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T Brown

Publications and source records attributed to T Brown.

At least 181 records · Page 10Linked to original sources
PubMed1996

Hydration of the RNA duplex r(CGCAAAUUUGCG)2 determined by NMR.

The so-called spine of hydration in the minor groove of AnTn tracts in DNA is thought to stabilise the structure, and kinetically bound water detected in the minor groove of such DNA species by NMR has been attributed to a narrow minor groove [Liepinsh, E., Leupin, W. and Otting, G. (1994) Nucleic Acids Res. 22, 2249-2254]. We report here an NMR study of hydration of an RNA dodecamer which has a wide, shallow minor groove. Complete assignments of exchangeable protons, and a large number of non-exchangeable protons in r(CGCAAAUUUGCG)2 have been obtained. In addition, ribose C2'-OH resonances have been detected, which are probably involved in hydrogen bonds. Hydration at different sites in the dodecamer has been measured using ROESY and NOESY experiments at 11.75 and 14.1 T. Base protons in both the major and minor grooves are in contact with water, with effective correlation times for the interaction of approximately 0.5 ns, indicating weak hydration, in contrast to the hydration of adenine C2H in the homologous DNA sequence. NOEs to H1' in the minor groove are consistent with hydration water present that is not observed in the analogous DNA sequence. Hydration kinetics in nucleic acids may be determined by chemical factors such as hydrogen-bonding more than by simple conformational factors such as groove width.

Base Sequence↗
PubMed1996

Comparison of the thermodynamic stabilities and solution conformations of DNA.RNA hybrids containing purine-rich and pyrimidine-rich strands with DNA and RNA duplexes.

The conformations and thermodynamic stabilities of duplexes containing purine-rich (GAA-GAGAAGC) and pyrimidine-rich (GCTTCTCTTC or GCUUCUCUUC) DNA and RNA strands have been measured by UV melting, electrophoresis, circular dichroism, and NMR spectroscopy. The free energies of stabilization (-delta G) were in the order rR.rY > rR.dY > dR.dY > dR.rY. The two DNA.RNA hybrid duplexes showed conformational properties intermediate between those of DNA.DNA and RNA.RNA duplexes and also different from one another. Differences between 1H chemical shifts of the DNA strands in the two hybrid duplexes and those of the DNA duplex were larger than analogous shift differences for the RNA protons, and the differences were larger for the purine than the pyrimidines in the DNA strands. Detailed analysis of the nucleotide conformations using both NOE and scalar coupling data showed that the sugar conformations of the ribonucleotides are all near C3'-endo. The deoxyribonucleotides were in the "S" domain, i.e., near C2'-endo in the DNA duplex, and C1'-exo to C2'-endo in the two hybrids. However, the deoxyriboses in the two hybrids appear more flexible than in the DNA duplex, with the fraction in the "N" (C3'-endo) state increasing in the order dR.dY < dR.rY < rR.dY. Globally, the pure DNA duplex was B-form and the pure RNA duplex A form. The two DNA.RNA hybrids were neither A nor B, but closer globally to the A than the B form. The less stable dR.rY duplex has a significantly different conformation from rR.dY both at the local nucleotide level and globally.

Carbohydrate Conformation↗
PubMed1996

Synthesis and properties of DNA-PNA chimeric oligomers.

Adenine, thymine and cytosine PNA monomers have been prepared using 3-amino-1,2-propanediol as a starting material. The benzoyl group was used to protect the exocyclic amines of the heterocyclic bases of A and C PNA monomers and the backbone primary amine was protected with the monomethoxytrityl group. The thymine and cytosine PNA monomers were used in conjunction with standard DNA synthesis monomers to produce chimeric PNA DNA (PDC) oligomers. Ultraviolet melting studies confirmed that these oligomers form stable hybrids with complementary DNA strands and that mismatches in the DNA but more so in the PNA sections lead to duplex destabilisation.

Chemistry, Organic↗
PubMed1996

1H-NMR characterization of L-tryptophan binding to TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis.

A 1H-NMR study of the binding of L-tryptophan to the trp RNA-binding attenuation protein of Bacillus subtilis (TRAP), an ondecamer (91.6 kDa), has been implemented. The assignment of the aromatic indole ring proton resonances of the bound tryptophan ligand has been successfully carried out by two-dimensional chemical exchange experiments. The observation of only a single set of chemical shifts of the bound ligand demonstrates that the tryptophan binding site is identical in all the 11 subunits of the protein. Further, the large change in ligand chemical shifts suggests that the conformation of tryptophan ligand undergoes a significant rearrangement after complex formation with TRAP. This is further substantiated by the extensive ligand-induced chemical shift changes observed to the protein resonances and identification of several strong ligand-protein intermolecular nuclear Overhauser effects. A correlation of these preliminary NMR data with the X-ray crystal structure of the TRAP-tryptophan complex also suggests, tentatively, that the observed changes to the NMR spectra of the protein might correspond to changes associated with residues surrounding the tryptophan binding pocket owing to complex formation.

Bacillus subtilis↗
PubMed1996

The crystal structure analysis of d(CGCGAASSCGCG)2, a synthetic DNA dodecamer duplex containing four 4'-thio-2'-deoxythymidine nucleotides.

The crystal structure refinement of the synthetic dodecamer d(CGCGAASSCGCG), where S = 4'-thio-2'-deoxythymidine, has converged at R=0.201 for 2605 reflections with F > 2sigma(F) in the resolution range 8.0-2.4 A for a model consisting of the dodecamer duplex and 66 water molecules. A comparison of its structure with that of the native dodecamer d(CGCGAATTCGCG) has revealed that the major differences between the two structures is a change in the conformation of the sugar-phosphate backbone in the regions at and adjacent to the positions of the modified nucleosides. Examination of the fine structural parameters for each of the structures reveals that the thiosugars adopt a C3'-exo conformation in d(CGCGAASSCGCG), rather than the approximate C1'-exo conformation found for the analogous sugars in the structure of d(CGCGAATTCGCG). The observed differences in structure between the two duplexes may help to explain the enhanced resistance to nuclease digestion of synthetic oligonucleotides containing 4'-thio-2'-deoxynucleotides.

Base Sequence↗
PubMed1996

An investigation of the mechanism responsible for fluoxetine-induced hypophagia in rats.

The effect of fluoxetine on feeding in p-chlorophenylalanine (PCPA) pretreated rats and the nature of its interaction with 5-HT2C receptors have been investigated. Animals that received 3 days PCPA (150 mg/kg i.p.) pretreatment and vehicle on the test day consumed a similar amount as control, saline pretreated animals under the test paradigm used in this study. Fluoxetine (20 and 30 mg/kg p.o.) significantly reduced food intake in PCPA and control pretreated animals to a similar extent, despite an approximately 90% reduction in the levels of brain 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in the PCPA-pretreated animals. Thus, hypophagia is unlikely to be caused by inhibition of 5-HT reuptake. In the pig choroid plexus in vitro, fluoxetine and norfluoxetine inhibited specific [3H] mesulergine binding with pKI's (+/- S.E.M.) of 6.45 +/- 0.09 (n = 4) and 6.05 +/- 0.05 (n = 3), and slope factors (+/- S.E.M.) of 1.06 +/- 0.14 and 0.99 +/- 0.13, respectively. In slices of piglet choroid plexus fluoxetine (1, 10 and 33 microM) caused a rightward shift in the dose-response curve produced by 5-HT with no effect on the maximal response, and a mean pKB of 5.94 +/- 0.09. Norfluoxetine (10 microM) also produced a rightward shift in the 5-HT dose-response curve with no effect on the maximal response, and a pKB of 6.20. Thus, both compounds acted as surmountable antagonists with no agonist efficacy at 5-HT2C receptors present in choroid plexus. The hypophagic effect of fluoxetine (20 mg/kg p.o.) was also unaffected by the non-specific 5-HT2C receptor antagonist metergoline (2 and 5 mg/kg i.p.). These findings suggest that the hypophagic effect of fluoxetine is not likely to be dependent upon intact brain 5-hydroxytryptaminergic presynaptic function, nor is it mediated by an agonist action at the 5-HT2C receptor, but by an additional, unknown mechanism.

Animals↗
PubMed1996

18F-desmethoxyfallypride: a fluorine-18 labeled radiotracer with properties similar to carbon-11 raclopride for PET imaging studies of dopamine D2 receptors.

We have developed (S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-18F-fluoropropyl)-2- methoxybenzamide (18F-desmethoxyfallypride) as a fluorine-18 radiotracer with properties analogous to that of 11C-raclopride. In vitro experiments in rat brain homogenates showed an association rate constant of 2.16 x 10(8) M(-1)min(-1) and a dissociation rate constant of 0.073 min(-1). High striatal uptake (up to 0.08% injected dose/cc) of 18F-desmethoxyfallypride in rhesus monkeys was observed in PET experiments. The radiotracer cleared from the striata with a dissociation rate of 1.80 x 10(-2) min(-1). Striatum to cerebellum ratios peaked at 3.0 in 30 min after which they decreased steadily. Intravenously administered haloperidol displaced specifically bound 18F-desmethoxyfallypride with a koff of 0.058 min(-1). Synaptic dopamine released by the treatment of the monkeys with d-amphetamine increased the dissociation rate of 18F-desmethoxyfallypride to 0.83 min(-1) thus reducing specifically bound 18F-desmethoxyfallypride by 56% over a period of 42 mins compared to a reduction of only 20% in controls during this time period. The sensitivity of 18F-desmethoxyfallypride towards competition with dopamine should make this radiotracer useful in PET studies to evaluate in vivo pharmacological effects of various agents that alter levels of endogenous dopamine.

Animals↗
PubMed1996

Amplification of antibody production by phosphorothioate oligodeoxynucleotides.

A phosphorothioate oligodeoxynucleotide that is complementary (antisense) to the initiation region of the rev gene of HIV-1 causes hypergammaglobulinemia and splenomegaly in mice, and it induces B cell proliferation and differentiation in mouse spleen mononuclear cells (SMNCs) and human peripheral blood mononuclear cells in vitro. The current studies were performed to investigate the specificity of these immunomodulatory effects. Both the sense and antisense rev oligomers stimulated tritiated thymidine incorporation and secretion of immunoglobulin M (IgM) and immunoglobulin G (IgG) by mouse SMNCs in a concentration-dependent fashion, but the antisense oligomer produced greater immune effects. Studies comparing phosphorothioate oligomers (anti-rev, c-myc, and c-myb) either methylated or unmethylated at CpG dinucleotides showed that methylation effectively abrogated the proliferative effect and tended to reduce the immunoglobulin secretory activity, but the latter was not statistically significant except in the case of IgG in anti-rev oligomer-treated cultures. Mice were injected with the sense or antisense rev oligomers singly or in combination. The animals then were immunized with tetanus toxoid and received a booster 21 days later. Oligodeoxynucleotide-treated mice had significantly higher levels of IgM antibodies on days 28 and 35 and of IgG antibodies on days 14 and 35 as compared with mice that were immunized but received vehicle alone. There was no evidence for additive, synergistic, or antagonistic interactions of the sense and antisense rev oligomers. These results indicate that the unmethylated anti-rev oligomer is the most potent of the phosphorothioate oligomers tested at activating lymphocyte proliferation and differentiation and that a single intravenous injection of this oligodeoxynucleotide augments antibody production to a specific antigen as long as 35 days later.

Animals↗
PubMed1996

Epidemiologic linkage of rodent and human hantavirus genomic sequences in case investigations of hantavirus pulmonary syndrome.

Sin Nombre virus (SNV) causes the zoonotic disease hantavirus pulmonary syndrome (HPS). Its mechanisms of transmission from rodent to human are poorly understood. It is possible that specific genetic signature sequences could be used to determine the probable site of each case-patient's exposure. Environmental assessments suggested 12 possible sites of rodent exposure for 6 HPS patients. Rodents were captured at 11 of the 12 sites and screened for SNV infection within 2 weeks of the patient's diagnosis. Viral sequences amplified from tissues of rodents at each site were compared with those from case-patients' tissues. Rodents bearing viruses with genetic sequence identity to case-patients' viruses across 2 genomic segments were identified in 4 investigations but never at >1 site. Indoor exposures to rodents were especially common at implicated sites. By distinguishing among multiple possible sites of exposure, viral genotyping studies can enhance understanding of the conditions associated with infection by SNV.

Animals↗
PubMed1996

Infectious mononucleosis and Hodgkin's disease--a similar seasonality.

The presentation of Hodgkin's disease and acute infectious mononucleosis (glandular fever) due to Epstein-Barr virus, have similar seasonal features with the peak incidence around March. The extent of seasonal variation is also similar. Seasonality of Hodgkin's disease is most obvious and also significant in adult age groups below the age of 40. Amongst those over 40 years, seasonality was no longer present in the 40-59 but returned over age 60. Seasonal similarity does not prove a relationship. However two speculations are made on possible mechanisms. Firstly glandular fever may accelerate presentation in young adults, destined to present with HD. Secondly the Epstein-Barr virus may have an inherent seasonal behaviour whether causing acute infectious mononucleosis or when latent and playing a role in the aetiology of Hodgkin's disease.

Adolescent↗
PubMed1996

Synthesis of oligonucleotides labelled with 2,4-dinitrophenyl groups at thymidine sites.

A phosphoramidite has been produced for labelling oligonucleotides with DNP groups at thymidine sites during solid-phase synthesis. The dinitrophenylamino group is attached via a caproamidopropargyl group to the 5-position of uracil. A related DNP-labelling phosphoramidite has been synthesised where the propargyl group is replaced by propyl. Both phosphoramidites have been used to synthesises DNP-labelled oligonucleotides. A related DNP-labelled deoxyuridine triphosphate has also been synthesised. DNP labelled oligonucleotide probes are valuable in diagnostic applications for the antibody-based detection of DNA and RNA.

Chromatography, High Pressure Liquid↗
PubMed1996

Antibody-mediated detection and physical properties of oligonucleotides labelled with multiple internal and terminal 2,4-dinitrophenyl groups.

DNP-labelled phosphoramidites have been used to synthesis oligonucleotides with multiple DNP reporter groups. The antibody-mediated detection and the stability of duplexes formed by these labelled oligonucleotides have been studied. A DNP-labelled deoxyuridine triphosphate has also been used to enzymatically incorporate DNP-labels into DNA via the polymerase chain reaction. The use of DNP-labelled primers in the PCR has also been investigated.

Antibodies↗
PubMed1995

A trigonal form of the idarubicin:d(CGATCG) complex; crystal and molecular structure at 2.0 A resolution.

The X-ray crystal structure of the complex between the anthracycline idarubicin and d(CGATCG) has been solved by molecular replacement and refined to a resolution of 2.0 A. The final R-factor is 0.19 for 3768 reflections with Fo > or = 2 sigma (Fo). The complex crystallizes in the trigonal space group P31 with unit cell parameters a = b = 52.996(4), c = 33.065(2) A, alpha = beta = 90 degree, gamma = 120 degree. The asymmetric unit consists of two duplexes, each one being complexed with two idarubicin drugs intercalated at the CpG steps, one spermine and 160 water molecules. The molecular packing underlines major groove-major groove interactions between neighbouring helices, and an unusually low value of the occupied fraction of the unit cell due to a large solvent channel of approximately 30 A diameter. This is the first trigonal crystal form of a DNA-anthracycline complex. The structure is compared with the previously reported structure of the same complex crystallizing in a tetragonal form. The geometry of both the double helices and the intercalation site are conserved as are the intramolecular interactions despite the different crystal forms.

Base Sequence↗
PubMed1995

Self-association of a DNA loop creates a quadruplex: crystal structure of d(GCATGCT) at 1.8 A resolution.

BACKGROUND: The flexibility of DNA enables it to adopt three interconvertible types of duplex termed the A-, B- and Z-forms. It can also produce hairpin loops, triplex structures and guanine-rich quadruplex structures. Conformational flexibility assists in the tight packaging of DNA, for example in chromosomes. This is important given the large quantity of genetic information that must be packaged efficiently. Moreover, the ability of DNA to specifically self-associate or interact with complementary sequences is fundamental to many biological processes. Structural studies provide information about DNA conformation and DNA-DNA interactions and suggest features that might be relevant to how the molecule performs its biological role. RESULTS: We have characterized the structure of a synthetic heptanucleotide that folds into a novel loop structure. The loop is stabilized by association with a cation, by intra-strand hydrogen bonds between guanine and cytosine that are distinct from the normal Watson-Crick hydrogen bonds, and by van der Waals interactions. Two loops associate through the formation of four G.C pairs that exhibit pronounced base-stacking interactions. The formation of a symmetric A.A base pair further stabilizes loop dimerization. Stacking of the A.A pair on a symmetry-related A.A pairing assists the formation of a four-stranded assembly. A T.T pairing is also observed between symmetry-related loops. CONCLUSIONS: This analysis provides a rare example of an experimentally determined non-duplex DNA structure. It provides conformational detail relevant to the tight packaging or folding of a DNA strand and illustrates how a cation might modulate phosphate-phosphate repulsion in a tightly packed structure. The observation of base quartets involving G.C base pairs suggests a further structure to be considered in DNA-DNA interactions. The structure also provides detailed geometries for A.A and T.T base pairs.

Base Composition↗