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Biomedical subjects

C Switzer

Publications and source records attributed to C Switzer.

16 recordsLinked to original sources
PubMed2006

The impact of a gluten-free diet on adults with coeliac disease: results of a national survey.

UNLABELLED: OBJECTIVE We sought to evaluate the impact of the gluten-free diet on the 5,240 members of the Canadian Celiac Association (CCA). Data are presented on 2,681 adults (>or=16 years) with biopsy-proven celiac disease (CD). METHODS: A mail-out survey was used. Quality of life was evaluated using the 'SF12', and celiac-specific questions. RESULTS: Mean age was 56 years, mean age at diagnosis was 45 years, and 75% were female. The 'SF12' summary scores were similar to normative Canadian data, but were significantly lower for females and newly diagnosed patients. Respondents reported: following a gluten-free (GF) diet (90%), improvement on the diet (83%), and difficulties following the diet (44%), which included: determining if foods were GF (85%), finding GF foods in stores (83%), avoiding restaurants (79%), and avoiding travel (38%). Most common reactions to consumed gluten (among 73%) included pain, diarrhea, bloating, fatigue, nausea, and headache. Excellent information on CD and its treatment was provided by the CCA (64%), gastroenterologists (28%), dietitians (26%) and family doctor (12%). CONCLUSIONS: Quality of life in those with CD could be increased with early diagnosis, increased availability of gluten-free foods, improved food labelling, and better dietary instruction. Education of physicians and dietitians about CD and its treatment is essential.

Canada↗
PubMed2001

Probing structure and function with alternative nucleic acids bearing 2',5'-linked, zwitterionic, and isocytosine-isoguanine components.

The incorporation of alternative functional components into nucleic acids can provide insight into what molecular features are necessary for an informational macromolecule to be successful. It can also provide a means to improve particular physical characteristics of nucleic acids for diagnostic and therapeutic purposes, or probe mechanisms. By testing the fitness of nucleic acid-like molecules derived by structural permutations of RNA, it may also prove possible to trace a path from simple prebiotic precursors to biotic molecules. This article describes the applications of 2',5'-phosphodiester linked, zwitterionic, and base-permuted nucleic acid derivatives.

Carbohydrates↗
PubMed2001

DNA bending by asymmetrically tethered cations: influence of tether flexibility.

BACKGROUND: We have been studying the proposal that laterally asymmetric charge neutralization along the DNA double helix can induce collapse toward the neutralized surface. Results of previous experiments implied that such a phenomenon can occur, suggesting a role for local interphosphate repulsive forces in DNA shape and rigidity. RESULTS: We now show that, whereas six ammonium ions tethered to one DNA face on flexible propyl chains can induce detectable DNA curvature, tethering of ammonium ions on rigid propynyl tethers does not induce DNA curvature. Molecular modeling indicates differing propensities for phosphate salt bridge formation between propyl- and propynyl-tethered ammonium ions. CONCLUSIONS: Ammonium ion localization is suggested as a key factor in induced bending. Rigidification of the double helix by stacking of propyne groups cannot be excluded.

Cations↗
PubMed2000

RecA protein promotes strand exchange with DNA substrates containing isoguanine and 5-methyl isocytosine.

The Escherichia coli RecA protein pairs homologous DNA molecules and promotes DNA strand exchange in vitro. We have examined DNA strand exchange between a 70 nucleotide ssDNA fragment and a 40 bp duplex, in which all G and C residues (at 18 positions distributed throughout the 40 bp exchanged region) were replaced with the nonstandard nucleosides 2'-deoxyisoguanosine (iG) and 2'-deoxy-5-methylisocytidine (MiC), respectively. We demonstrate that the nonstandard oligonucleotides are substrates for the RecA protein, permitting DNA strand exchange in vitro at a rate and efficiency comparable to exchange with normal DNA substrates. This observation provides an expanded experimental basis for discussions of potential roles for iG and MiC in a genetic code. Experiments of this type also provide another avenue for exploring RecA-facilitated DNA pairing mechanisms.

5-Methylcytosine↗
PubMed2000

Nonenzymatic oligomerization on templates containing phosphodiester-linked acyclic glycerol nucleic acid analogues.

Nonenzymatic oligomerization reactions represent a model for studying the prebiotic replication of informational macromolecules. To explore the fitness of acyclic oligonucleotides in these reactions, we have synthesized a series of DNA hairpins appended with templates incorporating atactic glyceryl cytosine residues. Atactic glyceryl cytosine units are found to impede, but not to block, template-directed oligomerization of guanosine 5'-phosphoro-2-methylimidazole (2-MeImpG). Evidence suggests that both D and L glyceryl nucleoside configurations at a given template position contribute to product formation. The stability of DNA duplexes bearing isolated glyceryl cytosine residues has also been investigated. Duplex thermal denaturation experiments indicate that an atactic glyC. dG base-pair is intermediate in stability between a dC. dG pair and a dT. dG mismatch.

Base Sequence↗
PubMed1999

A DNA pentaplex incorporating nucleobase quintets.

Supramolecular self-assembly is an integral step in the formation of many biological structures. Here we report a DNA pentaplex that derives from a metal-assisted, hydrogen bond-mediated self-assembly process. In particular, cesium ions are found to induce pentameric assembly of DNA bearing the nonstandard nucleobase iso-guanine. The pentaplex was designed by using a simple algorithm to predict nucleobase structural requirements within a quintet motif. The design principles are general and should extend to complexes beyond pentaplex. Structures exhibiting molecularities of five or more were previously accessible to peptides, but not nucleic acids.

Cations↗
PubMed1998

2'-Deoxyisoguanosine adopts more than one tautomer to form base pairs with thymidine observed by high-resolution crystal structure analysis.

The questions of whether different tautomeric forms of nucleic acid bases exist to any significant extent in DNA, or what their possible roles in mutation may be, are under intense scrutiny. 2'-Deoxyisoguanosine (iG) has been suggested to have a propensity to adopt the enol form. Isoguanine (also called 2-hydroxyadenine) can be found in oxidatively damaged DNA generated from treating DNA with a Fenton-type reactive oxygen-generating system and is known to cause mutation. We have analyzed the three-dimensional structure of the DNA dodecamer d(CGC[iG]AATTTGCG) (denoted iG-DODE) by X-ray crystallography and NMR. The crystal structure of the iG-DODE complexed with the minor groove binder Hoechst 33342, refined to 1.4 A resolution, showed that the two independent iG.T base pairs in the dodecamer duplex adopt different (one in Watson-Crick and the other in wobble) conformations. The high-resolution nature of the structure also affords unprecedented clear information about the conformation and interactions of the Hoechst drug. The Hoechst 33342 binds in the narrow minor groove at the iGAATT site, with the N-methylpiperazine ring near the iG4.T21 base pair. Three hydrogen bonds are found between the NH of the Hoechst ligand and T-O2 DNA atoms. In solution, the two iG.T base pairs in iG-DODE predominantly are in the wobble form at 2 degreesC. At higher temperatures, another duplex form (likely involving the enol form of iG) is in slow exchange with the keto form and becomes significantly populated, reaching approximately 40% at 40 degreesC. Our data support the conclusion that iG pairs with T in a Watson-Crick configuration to a significant extent at physiological temperature (37 degreesC), which may explain the facile incorporation rate of T across from an iG during in vitro DNA replication.

Adenosine↗
PubMed1998

Ribosome-catalyzed peptide-bond formation with an A-site substrate covalently linked to 23S ribosomal RNA.

In the ribosome, the aminoacyl-transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks photochemically with G2553 of 23S ribosomal RNA (rRNA). This covalently linked substrate reacts with a peptidyl-tRNA analog to form a peptide bond in a peptidyl transferase-catalyzed reaction. This result places the conserved 2555 loop of 23S rRNA at the peptidyl transferase A site and suggests that peptide bond formation can occur uncoupled from movement of the A-site tRNA. Crosslink formation depends on occupancy of the P site by a tRNA carrying an intact CCA acceptor end, indicating that peptidyl-tRNA, directly or indirectly, helps to create the peptidyl transferase A site.

Anti-Bacterial Agents↗
PubMed1997

Regioselective effect of zwitterionic DNA substitutions on DNA alkylation: evidence for a strong side chain orientational preference.

The incorporation of zwitterionic residues (5-substituted omega-aminoalkyl-2'-deoxypyrimidines) into DNA has been reported to bend DNA as measured by aberrant gel mobility [Strauss et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9515-9520]. Herein we report that DNA methylation by N-methyl-N-nitrosourea at N7-guanine is regioselectively inhibited by point substitutions of the zwitterionic residues 5-(6-aminohexyl)-2'-deoxycytidine, 5-(6-aminohexyl)-2'-deoxyuridine, or 5-(3-aminopropyl)-2'-deoxyuridine. No inhibition is observed for DNA methylation by dimethyl sulfate. On the basis of inhibition patterns for methylation with the different zwitterionic substitutions and the different length tethers, the omega-aminoalkyl side chains prefer to adopt a conformation that points them toward the 3'-base. Molecular modeling grid searches, coupled with energy minimizations, and simulated annealing molecular dynamics studies indicate that unfavorable steric interactions with the 5'-base and backbone, as well as stabilizing electrostatic interactions with electronegative atoms on the 3'-side, are responsible for the observed conformational preference. No evidence for association of the cationic side chain with the phosphate backbone is observed. The observed bending of DNA induced by the tethered ammonium ions may simply arise from their localization in the major groove.

Circular Dichroism↗
PubMed1997

Beyond guanine quartets: cation-induced formation of homogenous and chimeric DNA tetraplexes incorporating iso-guanine and guanine.

BACKGROUND: iso-Guanine (iso-G) is the purine component of an isomeric Watson-Crick base pair that may have existed prebiotically. By comparing the abiotic molecular recognition properties of iso-G and its complement, iso-cytosine (iso-C), with those of genomic nucleotide bases, it may be possible to explain the exclusion of the iso-G-iso-C base pair from modern genomes. Whether a nucleobase forms quartets may have a key role in determining its functionality. Biotically, nucleic acid tetraplexes have been implicated in cellular functions; prebiotically, tetraplexes would probably interfere with replication. Recently, in vitro selection has yielded receptors and catalysts that incorporate G quartets. The versatility of these structures could be enhanced by expanding the range of bases that can form the quartet motif. RESULTS: Native polyacrylamide gel electrophoresis of oligonucleotides bearing runs of iso-G provides strong support for tetraplex formation via cation-promoted DNA strand association. In particular, when strands of different lengths bearing the same iso-G tetrad recognition element were combined, five bands were observed after electrophoresis, corresponding to all possible heterotetraplexes with parallel strand alignment. An analogous experiment with a mixture of strands bearing iso-G or G tetrad recognition domains supports the existence of mixed iso-G/G tetraplexes with antiparallel strand alignment at chimeric junctions. iso-G tetraplex and quartet structure has also been probed by a photo-crosslinking experiment, ultra-violet spectroscopy and theoretical calculations. CONCLUSIONS: As iso-G and G both have a marked tendency to form tetraplexes, their tandem inclusion in genetic material may be problematic, leading to double-stranded DNA half composed of bases that have a tendency to auto-associate. The resulting density of 'selfish' bases could undermine Watson-Crick pair formation, especially in a prebiotic context devoid of enzymes. Nevertheless, the ability of iso-G to form mixed quartets with G may provide a basis for altering the properties of tetraplexes in the domain of artificial receptors or catalysts from in vitro selections.

Cations↗
PubMed1996

DNA bending by hexamethylene-tethered ammonium ions.

DNA is bent when complexed with certain proteins. We are exploring the hypothesis that asymmetric neutralization of phosphate charges will cause the DNA double helix to collapse toward the neutralized face. We have previously shown that DNA spontaneously bends toward one face of the double helix when it is partially substituted with neutral methylphosphonate linkages. We have now synthesized DNA duplexes in which cations are tethered by hexamethylene chains near specific phosphates. Electrophoretic phasing experiments demonstrate that tethering six ammonium ions on one helical face causes DNA to bend by approximately 5 degrees toward that face, in qualitative agreement with predictions. Ion pairing between tethered cations and DNA phosphates provides a new model for simulating the electrostatic consequences of phosphate neutralization by proteins.

Base Sequence↗
PubMed1996

DNA bending by a phantom protein.

BACKGROUND: Despite its stiffness, duplex DNA is extensively bent and folded during packaging and gene expression in biological systems. Modulation of the electrostatic repulsion between phosphates in the DNA backbone may be important in the bending of DNA by proteins. Here, we analyze the shape of DNA molecules that have been modified chemically to mimic the electrostatic consequences of a bound protein. RESULTS: We have simulated salt bridges between DNA phosphates and cationic amino acid sidechains of a phantom protein by tethering ammonium cations to one face of the DNA helix. Tethered ammonium cations, but not neutral acetylated controls, induce DNA to bend toward its neutralized surface. CONCLUSIONS: The shape of DNA molecules bearing a laterally-asymmetric distribution of tethered cations agrees qualitatively with theoretical predictions and with results previously obtained using neutral phosphate analogs. These data suggest principles that might be applied to the design of artificial DNA-bending proteins.

Base Sequence↗
PubMed1992

Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code.

One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role.

Amino Acid Sequence↗