Biomedical subjects

Paul M Smith

Publications and source records attributed to Paul M Smith.

8 recordsLinked to original sources

PubMed2004

Strategies for treating disorders of the mitochondrial genome.

Defects of the mitochondrial genome are a significant cause of disease. Patients suffer from a wide variety of clinical presentations, ranging from fatal infantile disease to mild muscle weakness. Most disorders, however, are characterized by inexorable progression. As mutations often cause defects in several components of the complexes that couple oxidative phosphorylation, this terminal state of oxidative metabolism cannot be readily bypassed by dietary means, leading to the search for novel therapies. In this article, we present the theory behind several concepts and report progress. We also discuss some of the recent difficulties encountered in the progress towards an antigenomc approach to treating mtDNA disorders.

Biological Transport↗

PubMed2004

Functional polypeptides can be synthesized from human mitochondrial transcripts lacking termination codons.

The human mitochondrial genome (mtDNA) is a small, circular DNA duplex found in multi-copy in the mitochondrial matrix. It is almost fully transcribed from both strands to produce large polycistronic RNA units that are processed and matured. The 13 mtDNA-encoded polypeptides are translated from mt-mRNAs that have been matured by polyadenylation of their free 3'-termini. A patient with clinical features consistent with an mtDNA disorder was recently shown to carry a microdeletion, resulting in the loss of the termination codon for MTATP6 and in its juxtaposition with MTCO3. Cell lines from this patient exhibited low steady-state levels of RNA14, the bi-cistronic transcript encoding subunits 6 and 8 of the F(o)F(1)-ATP synthase, complex V, consistent with a decreased stability. Recent reports of 'non-stop' mRNA decay systems in the cytosol have failed to determine the fate of gene products derived from transcripts lacking termination codons, although enhanced decay clearly required the 'non-stop' transcripts to be translated. We wished to determine whether functional translation products could still be expressed from non-stop transcripts in the human mitochondrion. Although a minor defect in complex V assembly was noted in the patient-derived cell lines, the steady-state level of ATPase 6 was similar to controls, consistent with the pattern of de novo mitochondrial protein synthesis. Moreover, no significant difference in ATP synthase activity could be detected. We conclude that, in the absence of a functional termination codon, although mitochondrial transcripts are more rapidly degraded, they are also translated to generate stable polypeptides that are successfully integrated into functional enzyme complexes.

Adenosine Triphosphatases↗

PubMed2004

Effects of caffeine ingestion on exercise testing: a meta-analysis.

This study used the meta-analytic approach to examine the effects of caffeine ingestion on exercise testing. Forty double-blind studies with 76 effect sizes (ES) met the inclusion criteria. The type of exercise test was classified as endurance, graded, or short-term. In comparison with placebo, caffeine improved test outcome by 12.3 % (95 % CI, 9.1 to 15.4), which was equivalent to an overall ES of 0.41 (95 % CI, 0.31 to 0.51). Endurance exercise significantly improved test outcome (P < 0.05) more than either graded or short-term exercise. When exercise protocol was examined, time-to-exhaustion (Tlim) protocols had a significantly greater (P < 0.05) ES than either the graded or the non-Tlim protocol(s). The results from this meta-analysis confirm the ergogenic effects of caffeine, particularly for endurance testing that use Tlim protocols.

Adult↗

PubMed2003

Bridging PNAs can bind preferentially to a deleted mitochondrial DNA template but replication by mitochondrial DNA polymerase gamma in vitro is not impaired.

Mutations in mitochondrial DNA (mtDNA) are an important cause of neurological and other human pathologies. In the vast majority of cases, supportive care only is available. Mutated and wild-type mtDNAs often coexist in the same cell. A strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Peptide nucleic acid molecules (PNAs) can be designed to selectively target pathogenic mtDNA with single point mutations. Molecules harbouring deletions present a complex problem. Deletions often occur between two short repeat sequences (4-13 residues), one of which is retained in the deleted molecule. With the more common large repeats, it is therefore difficult to design an antigenomic molecule that will bind selectively under physiological conditions. Following limited success with antigenomic oligodeoxynucleotides (ODNs), we have repeated these studies with a series of bridging PNAs. Molecules complementary to the sequence flanking either side of the 13 bp 'common deletion' were synthesised. The PNAs demonstrated markedly greater affinity for the delete than to the wild-type template. In runoff assays using Klenow fragment, these PNAs selectively inhibited replication of the delete template. However, no selective inhibition was observed when a polymerase gamma-containing mitochondrial fraction was used.

Base Sequence↗

PubMed2003

Specific targeting of a DNA-alkylating reagent to mitochondria. Synthesis and characterization of [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-8-oxy)butyl]-triphenylphosphonium iodide.

The selective manipulation of the expression and replication of mitochondrial DNA (mtDNA) within mammalian cells has proven difficult. In progressing towards this goal we synthesized a novel mitochondria-targeted DNA-alkylating reagent. The active alkylating moiety [(11aS)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (DC-81)], irreversibly alkylates guanine bases in DNA (with a preference for AGA triplets), preventing its expression and replication. To target this compound to mitochondria it was covalently coupled to the lipophilic triphenylphosphonium (TPP) cation to form a derivative referred to as mitoDC-81. Incorporation of this lipophilic cation led to the rapid uptake of mitoDC-81 by mitochondria, driven by the large membrane potential across the inner membrane. This compound efficiently alkylated isolated supercoiled, relaxed-circular or linear plasmid DNA and isolated mtDNA. However mitoDC-81 did not alkylate mtDNA within isolated mitochondria or cells, even though it accessed the mitochondrial matrix at concentrations up to 100-fold higher than those required to alkylate isolated DNA. This surprising finding suggests that mtDNA within intact mitochondria may not be accessible to this class of alkylating reagent. This inability to alkylate mtDNA in situ has significant implications for the design of therapies for mtDNA diseases and for studies on the packaging, expression and turnover of mtDNA in general.

Alkylating Agents↗

PubMed2002

The crystal structure of MT0146/CbiT suggests that the putative precorrin-8w decarboxylase is a methyltransferase.

The CbiT and CbiE enzymes participate in the biosynthesis of vitamin B12. They are fused together in some organisms to form a protein called CobL, which catalyzes two methylations and one decarboxylation on a precorrin intermediate. Because CbiE has sequence homology to canonical precorrin methyltransferases, CbiT was hypothesized to catalyze the decarboxylation. We herein present the crystal structure of MT0146, the CbiT homolog from Methanobacterium thermoautotrophicum. The protein shows structural similarity to Rossmann-like S-adenosyl-methionine-dependent methyltransferases, and our 1.9 A cocrystal structure shows that it binds S-adenosyl-methionine in standard geometry near a binding pocket that could accommodate a precorrin substrate. Therefore, MT0146/CbiT probably functions as a precorrin methyltransferase and represents the first enzyme identified with this activity that does not have the canonical precorrin methyltransferase fold.

Amino Acid Sequence↗

PubMed2002

Caffeine is ergogenic after supplementation of oral creatine monohydrate.

PURPOSE: The purpose of this investigation was to assess the acute effects of caffeine ingestion on short-term, high-intensity exercise (ST) after a period of oral creatine supplementation and caffeine abstinence. METHODS: Fourteen trained male subjects performed treadmill running to volitional exhaustion (T(lim)) at an exercise intensity equivalent to 125% VO(2max). Three trials were performed, one before 6 d of creatine loading (0.3 g x kg x d(-1) baseline), and two further trials after the loading period. One hour before the postloading trials, caffeine (5 mg x kg(-1)) or placebo was orally ingested in a cross-over, double-blind fashion. Four measurements of rating of perceived exertion were taken, one every 30 s, during the first 120 s of the exercise. Blood samples were assayed for lactate, glucose, potassium, and catecholamines, immediately before and after exercise. RESULTS: Body mass increased (P < 0.05) over the creatine supplementation period, and this increase was maintained for both caffeine and placebo trials. There was no increase in the maximal accumulated oxygen deficit between trials; however, total VO(2) was significantly increased in the caffeine trial in comparison with the placebo trial (13.35 +/- 3.89 L vs 11.67 +/- 3.61 L). In addition, caffeine T(lim) (222.1 +/- 48.9 s) was significantly greater (P < 0.05) than both baseline (200.8 +/- 33.4 s) and placebo (198.3 +/- 45.4 s) T(lim). RPE was also lower at 90 s in the caffeine treatment (13.8 +/- 1.8 RPE points) in comparison with baseline (14.6 +/- 1.9 RPE points). CONCLUSION: As indicated by a greater T(lim), acute caffeine ingestion was found to be ergogenic after 6-d of creatine supplementation and caffeine abstinence.

Adult↗

PubMedDate not supplied

Synthesis of trifunctional PNA-benzophenone derivatives for mitochondrial targeting, selective DNA binding, and photo-cross-linking.

Mutations in mitochondrial DNA (mtDNA) cause a variety of human pathologies. In many patients, mutated and wild-type mtDNAs coexist in the same cell, a situation termed mtDNA heteroplasmy. In the absence of standard therapies for these disorders, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by the selective hybridization of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated defects. To allow for selective binding under physiological conditions, peptide nucleic acids (PNA) are being used. Two other problems, however, have to be resolved: mitochondrial import and attachment of the PNA to the target DNA to inhibit replication. Mitochondrial localization can be achieved by the addition of a caged lipophilic cation and addition of a photo-cross-linking reagent should facilitate covalent attachment. We therefore report the synthesis of benzophenone-PNA derivatives carrying a triphenylphosphonium moiety and demonstrate irreversible binding selectivity between two DNA molecules that differ by a single nucleotide.

Benzophenones↗