Biomedical subjects

Michael T Howard

Publications and source records attributed to Michael T Howard.

10 recordsLinked to original sources

PubMed2006

Polyamine analogs with xylene rings induce antizyme frameshifting, reduce ODC activity, and deplete cellular polyamines.

Numerous studies have correlated elevated polyamine levels with abnormal or rapid cell growth. One therapeutic strategy to treat diseases with increased cellular proliferation rates, most obviously cancer, has been to identify compounds which lower cellular polyamine levels. An ideal target for this strategy is the protein antizyme-a negative regulator of polyamine biosynthesis and import, and a positive regulator of polyamine export. In this study, we have optimized two tissue-culture assays in 96-well format, to allow the rapid screening of a 750-member polyamine analog library for compounds which induce antizyme frameshifting and fail to substitute for the natural polyamines in growth. Five analogs (MQTPA1-5) containing xylene (1,4-dimethyl benzene) were found to be equal to or better than spermidine at stimulating antizyme frameshifting and were inefficient at rescuing cell growth following polyamine depletion. These compounds were further characterized for effects on natural polyamine levels and enzymes involved in polyamine metabolism. Finally, direct measurements of antizyme induction in cells treated with two of the lead compounds revealed an 8- to 15-fold increase in antizyme protein over untreated cells. The impact of the xylene moiety and the distance between the positively charged amino groups on antizyme frameshifting and cell growth are discussed.

Acetyltransferases↗

PubMed2006

Antisense-induced ribosomal frameshifting.

Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1-ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem-loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5' of the shift site and to polyamine levels.

Animals↗

PubMed2006

Epitopes derived by incidental translational frameshifting give rise to a protective CTL response.

Aberrant gene expression can be caused by several different mechanisms at the transcriptional, RNA processing, and translational level. Although most of the resulting proteins may have no significant biological function, they can be meaningful for the immune system, which is sensitive to extremely low levels of Ag. We have tested this possibility by investigating the ability of CD8+ T cells (TCD8+) to respond to an epitope whose expression results from incidental ribosomal frameshifting at a sequence element within the HSV thymidine kinase gene. This element, with no apparent functional significance, has been identified due to its ability to facilitate escape from the antiviral compound acyclovir. Using a recombinant vaccinia virus expression system, we find that in vitro and in vivo TCD8+ responses to the frameshift-dependent epitope are easily discernible. Furthermore, the in vivo response is at a sufficient level to mediate protection from a tumor challenge. Thus, the targets of immune responses to infectious agents can extend beyond the products of conventional open reading frames. On a per-cell basis, responses to such minimally expressed epitopes may be exceedingly effective due to the selective expansion of high avidity TCD8+.

Animals↗

PubMed2006

Independent evolution of bitter-taste sensitivity in humans and chimpanzees.

It was reported over 65 years ago that chimpanzees, like humans, vary in taste sensitivity to the bitter compound phenylthiocarbamide (PTC). This was suggested to be the result of a shared balanced polymorphism, defining the first, and now classic, example of the effects of balancing selection in great apes. In humans, variable PTC sensitivity is largely controlled by the segregation of two common alleles at the TAS2R38 locus, which encode receptor variants with different ligand affinities. Here we show that PTC taste sensitivity in chimpanzees is also controlled by two common alleles of TAS2R38; however, neither of these alleles is shared with humans. Instead, a mutation of the initiation codon results in the use of an alternative downstream start codon and production of a truncated receptor variant that fails to respond to PTC in vitro. Association testing of PTC sensitivity in a cohort of captive chimpanzees confirmed that chimpanzee TAS2R38 genotype accurately predicts taster status in vivo. Therefore, although Fisher et al.'s observations were accurate, their explanation was wrong. Humans and chimpanzees share variable taste sensitivity to bitter compounds mediated by PTC receptor variants, but the molecular basis of this variation has arisen twice, independently, in the two species.

Alleles↗

PubMed2005

Polyamine sensing during antizyme mRNA programmed frameshifting.

A key regulator of cellular polyamine levels from yeasts to mammals is the protein antizyme. The antizyme gene consists of two overlapping reading frames with ORF2 in the +1 frame relative to ORF1. A programmed +1 ribosomal frameshift occurs at the last codon of ORF1 and results in the production of full-length antizyme protein. The efficiency of frameshifting is proportional to the concentration of polyamines, thus creating an autoregulatory circuit for controlling polyamine levels. The mRNA recoding signals for frameshifting include an element 5' and a pseudoknot 3' of the shift site. The present work illustrates that the ORF1 stop codon and the 5' element are critical for polyamine sensing, whereas the 3' pseudoknot acts to stimulate frameshifting in a polyamine independent manner. We also demonstrate that polyamines are required to stimulate stop codon readthrough at the MuLV redefinition site required for normal expression of the GagPol precursor protein.

Animals↗

PubMed2005

Recoding elements located adjacent to a subset of eukaryal selenocysteine-specifying UGA codons.

Incorporation of the 21st amino acid, selenocysteine, into proteins is specified in all three domains of life by dynamic translational redefinition of UGA codons. In eukarya and archaea, selenocysteine insertion requires a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 3'UTR of selenoprotein mRNAs. Here we present comparative sequence analysis and experimental data supporting the presence of a second stop codon redefinition element located adjacent to a selenocysteine-encoding UGA codon in the eukaryal gene, SEPN1. This element is sufficient to stimulate high-level (6%) translational redefinition of the SEPN1 UGA codon in human cells. Readthrough levels further increased to 12% when tested in the presence of the SEPN1 3'UTR SECIS. Directed mutagenesis and phylogeny of the sequence context strongly supports the importance of a stem loop starting six nucleotides 3' of the UGA codon. Sequences capable of forming strong RNA structures were also identified 3' adjacent to, or near, selenocysteine-encoding UGA codons in the Sps2, SelH, SelO, and SelT selenoprotein genes.

Animals↗

PubMed2005

Programmed ribosomal frameshifting in decoding the SARS-CoV genome.

Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses. Comparative analysis of the frameshift region reveals a universal shift site U_UUA_AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops. Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry. Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U_UUA_AAC. Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I-stem II junction in SARS-CoV are important for frameshift stimulation. These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.

Base Sequence↗

PubMed2004

Readthrough of dystrophin stop codon mutations induced by aminoglycosides.

We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchenne's and Becker's muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (< 2% stop codon readthrough); and two appear refractory to aminoglycoside treatment. Readthrough levels do not substantially vary between different sources of gentamicin, and, for this set of mutations, the efficiency of termination at the premature stop codon mutation does not appear to correlate with disease severity.

Aminoglycosides↗

PubMed2004

Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides.

Evidence is presented that morpholino, 2'-O-methyl, phosphorothioate, and RNA antisense oligonucleotides can direct site-specific -1 translational frameshifting when annealed to mRNA downstream from sequences where the P- and A-site tRNAs are both capable of repairing with -1 frame codons. The efficiency of ribosomes shifting into the new frame can be as high as 40%, determined by the sequence of the frameshift site, as well as the location, sequence composition, and modification of the antisense oligonucleotide. These results demonstrate that a perfect duplex formed by complementary oligonucleotides is sufficient to induce high level -1 frameshifting. The implications for the mechanism of action of natural programmed translational frameshift stimulators are discussed.

Animals↗

PubMedDate not supplied

The Blue Obelisk-interoperability in chemical informatics.

The Blue Obelisk Movement (http://www.blueobelisk.org/) is the name used by a diverse Internet group promoting reusable chemistry via open source software development, consistent and complimentary chemoinformatics research, open data, and open standards. We outline recent examples of cooperation in the Blue Obelisk group: a shared dictionary of algorithms and implementations in chemoinformatics algorithms drawing from our various software projects; a shared repository of chemoinformatics data including elemental properties, atomic radii, isotopes, atom typing rules, and so forth; and Web services for the platform-independent use of chemoinformatics programs.

Algorithms↗