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Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 55 records · Page 3Linked to original sources
PubMed1999

Structure-function analysis of the Z-DNA-binding domain Zalpha of dsRNA adenosine deaminase type I reveals similarity to the (alpha + beta) family of helix-turn-helix proteins.

RNA editing alters pre-mRNA through site-selective adenosine deamination, which results in codon changes that lead to the production of novel proteins. An enzyme that catalyzes this reaction, double-stranded RNA adenosine deaminase (ADAR1), contains two N-terminal Z-DNA-binding motifs, Zalpha and Zbeta, the function of which is as yet unknown. In this study, multidimensional NMR spectroscopy was used to show that the topology of Zalpha is alpha1beta1alpha2alpha3beta2beta3. Long-range NOEs indicate that beta1 and beta3 interact with each other. Site-directed mutagenesis was used to identify residues in alpha3, beta3 and the loop connecting beta2 to beta3 that affect Z-DNA binding. Also identified were 11 hydrophobic residues that are essential for protein stability. Comparison with known structures reveals some similarity between Zalpha and (alpha + beta) helix-turn-helix proteins, such as histone 5 and the family of hepatocyte nuclear factor-3 winged-helix-turn-helix transcription factors. Taken together, the structural and functional data suggest that recognition of Z-DNA by Zalpha involves residues in both the alpha3 helix and the C-terminal beta-sheet.

Adenosine Deaminase↗
PubMed1999

Assessment of GABA(A)benzodiazepine receptor (GBzR) sensitivity in patients on benzodiazepines.

OBJECTIVES: To measure GABA(A) benzodiazepine receptor sensitivity in patients taking benzodiazepines and compare with matched controls. METHODS: Seven patients who were on prescribed benzodiazepines for an anxiety disorder or insomnia were recruited from general practice and an adult mental health service outpatient clinic. They were matched with seven volunteers. All subjects received an intravenous injection of midazolam 50 microgram/kg in 10 ml normal saline over 10 min. Objective responses to midazolam were assessed using saccadic eye movement velocity slowing and subjective assessments using visual analogue scales. Measurements were recorded for 120 min and plasma midazolam concentrations obtained at 15-min intervals post-infusion to 120 min. Ratios of pharmacodynamic/pharmacokinetic effects were obtained for each individual to estimate GABA(A) benzodiazepine receptor sensitivity. RESULTS: Patients had an attenuated response to midazolam on both subjective and objective measures. GABA(A) benzodiazepine receptor sensitivity was significantly reduced in the patient group. CONCLUSIONS: Chronic treatment with benzodiazepines was associated with reduced effects of midazolam. Saccadic eye movement velocity was especially sensitive as a measure of attenuated response.

Adult↗
PubMed1999

A mechanosensitive calcium channel in human intestinal smooth muscle cells.

BACKGROUND & AIMS: Gastrointestinal smooth muscle strips devoid of enteric nerve cells can contract in response to stretch, suggesting that mechanosensitivity and mechanotransduction can occur at the level of the smooth muscle cell. The aim of this study was to determine whether stretch-activated calcium channels are present in gastrointestinal smooth muscle cells. METHODS: Whole-cell and single-channel calcium currents were measured from human jejunal circular smooth muscle cells in response to increased intracellular pressure, bath perfusion, and membrane stretch. RESULTS: At 10 mm Hg positive pressure, peak calcium current increased from -36 +/- 10 pA to -53 +/- 13 pA. Bath perfusion at 10 mL/min increased calcium current from -97.7 +/- 14 pA to -122 +/- 16 pA. Single-channel open probability increased in response to negative pipette pressure. All increases were blocked by nifedipine. CONCLUSIONS: A stretch-activated, nifedipine-sensitive calcium channel is present in human jejunal circular smooth muscle cells. The channel is activated by both an increase in intracellular pressure and by external shear forces. The presence of a stretch-activated calcium channel in gastrointestinal smooth muscle cells may allow the smooth muscle cells to act directly as mechanotransducers and to participate in the regulation of smooth muscle tone and intestinal motility.

Calcium Channels↗
PubMed1999

Biological surface engineering: a simple system for cell pattern formation.

Biological surface engineering using synthetic biological materials has a great potential for advances in our understanding of complex biological phenomena. We developed a simple system to engineer biologically relevant surfaces using a combination of self-assembling oligopeptide monolayers and microcontact printing (muCP). We designed and synthesized two oligopeptides containing a cell adhesion motif (RADS)n (n = 2 and 3) at the N-terminus, followed by an oligo(alanine) linker and a cysteine residue at the C-terminus. The thiol group of cysteine allows the oligopeptides to attach covalently onto a gold-coated surface to form monolayers. We then microfabricated a variety of surface patterns using the cell adhesion peptides in combination with hexa-ethylene glycol thiolate which resist non-specific adsorption of proteins and cells. The resulting patterns consist of areas either supporting or inhibiting cell adhesion, thus they are capable of aligning cells in a well-defined manner, leading to specific cell array and pattern formations.

3T3 Cells↗
PubMed1999

Mechanisms of action of selective serotonin reuptake inhibitors in the treatment of psychiatric disorders.

Selective serotonin reuptake inhibitors (SSRIs) have demonstrated efficacy in depression and anxiety disorders. This raises the question of how the single action of serotonin reuptake inhibition can improve several psychiatric conditions. In order to understand this apparent paradox it is necessary to consider where SSRIs act in the pathogenic process underlying depression or anxiety disorders. Tryptophan depletion has been used extensively in research into depression and has shown that, in patients receiving an SSRI whose depression is in remission, depleting serotonin leads to recurrence of the disorder. Similar results have been found for panic disorder. This suggests that increased levels of serotonin are necessary in the synapse for the SSRI to be effective in the treatment of depression and panic disorder. In obsessive compulsive disorder, depletion of serotonin in patients recovered on an SSRI does not cause relapse; receptor adaptation may be more important. Variations within the SSRI drug class, such as the selectivity ratios for serotonin versus noradrenaline uptake, elimination half-life, and affinity for the 5-HT2 receptor have been identified and may be important determinants of efficacy, side effects and clinical use.

Anxiety Disorders↗
PubMed1999

Left-handed Z-DNA: structure and function.

Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

Adenosine Deaminase↗
PubMed1999

Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot.

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

Crystallography, X-Ray↗
PubMed1999

RNA processing and the evolution of eukaryotes.

In eukaryotes, RNA processing events, including alternative splicing and RNA editing, can generate many different messages from a single gene. As a consequence, the RNA pool, which we refer to here as the 'ribotype', has a different information content from the genotype and can vary as circumstances change. The outcome of a single RNA processing event often regulates the outcome of another, giving rise to networks that affect the composition and expression of a particular ribotype. Successful ribotypes are determined by natural selection, and can be incorporated into the genome over time by reverse transcription. Eukaryotic evolution is therefore influenced by the alternate ways in which RNAs are processed and the continual interplay between RNA and DNA.

Animals↗
PubMed1999

Crystallization and preliminary studies of the DNA-binding domain Za from ADAR1 complexed to left-handed DNA.

The proteolytically defined Z-DNA binding domain Za of human adenosine deaminase type 1 (hADAR1) has been crystallized in complex with the DNA oligomer d(TCGCGCG). The crystals were obtained from a solution containing ammonium sulfate as precipitating agent and belong to the tetragonal space group P4212. A complete diffraction data set has been collected to a resolution of 2.4 A. The unit-cell dimensions are a = b = 85.9, c = 71.3 A. A Raman spectrum of the complex indicates that the DNA in the complex adopts the left-handed Z conformation.

Adenosine Deaminase↗
PubMed1999

Effects of melatonin on ionic currents in cultured ocular tissues.

The effects of melatonin on ionic conductances in a cultured mouse lens epithelial cell line (alpha-TN4) and in cultured human trabecular meshwork (HTM) cells were measured using the amphotericin perforated patch whole cell voltage-clamp technique. Melatonin stimulated a voltage-dependent Na+-selective current in lens epithelial cells and trabecular meshwork cells. The effects of melatonin were observed at 50 pM and were maximal at 100 microM. Melatonin enhanced activation and inactivation kinetics, but no change was observed in the voltage dependence of activation. The results are consistent with an increase in the total number of ion channels available for activation by membrane depolarization. Melatonin was also found to stimulate a K+-selective current at high doses (1 mM). Melatonin did not affect the inwardly rectifying K+ current or the delayed rectifier type K+ current that has been described in cultured mouse lens epithelial cells. The results show that melatonin specifically stimulated the TTX-insensitive voltage-dependent Na+ current by an apparently novel mechanism.

Animals↗
PubMed1999

Combined effects of argon laser irradiation and fluoride treatments in prevention of caries-like lesion formation in enamel: an in vitro study.

The sample of this study consisted of 10 human permanent molars, which were sectioned into tooth quarters using slow speed diamond saw (Isomet), then a quarter from each tooth was assigned to one of four treatment groups: A) control (no treatment); B) argon laser only; C) argon laser plus neutral sodium fluoride for 4 minutes; D) argon laser with zinc fluoride for 4 minutes; each tooth quarter was coated with acid resistant varnish, leaving a window of 2 mm x 3 mm of sound enamel exposed. The results were that teeth treated with argon laser and then with zinc fluoride for four minutes have significantly reduced white spotting or etching. Zinc fluoride and argon laser combination are particularly effective in compensating for carbonate inclusion. It has a property of stabilizing hydroxyapetite crystal and restoring the structural defects of this crystal. Caries detection dye is a reliable diagnostic tool for white spot lesions. It reduces the false positive and false negative results by 60%, when compared with visual 16x magnification.

Argon↗
PubMed1998

Intercalated cytosine motif and novel adenine clusters in the crystal structure of the Tetrahymena telomere.

The cytosine-rich strand of the Tetrahymena telomere consists of multiple repeats of sequence d(AACCCC). We have solved the crystal structure of the crystalline repeat sequence at 2.5 A resolution. The adenines form two different and previously unknown clusters (A clusters) in orthogonal directions with their counterparts from other strands, each containing a total of eight adenines. The clusters appear to be stable aggregates held together by base stacking and three different base-pairing modes. Two different types of cytosine tetraplexes are found in the crystal. Each four-stranded complex is composed of two intercalated parallel-stranded duplexes pointing in opposite directions, with hemiprotonated cytosine-cytosine (C.C+) base pairs. The outermost C.C+base pairs are from the 5'-end of each strand in one cytosine tetraplex and from the 3'-end of each strand in the other. The A clusters and the cytosine tetraplexes form two alternating stacking patterns, creating continuous base stacking in two perpendicular directions along the x - and z -axes. The adenine clusters could be organizational motifs for macromolecular RNA.

Adenine↗
PubMed1998

Spectroscopic characterization of a DNA-binding domain, Z alpha, from the editing enzyme, dsRNA adenosine deaminase: evidence for left-handed Z-DNA in the Z alpha-DNA complex.

Double-stranded RNA adenosine deaminase (ADAR1) is an ubiquitous enzyme in metazoa that edits pre-mRNA changing adenosine to inosine in regions of double-stranded RNA. Zalpha, an N-terminal domain of human ADAR1 encompassing 76 amino acid residues, shows apparent specificity for the left-handed Z-DNA conformation adopted by alternating (dGdC) polymers modified by bromination or methylation, as well as for (dGdC)13 inserts present in supercoiled plasmids. Here, a combination of circular dichroism, fluorescence, and gel-retardation studies is utilized to characterize recombinant Zalpha peptide and to examine its interaction with DNA. Results from laser-Raman spectroscopy experiments provide direct evidence for the existence of Z-DNA in peptide-DNA complexes.

Adenosine Deaminase↗
PubMed1998

The Zalpha domain from human ADAR1 binds to the Z-DNA conformer of many different sequences.

Z-DNA, the left-handed conformer of DNA, is stabilized by the negative supercoiling generated during the movement of an RNA polymerase through a gene. Recently, we have shown that the editing enzyme ADAR1 (double-stranded RNA adenosine deaminase, type 1) has two Z-DNA binding motifs, Zalpha and Zbeta, the function of which is currently unknown. Here we show that a peptide containing the Zalpha motif binds with high affinity to Z-DNA as a dimer, that the binding site is no larger than 6 bp and that the Zalpha domain can flip a range of sequences, including d(TA)3, into the Z-DNAconformation. Evidence is also presented to show that Zalpha and Zbeta interact to form a functional DNA binding site. Studies with atomic force microscopy reveal that binding of Zalpha to supercoiled plasmids is associated with relaxation of the plasmid. Pronounced kinking of DNA is observed, and appears to be induced by binding of Zalpha. The results reported here support a model where the Z-DNA binding motifs target ADAR1 to regions of negative supercoiling in actively transcribing genes. In this situation, binding by Zalpha would be dependent upon the local level of negative superhelicity rather than the presence of any particular sequence.

Adenosine Deaminase↗
PubMed1998

Double-stranded RNA-specific adenosine deaminase: nucleic acid binding properties.

The RNA-specific adenosine deaminase (ADAR1, herein referred to as ADAR) is an interferon-inducible RNA-editing enzyme. ADAR catalyzes the C-6 deamination of adenosine in double-stranded (ds) structures present in viral RNAs and cellular pre-mRNAs as well as synthetic dsRNA substrates. ADAR possesses three functionally distinct copies of the highly conserved double-stranded RNA binding R motif (RI, RII, RIII) implicated in the recognition of dsRNA structures within the substrate RNAs. ADAR is also a Z-DNA-binding protein. Two Z-DNA binding motifs (Zalpha and Zbeta) present in ADAR correspond to repeated regions homologous to the N-terminal region of the vaccinia virus E3L protein. Here we describe assay methods for measurement of ADAR enzymatic activity, dsRNA binding activity, and Z-DNA binding activity.

Adenosine Deaminase↗