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Biomedical subjects

C R Geyer

Publications and source records attributed to C R Geyer.

9 recordsLinked to original sources
PubMed2006

RIZ1 repression is associated with insulin-like growth factor-1 signaling activation in chronic myeloid leukemia cell lines.

RIZ1 is a histone methyltransferase whose expression and activity are reduced in many cancers. In chronic myelogenous leukemia (CML), blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located and with decreased RIZ1 expression. Forced RIZ1 expression in model CML blast crisis (BC) cell lines decreases proliferation, increases apoptosis and enhances differentiation. We characterized molecular mechanisms that may contribute to potential CML tumor suppressor properties of RIZ1. Several RIZ1-regulated genes involved in insulin-like growth factor-1 (IGF-1) signaling were identified using cDNA microarrays. RIZ1 was shown to associate with promoter regions of IGF-1 and to increase histone H3 lysine 9 methylation using chromatin immunoprecipitation assays. IGF-1-blocking antibody was used to demonstrate the importance of autocrine IGF-1 signaling in CML-BC cell line viability. Forced RIZ1 expression in CML-BC cell lines decreases IGF-1 receptor activation and activation of downstream signaling components extracellular signal-regulated kinase 1/2 and AKT. These results highlight the therapeutic potential of inhibiting IGF-1 pathway in the acute phase of CML.

DNA-Binding Proteins↗
PubMed2000

Use of intrinsic binding energy for catalysis by a cofactor-independent DNA enzyme.

The concept of the Circe effect, according to which an enzyme's substrate-binding energy is utilized to destabilize the substrate towards the reaction transition state, has been shown to be a relevant catalytic strategy for naturally occurring protein enzymes and for two ribozymes that use nucleotide-based substrates and metal ion cofactors. We wished to investigate whether such a catalytic strategy extends even to divergent and unevolved catalysts constructed from biopolymers. We examined the properties of a small, in vitro selected, and cofactor-independent DNA enzyme, PS5.M, which catalyzes porphyrin metallation. The metallation reaction is unique, in that the energies for binding and for metallation of both the substrate and of a transition-state analogue (TSA) can be measured. We report that PS5.M, originally selected for binding to the TSA, displays the Circe effect in channeling a significant component of entropy-rich "intrinsic" binding energy to distort and to alter the basicity of the bound substrate. The study demonstrates that nucleic acids are, by themselves, capable of creating active sites for the catalysis of chemical reactions involving non-nucleotide substrates. Furthermore, the study of the metallation of the TSA provides a quantitative estimate of the effectiveness of such a compound in mimicking the true transition state for porphyrin metallation.

Binding Sites↗
PubMed1999

"Mutagenesis" by peptide aptamers identifies genetic network members and pathway connections.

We selected peptide aptamers from combinatorial libraries that disrupted cell-cycle arrest caused by mating pheromone in yeast. We used these aptamers as baits in two-hybrid hunts to identify genes involved in cell-cycle arrest. These experiments identified genes known to function in the pathway, as well as a protein kinase, the CBK1 product, whose function was not known. We used a modified two-hybrid system to identify specific interactions disrupted by these aptamers. These experiments demonstrate a means to perform "genetics" on the protein complement of a cell without altering its genetic material. Peptide aptamers can be identified that disrupt a process. These aptamers can then be used as affinity reagents to identify individual proteins and protein interactions needed for the process. Forward genetic analysis with peptide aptamer "mutagens" should be particularly useful in elucidating genetic networks in organisms and processes for which classical genetics is not feasible.

Amino Acid Sequence↗
PubMed1998

Lanthanide probes for a phosphodiester-cleaving, lead-dependent, DNAzyme.

Lanthanides are useful probes for studying metal ion interactions in biological systems. The trivalent cations of the lanthanide metals are unique in that their ionic radii and the first pKa values of bound water molecules vary monotonically along the period. In addition, the europium and terbium cations have the useful property that their luminescence is enhanced when bound to nucleic acids. We have found that lanthanide ions can function as effective co-factors for a lead-dependent, phosphodiester-cleaving catalytic DNA (DNAzyme). We used the unique properties of the lanthanide co-factors to study the metal binding site as well as the catalytic mechanism of the DNAzyme. The catalyzed lanthanide-mediated cleavage occurred at neutral pH and at room temperature, with multiple turnovers of substrate. A range of lanthanide ions could act as co-factors, but differentially, with the smaller lanthanides (Tb, Tm, Lu) being the most effective. The rate of cleavage of the phosphodiester did not vary linearly with either the ionic radius or the first pKa of lanthanide-coordinated water molecules. The DNAzyme appeared to use only a single bound lanthanide ion as co-factor. Luminescence spectroscopy with terbium revealed the importance of the 2' hydroxyl group at the cleavage site in lanthanide ion binding, and the substrate molecule alone appeared to generate substantially the catalytically relevant metal-binding site. This model system demonstrated further the utility of complexing lanthanide ions directly to DNA molecules for catalytic purposes. The use of lanthanide ions also provides a means for investigating the metal ion binding sites of nucleic acid enzymes in general.

Binding Sites↗
PubMed1998

DNA enzymes.

The past year has seen a coming-of-age in DNA enzyme research. Far from being laboratory curiosities, the activities of new DNA enzymes have broadened the known catalytic repertoire of nucleic acid enzymes, provided valuable insights into different mechanistic possibilities open to nucleic acid catalysts, and explored the importance for catalysis of native functionalities within DNA and RNA, as well as of a diversity of extrinsic cofactors. Thus, the first amino acid cofactor-utilizing DNA enzyme has been described, as well as DNA enzymes that cleave RNA without the assistance of any external cofactor. On the practical side, the most efficient RNA-cleaving nucleic acid enzyme described to date is a DNA enzyme.

DNA↗
PubMed1997

Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme.

BACKGROUND: RNA and DNA are polymers that lack the diversity of chemical functionalities that make proteins so suited to biological catalysis. All naturally occurring ribozymes (RNA catalysts) that catalyze the formation, transfer and hydrolysis of phosphodiesters require metal-ion cofactors for their catalytic activity. We wished to investigate whether, and to what extent, DNA molecules could catalyze the cleavage (by either hydrolysis or transesterification) of a ribonucleotide phosphodiester in the absence of divalent or higher-valent metal ions or, indeed, any other cofactors. RESULTS: We performed in vitro selection and amplification experiments on a library of random-sequence DNA that incorporated a single ribonucleotide, a suitable site for cleavage. Following 12 cycles of selection and amplification, a 'first generation' of DNA enzymes (DNAzymes) cleaved their internal ribonucleotide phosphodiesters at rates approximately 10(7)-fold faster than the spontaneous rate of cleavage of the dinucleotide ApA in the absence of divalent cations. Re-selection from a partially randomized DNA pool yielded 'second generation' DNAzymes that self-cleaved at rates of approximately 0.01 min-1 (a 10(8)-fold rate enhancement over the cleavage rate of ApA). The properties of these selected catalysts were different in key respects from those of metal-utilizing ribozymes and DNAzymes. The catalyzed cleavage took place in the presence of different chelators and ribonuclease inhibitors. Trace-metal analysis of the reaction buffer (containing very high purity reagents) by inductively coupled plasma-optical emission spectrophotometry indicated that divalent or higher-valent metal ions do not mediate catalysis by the DNAzymes. CONCLUSIONS: Our results indicate that, although ribozymes are sometimes regarded generically to be metalloenzymes, the nucleic acid components of ribozymes may play a substantial role in the overall catalysis. Given that metal cofactors increase the rate of catalysis by ribozymes only approximately 10(2)-10(3)-fold above that of the DNAzyme described in this paper, it is conceivable that substrate positioning, transition-state stabilization or general acid/base catalysis by the nucleic acid components of ribozymes and DNAzymes may contribute significantly to their overall catalytic performance.

Base Sequence↗
PubMed1996

Recognition of anionic porphyrins by DNA aptamers.

DNA sequences were isolated by in vitro selection for binding to N-methylmesoporphyrin IX (NMM), a molecule that behaves as a stable transition-state analogue for porphyrin chelatases. Clones approximately 280 and approximately 120 nucleotides long were obtained, which bound to NMM with sub-micromolar affinity but bound mesoporphyrin IX (MPIX), as well as various metalloderivatives of MPIX, with lower affinity. Footprinting experiments with dimethyl sulfate, DNase I, and bound hemin molecules activated by superoxide identified a series of short guanine-rich motifs to be the binding sites for the various porphyrins. One clone, PS2, examined in depth, gave a methylation footprint with bound NMM but not with bound MPIX nor with a number of metalloporphyrins. The binding domain PS2, synthesized as a short oligonucleotide, itself showed high-affinity binding to NMM. The binding sequences from different clones were loosely homologous, and the footprinting data were consistent with their folding to form one or more guanine quartets in the presence of NMM. Ultraviolet--visible absorption and circular dichroism spectroscopy of the DNA--NMM complexes indicates, however, that the interaction is not primarily intercalative in nature. The preferential binding of NMM by these aptamers raises the possibility of their being able to catalyze the chelation of metal ions by the porphyrin MPIX.

Base Sequence↗
PubMed1989

Promoting the quality of life in nursing homes: can regulation succeed?

It has been assumed that the best policy for promoting quality of life in nursing homes is direct regulation. In this paper it is argued that if our experience in regulating quality of care is any indication, we may not possess the political will to successfully regulate quality of life. Moreover, from a legal perspective, the less concrete nature of the concept of quality of life may make it more difficult to regulate than quality of care. Finally, although regulation would probably be necessary if potential nursing home residents (and their agents) lacked the information or rationality to make choices that promoted their interests, this has never been shown to be the case empirically. Therefore, we may not be forced to choose regulation to achieve an adequate quality of life. Alternative--and perhaps better--policies may be available.

Cost Control↗