Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 37 records · Page 2Linked to original sources

PubMed2000

The zalpha domain of the editing enzyme dsRNA adenosine deaminase binds left-handed Z-RNA as well as Z-DNA.

The Zalpha domain of human double-stranded RNA adenosine deaminase 1 binds specifically to left-handed Z-DNA and stabilizes the Z-conformation. Here we report spectroscopic and analytical results that demonstrate that Zalpha can also stabilize the left-handed Z-conformation in double-stranded RNA. Zalpha induces a slow transition from the right-handed A-conformation to the Z-form in duplex r(CG)(6), with an activation energy of 38 kcal mol(-1). We conclude that Z-RNA as well as Z-DNA can be accommodated in the tailored binding site of Zalpha. The specific binding of Z-RNA by Zalpha may be involved in targeting double-stranded RNA adenosine deaminase 1 for a role in hypermutation of RNA viruses.

Adenosine Deaminase↗

PubMed2000

The zab domain of the human RNA editing enzyme ADAR1 recognizes Z-DNA when surrounded by B-DNA.

The Zab domain of the editing enzyme ADAR1 binds tightly and specifically to Z-DNA stabilized by bromination or supercoiling. A stoichiometric amount of protein has been shown to convert a substrate of suitable sequence to the Z form, as demonstrated by a characteristic change in the CD spectrum of the DNA. Now we show that Zab can bind not only to isolated Z-forming d(CG)(n) sequences but also to d(CG)(n) embedded in B-DNA. The binding of Zab to such sequences results in a complex including Z-DNA, B-DNA, and two B-Z junctions. In this complex, the d(CG)(n) sequence, but not the flanking region, is in the Z conformation. The presence of Z-DNA was detected by cleavage with a Z-DNA specific nuclease, by undermethylation using Z-DNA sensitive SssI methylase, and by circular dichroism. It is possible that Zab binds to B-DNA with low affinity and flips any favorable sequence into Z-DNA, resulting in a high affinity complex. Alternatively, Zab may capture Z-DNA that exists transiently in solution. The binding of Zab to potential as well as established Z-DNA segments suggests that the range of biological substrates might be wider than previously thought.

Adenosine Deaminase↗

PubMed2000

Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds.

A new type of self-assembling peptide (sapeptide) scaffolds that serve as substrates for neurite outgrowth and synapse formation is described. These peptide-based scaffolds are amenable to molecular design by using chemical or biotechnological syntheses. They can be tailored to a variety of applications. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary beta-sheet oligopeptides under physiological conditions, producing a hydrogel material. The scaffolds can support neuronal cell attachment and differentiation as well as extensive neurite outgrowth. Furthermore, they are permissive substrates for functional synapse formation between the attached neurons. That primary rat neurons form active synapses on such scaffold surfaces in situ suggests these scaffolds could be useful for tissue engineering applications. The buoyant sapeptide scaffolds with attached cells in culture can be transported readily from one environment to another. Furthermore, these peptides did not elicit a measurable immune response or tissue inflammation when introduced into animals. These biological materials created through molecular design and self assembly may be developed as a biologically compatible scaffold for tissue repair and tissue engineering.

Amino Acid Sequence↗

PubMed2000

Sequence, genomic organization and functional expression of the murine tRNA-specific adenosine deaminase ADAT1.

We have recently identified the first mammalian tRNA-specific adenosine deaminase human ADAT1, a member of the ADAR family of RNA editing enzymes. This protein is responsible for the first step of the unique A(37) to m(1)I(37) modification in eukaryotic tRNA(Ala). Here, we present the genomic structure of murine ADAT1 and the functional expression of mADAT1 cDNA. In mouse, as well as in human, ADAT1 is expressed from a single copy gene. The coding region of the mADAT1 gene is spread over nine exons, covering approximately 30kb of genomic DNA and encodes a protein of 499 amino acids. Overall, mADAT1 shares 81% nucleotide homology and 87.5% protein homology with the human ortholog. The recombinant mouse protein is active specifically and with a high efficiency on human tRNA(Ala) in vitro. Its genomic organization is compared to the structures of the sequence-related, pre-mRNA specific adenosine deaminases ADAR1 and ADAR2.

Adenosine Deaminase↗

PubMed2000

Changing genetic information through RNA editing.

RNA editing, the post-transcriptional alteration of a gene-encoded sequence, is a widespread phenomenon in eukaryotes. As a consequence of RNA editing, functionally distinct proteins can be produced from a single gene. The molecular mechanisms involved include single or multiple base insertions or deletions as well as base substitutions. In mammals, one type of substitutional RNA editing, characterized by site-specific base-modification, was shown to modulate important physiological processes. The underlying reaction mechanism of substitutional RNA editing involves hydrolytic deamination of cytosine or adenosine bases to uracil or inosine, respectively. Protein factors have been characterized that are able to induce RNA editing in vitro. A supergene family of RNA-dependent deaminases has emerged with the recent addition of adenosine deaminases specific for tRNA. Here we review the developments that have substantially increased our understanding of base-modification RNA editing over the past few years, with an emphasis on mechanistic differences, evolutionary aspects and the first insights into the regulation of editing activity.

Animals↗

PubMed2000

Failure to detect changes in color for lines rotating in depth: the effects of grouping and type of color change.

A new technique for measuring change detection was introduced in which contours rotating in depth around a vertical axis (in a computer display) could be altered in color as they passed through their point of minimum extension (the median plane) where a thin static vertical occluder hid the change. Sets of five or six contours were either strongly grouped (similar in length, orientation and spacing) or weakly grouped (of variable length, orientation and spacing). Changes consisted of one line changing to a new color or else two lines swapping colors. The measure was the proportion of missed changes. When subjects were not instructed to look for change almost no changes were reported although subjects were told beforehand that they would have to describe the configuration after viewing it. When subjects were instructed to look for changes, it was found that detection of color change was significantly better for strongly grouped lines. It is proposed that grouping, by reducing redundancy, also reduces attentional demands with respect to the properties on which it is based, making it easier to attend to and therefore detect changes in other properties. We found that it was much easier to detect the introduction of a new color than to detect a swap between two existing colors. It is hypothesized that swap-type changes were harder to detect because they required attention to a conjunction of position and color.

Adult↗

PubMed2000

Mutational study reveals that tertiary interactions are conserved in ribosomal frameshifting pseudoknots of two luteoviruses.

Expression of the putative replicase of potato leafroll virus (PLRV) is regulated by -1 ribosomal frameshifting in which a primary viral transcript has two overlapping open reading frames (ORFs). A region of 39 nt at the junction of the two ORFs is essential for frameshifting to occur. It has been shown to harbor two signals, one active on the level of the primary structure, termed the slippery sequence, and one component that forms a secondary or tertiary level structure, described as either a pseudoknot or a stem-loop motif. We have performed extensive site-directed mutagenesis of the frameshifting region and analyzed individual mutants for their ability to promote -1 frameshifting in vitro. Detailed comparison of our results with analogous mutants in the frameshifting region of the evolutionarily related beet western yellow virus, for which a crystal structure is available, unequivocally argues for the pseudoknot to be the structural motif necessary for the frameshifting function in PLRV transcripts. Mutations in PLRV that affect putative pseudoknot-specific tertiary-base interactions drastically affect frameshifting activity. In addition, a specific deletion mutant was identified that displayed PLRV wild-type frameshifting activity with only 22 nt available for pseudoknot formation.

Base Sequence↗

PubMed2000

Conformational behavior of ionic self-complementary peptides.

Several de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable beta-sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N-terminus and positively charged arginine or lysine residues located toward the C-terminus. This arrangement of charge balances the alpha-helical dipole moment (C --> N), resulting in a strong tendency to form stable alpha-helices as well. Therefore, these peptides can form both stable alpha-helices and beta-sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable beta-sheet and alpha-helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.

Amino Acid Sequence↗

PubMed2000

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel.

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.

Calcium Channel Blockers↗

PubMed1999

Specific mutations in a viral RNA pseudoknot drastically change ribosomal frameshifting efficiency.

Many viruses regulate protein synthesis by -1 ribosomal frameshifting using an RNA pseudoknot. Frameshifting is vital for viral reproduction. Using the information gained from the recent high-resolution crystal structure of the beet western yellow virus pseudoknot, a systematic mutational analysis has been carried out in vitro and in vivo. We find that specific nucleotide tertiary interactions at the junction between the two stems of the pseudoknot are crucial. A triplex is found between stem 1 and loop 2, and triplex interactions are required for frameshifting function. For some mutations, loss of one hydrogen bond is sufficient to abolish frameshifting. Furthermore, mutations near the 5' end of the pseudoknot can increase frameshifting by nearly 300%, possibly by modifying ribosomal contacts. It is likely that the selection of suitable mutations can thus allow viruses to adjust frameshifting efficiencies and thereby regulate protein synthesis in response to environmental change.

Cells, Cultured↗

PubMed1999

Visualization of interstitial cells of Cajal in living, intact tissues.

Interstitial cells of Cajal (ICC) appear to be a major element in pacing and signal transmission in the gastrointestinal tract. A prominent problem in the study of ICC has been the difficulty in observing them in intact tissues. We used several methods to visualize living ICC in freshly-dissected tissues: (1) Placing small crystals of the lipophilic dye DiI in the submucosal-circular muscle border in the mouse colon resulted in the labeling of living ICC-like cells. Two main morphological cell types, bipolar and multipolar, were noted. The DiI stain could be converted into a stable, electron-opaque product. Electron-microscopic observations showed that the labeled cells had the typical appearance of ICC reported in previous studies. (2) Living ICC in the region of the myenteric plexus (ICC-MP) in the small intestines of mice and guinea-pigs were observed with Nomarski optics. This enabled the visualization of ICC in living tissues, and the impalement of the cells with Lucifer yellow-filled microelectrodes. The dye-labeled cells had the morphological features of ICC-MP, and about 30% of them were found to be dye coupled to 1-21 other ICC. The identity of the cells as ICC was verified by electron-microscopy following photoconversion, and by c-kit immunohistochemistry. (3) Living ICC were labeled with a c-kit antibody that does not require tissue fixation. This resulted in the fluorescent staining of the entire ICC network. Single cells were labeled by dye injection, which provided a detailed picture of ICC morphology. This method was found to be suitable for a wide range of tissues. We expect that these three methods for identifying ICC in intact, living tissues will be useful for physiological and pharmacological investigations of ICC in a variety of gastrointestinal tissues.

Animals↗

PubMed1999

The solution structure of the Zalpha domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA.

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zalpha. Here we report the solution structure of free Zalpha and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zalpha)(2)/Z-DNA complex shows that most Z-DNA contacting residues in free Zalpha are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (alpha+beta)helix-turn-helix/B-DNA complexes suggests that binding of Zalpha to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.

Adenosine Deaminase↗

PubMed1999

A 6 bp Z-DNA hairpin binds two Z alpha domains from the human RNA editing enzyme ADAR1.

The Z alpha domain of the human RNA editing enzyme double-stranded RNA deaminase I (ADAR1) binds to left-handed Z-DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Z alpha domains bind to one d(CG)3T4(CG)3 hairpin which contains a stem of six base pairs in the Z-DNA conformation. Both wild-type Z alpha and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (> or = 6 bp) segments of Z-DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.

Adenosine Deaminase↗

PubMed1999

Identification and characterization of a human tRNA-specific adenosine deaminase related to the ADAR family of pre-mRNA editing enzymes.

The mammalian adenosine deaminases acting on RNA (ADARs) constitute a family of sequence-related proteins involved in pre-mRNA editing of nuclear transcripts through site-specific adenosine modification. We report here the identification and characterization of a human ADAR protein, hADAT1, that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala). It represents the functional homologue of the recently identified yeast protein Tad1p [Gerber, A., Grosjean, H., Melcher, T. & Keller, W. (1998) EMBO J. 17, 4780-4789]. The hADAT1 cDNA predicts a protein of 502 aa whose sequence displays strongest overall homology to a Drosophila melanogaster ORF (50% similarity, 32% identity), and the catalytic domain is closely related to the other ADAR proteins. In vitro, the recombinantly expressed and purified hADAT1 protein efficiently and specifically deaminates A(37) in the anticodon loop of tRNA(Ala) from higher eukaryotes and with lower efficiency from lower eukaryotes. It does not modify adenosines residing in double-stranded RNA or in pre-mRNAs that serve as substrates for ADAR1 or ADAR2. The anticodon stem-loop of tRNA(Ala) alone is not a functional substrate for hADAT1. The enzyme is expressed ubiquitously in human tissues and is represented by a single gene. The identification and cloning of hADAT1 should help to elucidate the physiological significance of this unique modification in tRNA(Ala), which is conserved from yeast to man.

Adenosine Deaminase↗

PubMed1999

The interaction between Z-DNA and the Zab domain of double-stranded RNA adenosine deaminase characterized using fusion nucleases.

Zab is a structurally defined protein domain that binds specifically to DNA in the Z conformation. It consists of amino acids 133-368 from the N terminus of human double-stranded RNA adenosine deaminase, which is implicated in RNA editing. Zab contains two motifs with related sequence, Zalpha and Zbeta. Zalpha alone is capable of binding Z-DNA with high affinity, whereas Zbeta alone has little DNA binding activity. Instead, Zbeta modulates Zalpha binding, resulting in increased sequence specificity for alternating (dCdG)n as compared with (dCdA/dTdG)n. This relative specificity has previously been demonstrated with short oligonucleotides. Here we demonstrate that Zab can also bind tightly to (dCdG)n stabilized in the Z form in supercoiled plasmids. Binding was assayed by monitoring cleavage of the plasmids using fusion nucleases, in which Z-DNA-binding peptides from the N terminus of double-stranded RNA adenosine deaminase are linked to the nuclease domain of FokI. A fusion nuclease containing Zalpha shows less sequence specificity, as well as less conformation specificity, than one containing Zab. Further, a construct in which Zbeta has been replaced in Zab with Zalpha, cleaves Z-DNA regions in supercoiled plasmids more efficiently than the wild type but with little sequence specificity. We conclude that in the Zab domain, both Zalpha and Zbeta contact DNA. Zalpha contributes contacts that produce conformation specificity but not sequence specificity. In contrast, Zbeta contributes weakly to binding affinity but discriminates between sequences of Z-DNAs.

Adenosine Deaminase↗

PubMed1999

Crystal structure of the Zalpha domain of the human editing enzyme ADAR1 bound to left-handed Z-DNA.

The editing enzyme double-stranded RNA adenosine deaminase includes a DNA binding domain, Zalpha, which is specific for left-handed Z-DNA. The 2.1 angstrom crystal structure of Zalpha complexed to DNA reveals that the substrate is in the left-handed Z conformation. The contacts between Zalpha and Z-DNA are made primarily with the "zigzag" sugar-phosphate backbone, which provides a basis for the specificity for the Z conformation. A single base contact is observed to guanine in the syn conformation, characteristic of Z-DNA. Intriguingly, the helix-turn-helix motif, frequently used to recognize B-DNA, is used by Zalpha to contact Z-DNA.

Adenosine Deaminase↗

PubMed1999

RNA processing in evolution. The logic of soft-wired genomes.

Direct read-out of information from DNA into RNA allows the genome to be faithfully reproduced in RNA. This outcome occurs in what may be called "hard-wired" organisms. On the other hand, in what we refer to as "soft-wired" organisms, RNA is processed extensively, allowing a number of different messages to be produced from the same gene. As a consequence, the nucleotide sequences present in RNA (referred to here as the ribotype) differ from those present in DNA (the genotype). In soft-wired organisms, RNA processing can be thought of as a series of steps, one or more of which have two mutually exclusive outcomes: a "default" outcome and an "alternative" outcome. In the presence of appropriate regulatory signals, the RNA is processed using the alternative pathway, while the default pathway is used in their absence. The setup is functionally equivalent to that found in binary "logic gates." In both cases, "logical operations" are implemented by using regulatory signals to establish a conditional relationship between input and output and can be described using the Boolean operators AND, OR, and NOT. In the case of RNA processing events, the outcomes can be used either to directly regulate cellular responses or to control other RNA processing events. In the latter case, "networks" are established that make processing of one RNA contingent on another. Such networks allow cells to respond to their surroundings by changing the connectivity between different RNA processing events, using RNA as a substrate to compute an appropriate response. As such logical operations impact phenotype, they are subject to natural selection. Through reverse transcription, successful outcomes can be incorporated into the genome.

Animals↗

PubMed1999

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1.

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Adenosine Deaminase↗