is incorporated opposite the damage. The modifications of the DNA duplex conformation provoked by 7,8-dihydro-8-oxoadenine are minor. 1H-NMR spectroscopy shows that the duplex is in a B form, the sugar in a normal position in the helix and the modified base in the anti position. NMR confirms that 7,8-dihydro-8-oxoadenine exists predominantly in the keto form.
Adenine↗
Immunohistologic localization of alpha, mu, and pi class glutathione S-transferases in human tissues.
Human alpha, pi, and mu class glutathione S-transferases (GSH S-T) have been localized immunohistologically in a variety of organs. Alpha GSH S-T are found principally in hepatocytes, proximal convoluted tubules of kidney, the deep reticular layer of the adrenal gland, interstitial cells of the testis, and oxyntic cells of the stomach. The pi GSH S-T are present in relative abundance in ductular, as opposed to parenchymal cells in the liver, pancreas, salivary glands, and kidney. The presence of mu GSH S-T in the tissues of certain patients and its absence in the same tissues from other patients has been demonstrated. The pi GSH S-T seems to be most persistently and strongly expressed in tumors but alpha GSH S-T are also found in some neoplasms whereas the mu GSH S-T are occasionally present when the other two transferases are weak or absent.
Adrenal Glands↗
Feasibility study of monitoring temperature rise in muscle phantoms by the Electrical Impedance Tomography system during hyperthermia treatment.
A simulation study of monitoring temperature rises during hyperthermia treatment was conducted on an Electrical Impedance Tomography (EIT) system developed at the University of Washington. A 2,450 MHz microwave generator was used to induce heat on muscle phantoms. Reconstructed temperature distributions by the EIT system showed hot spots with temperature rises of 2 to 8 degrees in the phantoms. The locations of the temperature rises were verified by infrared pictures and a thermistor probe. From repeated experiments, the results consistently revealed comparable temperature rises within the area underneath the rectangular aperture of the microwave applicator. These results are encouraging, and warrant further study of using the EIT technique to monitor the temperature patterns in actual clinical hyperthermia treatment.
Body Temperature↗
The GTm6AC sequence is overwound and bent.
By a combination of distance constraints obtained from NMR spectra and molecular mechanics calculations we have determined the three dimensional structure of the self-complementary decanucleotide d(CGCGTm6ACGCG). Methylation of an adenine at a position 3' to T induces significant conformational changes relative to B-DNA. This arises from the close proximity of the four methyl groups in the large groove in the centre of the sequence. The helical twist between the two T.m6A base pairs is found to be 45 degrees, as for D-DNA, and is accompanied by a high negative value of the wedge roll angle between these base pairs. The overall nonzero wedge roll observed shows that the helix is bent. These constraints appear to be material for the absence of the sequence T-m6A in natural DNAs.
Adenine↗
Measurement of the radiosensitivity of a radiation-induced adenine defect in a single stranded DNA chain.
To determine the radiation sensitivity of an adenine defect inside the DNA chain, an oligonucleotide bearing 7,8-dihydro-8-oxoadenine was chemically synthesized by the phosphoramidite method. This damaged oligonucleotide 32P-labelled at one end was gamma irradiated in an aerated aqueous solution, and heated with piperidine. The fragments obtained were separated by polyacrylamide sequencing gel electrophoresis, according to their chain lengths. Mean radiosensitivity coefficients for the four natural nucleotides and the relative radiation sensitivity of 7,8-dihydro-8-oxoadenine leading to chain rupture in alkaline medium, could be obtained from the band intensities by computer calculation. An unexpectedly high value was obtained for the damaged adenine residue. Determinations were performed for different doses of irradiation.
Adenine↗
The 45 kilodalton molecule of Mycobacterium tuberculosis identified by immunoblotting and monoclonal antibodies as antigenic in patients with tuberculosis.
The object of this study was to discover new M. tuberculosis antigens which are recognized by patients with tuberculosis, because effective serodiagnostic tests are likely to require combinations of different antigens. In our early experiments using immunoblotting, the findings suggested that human sera from smear-negative tuberculosis patients bound to an antigen in the 45 kDa region. Subsequently, estimates of molecular weight in the immunoblots confirmed that the murine monoclonal antibody (MAB) HGT-6 and sera from patients both recognized the same 45 kDa molecule. An antibody-antibody competition assay between MAB HGT-6 and sera from smear-positive tuberculosis patients yielded a positive result in 23 out of 43 sera from patients, but in only four out of 23 from controls. This is further evidence that the 45 kDa antigen is recognized by tuberculous patients. We analysed whether a combination of the 45 kDa antigen results and those of known antigens might better discriminate between minimal smear-negative disease and healthy controls than could test with single antigens. There is no clinically useful laboratory test for smear-negative tuberculosis. In immunoblotting, combining the results with the 65, 45, 38 and 10 kDa antigens gave the best discrimination. This suggests that future serodiagnostic tests for minimal disease, such as the antibody-antibody competition assay, should contain a MAB against the 45 kDa antigen and possibly also against the 10 kDa antigen.
Antibodies, Monoclonal↗
Synthesis of DNA fragments bearing 7,8-dihydro-8-oxo-adenine: a gamma radiation product.
In order to investigate the mutagenic effects and the repair of DNA lesions induced by gamma rays, 7,8-dihydro-8-oxo-adenine was introduced in DNA fragments using cyanoethyl phosphoroamidite method. The modified oligonucleotides were purified by HPLC and the presence of the defect in the final product was confirmed by DNA sequencing, enzymatic hydrolysis and FAB mass spectrometry.
Adenine↗
A two-dimensional 1H-NMR study of the dam methylase site: comparison between the hemimethylated GATC sequence, its unmethylated analogue and a hemimethylated CATG sequence. The sequence dependence of methylation upon base-pair lifetimes.
We report two-dimensional NOE (NOESY) spectra on the sequence d(GCGATCATGG).d(CCATGATCGC) which contains the unmethylated dam site. As expected the DNA adopts a B-form conformation but appears to be distorted at the TG step of the second strand. This distorsion, probably bending, is not seen on the opposite strand. When the first strand is methylated on adenine in the GATC or CATG sequence the NOESY spectra indicate little or no change in the conformation. However the single strand-duplex exchange is slowed down to the slow-exchange region on a proton NMR time scale. We have assigned the exchangeable imino and cytidine amino resonances of the three duplexes. From the imino linewidths as a function of temperature, we observe that the unmethylated and the hemimethylated Gm6ATC duplexes melt normally from the ends. However, this is not so for the hemimethylated Cm6ATG duplex which, apart from the terminal base pairs, melts cooperatively and at higher temperature. In spectra recorded in H2O a second duplex is observed, for the Gm6ATC sequence, which we have not been able to identify. It is however unlikely to be a hairpin structure. Ultraviolet-melting curves also indicate the presence of two transitions for this duplex. The effect of methylation upon base-pair lifetimes has been studied by comparing the above three duplexes. Little effect is observed upon methylation in the GATC sequence but a drastic increase in the lifetimes of all base pairs is observed upon methylation in the CATG sequence.
Base Composition↗
An NMR study of A-T base pair opening rates in oligonucleotides. Influence of sequence and of adenine methylation.
We report relaxation time measurements by semi-selective and totally selective NMR techniques on the thymidine imino protons of d(GGATATCC) and d(GGm6ATATCC). For these oligonucleotides helix fraying, rather than single base pair opening, is the major exchange mechanism even 25 degrees C below the Tm. We have therefore applied a new saturation transfer technique to measure exchange rates at temperatures where fraying has a very small or negligible contribution. Measurements of exchange rates as a function of temperature give significantly different activation energies for base pairs 3 and 4 in d(GGATATCC). Adenine methylation results in a slowing down of the opening rate for the m6A-T base pair but surprisingly has an even greater effect upon the adjacent non-methylated A-T base pair.
Adenine↗
Consequences of methylation on the amino group of adenine. A proton two-dimensional NMR study of d(GGATATCC) and d(GGm6ATATCC).
A two-dimensional 500-MHz 1H-NMR study of two oligonucleotides, d(GGATATCC) and d(GGm6ATATCC), is presented in which we have investigated the effects of adenine methylation. The two-dimensional nuclear Overhauser spectra (NOESY) show that both oligonucleotides adopt a normal right-handed B-type helix and one-dimensional nuclear Overhauser enhancement (NOE) studies demonstrate that any difference in conformation must be small. However methylation drastically slows down the helix in equilibrium coil exchange which becomes slow on a proton NMR time scale. While d(GGATATCC) fits a two-site exchange model, d(GGm6ATATCC) does not and we invoke the presence of a third species which may be an intermediate in helix formation. NMR and ultraviolet spectroscopy show that methylation destabilizes the helix, measured by the melting temperature and enthalpy of dissociation.
Adenine↗
NMR studies on oligodeoxyribonucleotides containing the dam methylation site GATC. Comparison between d(GGATCC) and d(GGm6ATCC).
The conformation of two hexanucleotides, d(GGATCC) and d(GGm6ATCC), has been studied by proton nuclear magnetic resonance. Nuclear Overhauser effect (NOE) measurements on d(GGATCC) are in agreement with a normal B form right-handed helical structure. The single- and double-strand resonances are in fast exchange on a proton NMR time scale. The exchange is observed to be slow for d(GGm6ATCC); up to the Tm, separate resonances are observed for each state, though above the Tm exchange becomes more rapid. The preferred orientation of the adenosine methylamino group (methyl cis to N1) hinders base-pair formation. At 0 degree C irradiation of the m6A-T imino proton gives an NOE to AH2, showing that base pairing is Watson-Crick. Intra- and interresidue NOEs show that the helix is right handed and in the B form. Comparing results on the two oligomers demonstrates that adenosine methylation induces little or no change in the conformation of the helix but reduces the Tm from 45 to 32 degrees C. All of the amino proton resonances, as well as the imino resonances, have been assigned. From NOE experiments on the unmethylated oligomer we have located the Watson-Crick and non-Watson-Crick adenosine amino protons. At 0 degree C these resonances show broadening due to rotation of the amino group, and their rotation is slightly slower than for the adjacent guanosine amino group, though both these amino groups have lifetimes of less than 10 ms at 0 degree C. The imino protons show normal behavior, disappearing from the spectra ca. 20 degrees C below the Tm.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗