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Biomedical subjects

A McGregor

Publications and source records attributed to A McGregor.

At least 19 recordsLinked to original sources
PubMed2001

A larger hippocampus is associated with longer-lasting spatial memory.

Volumetric studies in a range of animals (London taxi-drivers, polygynous male voles, nest-parasitic female cowbirds, and a number of food-storing birds) have shown that the size of the hippocampus, a brain region essential to learning and memory, is correlated with tasks involving an extra demand for spatial learning and memory. In this paper, we report the quantitative advantage that food storers gain from such an enlargement. Coal tits (Parus ater) a food-storing species, performed better than great tits (Parus major), a nonstoring species, on a task that assessed memory persistence but not on a task that assessed memory resolution or on one that tested memory capacity. These results show that the advantage to the food-storing species associated with an enlarged hippocampus is one of memory persistence.

Animals↗
PubMed2001

Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence.

The two prohibitin proteins, Phb1p and Phb2p(BAP37), have been ascribed various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging. We show that the mammalian prohibitins are present in the inner mitochondrial membrane and are always bound to each other, with no free protein detectable. They are coexpressed during development and in adult mammalian tissues, and expression levels are indicative of a role in mitochondrial metabolism, but are not compatible with roles in the regulation of cellular proliferation or apoptosis. High level expression of the proteins is consistently seen in primary human tumors, while cellular senescence of human and chick fibroblasts is accompanied by heterogeneous decreases in both proteins. The two proteins are induced by metabolic stress caused by an imbalance in the synthesis of mitochondrial- and nuclear-encoded mitochondrial proteins, but do not respond to oxidative stress, heat shock, or other cellular stresses. The gene promoter sequences contain binding sites for the Myc oncoprotein and overexpression of Myc induces expression of the prohibitins. The data support conserved roles for the prohibitins in regulating mitochondrial respiratory activity and in aging.

Animals↗
PubMed2001

Inhibition of mitochondrial protein synthesis promotes autonomous regulation of mtDNA expression and generation of a new mitochondrial RNA species.

Mammalian mitochondria are known to proliferate in response to several stimuli. Proliferation requires an increase in expression of genes encoding proteins involved in mitochondrial biogenesis, as well as in the replication and expression of mitochondrial DNA (mtDNA). In contrast, we report that inhibiting mitochondrial protein synthesis causes a modulation in mtDNA gene expression without the concomitant increase in proliferative markers. Further, inhibition results in the production of a previously unidentified light-strand mitochondrial RNA that spans the entire displacement loop, the function of which is currently unknown.

Base Sequence↗
PubMed2001

Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli.

Since cytomegalovirus (CMV) infection is highly species-specific, it is necessary to study animal cytomegaloviruses to assess viral factors which contribute to pathogenesis. The generation of recombinant viruses carrying reporter genes would provide useful tools for studying the genetics of CMV pathogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPCMV) was amenable to such manipulation. Metabolic selection using the guanosylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombinant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) cassette introduced into the HindIII "N" region of the viral genome. This virus had replication kinetics identical to wild-type virus. We next attempted to clone the GPCMV genome as a bacterial artificial chromosome (BAC). A BAC plasmid containing a gpt/eGFP cassette and the chloramphenicol resistance marker was introduced into HindIII "N" to generate another GPCMV recombinant, vAMBGPCMV. Circular viral DNA isolated from vAMBGPCMV-infected cells was used to transform Escherichia coli. Restriction profiles revealed that the GPCMV genome had been cloned as a BAC plasmid, and transfection of BAC plasmid DNA confirmed that the BAC clone was infectious. A novel strategy based on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid. Recombinants retained the capability to replicate and express reporter genes in guinea pigs, suggesting that these viruses will be useful for in vivo pathogenesis studies.

Animals↗
PubMed2001

Recent advances in herpesvirus genetics using bacterial artificial chromosomes.

Although the members of the Herpesvirus family are responsible for a wide variety of human diseases, advances in the understanding of viral molecular mechanisms of pathogenesis have been hampered by the large size of herpesvirus genomes, rendering the viruses difficult to experimentally manipulate. Better techniques have been needed to facilitate mutagenesis of herpesvirus genomes, allowing for the assessment of the role of specific viral gene products in replication, immunity, and pathogenesis. Homologous recombination with plasmids containing genes of interest flanked by selectable markers has been a successful method for generating viral mutants, as has the generation of recombinant virus from transfection of cosmid clones. Although these efforts to generate recombinant viruses have met with modest success, the protocols have been cumbersome. More recently, a novel technique for the manipulation of herpesvirus genomes has been developed. This technology utilizes bacterial F plasmids, and allows for the stable cloning of herpesvirus genomes as bacterial artificial chromosomes (BACs) in Escherichia coli. Once cloned, such BACs are stable, and DNA purified from E. coli is infectious, fully capable of reproducing replication-competent virus. Manipulation of herpesvirus genomes is now feasible using the powerful techniques of bacterial genetics, and should facilitate a better understanding of the molecular pathogenesis of herpesvirus infections.

Chromosomes, Artificial, Bacterial↗
PubMed2001

Absence of expression from RNA internalised into electroporated mammalian mitochondria.

Transfection of mammalian mitochondria has proved to be notoriously difficult. Whilst there have been sporadic reports of import of foreign nucleic acids into isolated organelles, these imported nucleic acids have never been demonstrated to be functional. Inability to manipulate mitochondrial gene expression has hampered our understanding of RNA processing, maturation and translation in mitochondria. In an attempt to establish a model system for mt-RNA expression, we have electroporated rat liver mitochondria and mitoplasts in the presence of various RNA constructs built around the mitochondrial reporter gene mt-luciferase. Following electroporation, a fraction of the RNA was shown to be stably maintained, mitochondria remained coupled for oxidative phosphorylation and intramitochondrial protein synthesis was unaffected. In no case, however, was this RNA translated.

Animals↗
PubMed2001

Anterior cruciate ligament rupture: reconstruction surgery and rehabilitation. A nation-wide survey of current practice.

A database of the current practice of anterior cruciate ligament (ACL) reconstruction surgery in Great Britain was collated from a postal survey. This survey revealed that clear trends are emerging in the United Kingdom (UK) with regard to ACL reconstruction surgery (such as the type of graft used and timing of surgery) and rehabilitation. Certain issues, however, remain controversial, including bracing and knee range of movement. Clinical outcome measures are consistently used to judge operative success, but few specialists use defined objective measurements such as the IKDC scoring system.

Anterior Cruciate Ligament↗
PubMed2001

Fibre-optic bronchoscopy in adults: a position paper of The Thoracic Society of Australia and New Zealand.

Fibre-optic bronchoscopy in adults is a common procedure in clinical respiratory practice. Under controlled conditions it is safe, resulting in relatively few significant adverse events. The present position paper updates guidelines previously published by The Thoracic Society of Australia and New Zealand and is based on evidence obtained by searching the Medline and Embase databases. The level of evidence to support recommendations is indicated in the text. Where no evidence has been found, the guidelines reflect the opinions of the authors. Specific recommendations are made regarding sedation and anaesthesia, the cleaning of bronchoscopes and the training of bronchoscopists.

Adult↗
PubMed2001

A comparison of dermal and cadaveric pericardial grafts in the modified Horton-Devine procedure for Peyronie's disease.

PURPOSE: Penile curvature due to Peyronie's disease is often treated with plaque incision or excision and grafting. The advantages of various graft materials have long been debated. We studied the outcomes of dermal and cadaveric pericardial grafts after plaque incision or excision for Peyronie's disease. MATERIALS AND METHODS: We retrospectively evaluated all modified Horton-Devine procedures performed between February 1998 and April 2000. Dermal graft harvested from the lower abdomen or commercially available cadaveric pericardium was used to cover the corporeal defect. A followup telephone questionnaire was administered to determine patient satisfaction and function. RESULTS: We reviewed the records of 27 men, including 18 and 9 who received a dermal and pericardial graft, respectively. We successfully contacted 24 patients, including 15 with a dermal and 9 with a pericardial graft, to evaluate satisfaction. Average postoperative followup was 10 months. Of the 24 patients, 22 reported successful coitus and 15 (63%) needed assistance to achieve erection rigid enough for satisfactory coitus. Eight patients reported recurrent curvature, which was minimal and did not hamper sexual function. Overall 11 of the 15 patients with a dermal and 8 of the 9 with a pericardial graft would undergo the surgery again or recommend it to others. CONCLUSIONS: The results of the dermal and cadaveric pericardial grafts were comparable. The minimal preoperative preparation, decreased patient morbidity and pliability of cadaveric pericardium make it an attractive suitable graft substitute in the modified Horton-Devine procedure. Longer followup and larger patient series are needed to confirm the advantages of cadaveric pericardium.

Adult↗
PubMed2000

HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity.

Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the identification of small oligonucleotides that bind with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of potential chemotherapeutics that could block the enzymatic activity of pathologically relevant proteins. We have conducted a detailed biochemical study of the interaction of human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) with a SELEX-derived pseudoknot RNA aptamer. Electron paramagnetic resonance spectroscopy of site-directed spin-labeled RT mutants revealed that this aptamer was selected for binding to the "closed" conformation of the enzyme. Kinetic analysis showed that the RNA inhibitor bound to HIV RT in a two-step process, with association rates similar to those described for model DNA/DNA and DNA/RNA substrates. However, the dissociation of the pseudoknot RNA from RT was dramatically slower than observed for model substrates. Equilibrium binding studies revealed an extraordinarily low K(d), of about 25 pm, for the enzyme-aptamer interaction, presumably a consequence of the slow off-rates. Additionally, this pseudoknot aptamer is highly specific for HIV-1 RT, with the closely related HIV-2 enzyme showing a binding affinity close to 4 orders of magnitude lower.

Crystallography, X-Ray↗
PubMed1999

Conversion of a reporter gene for mitochondrial gene expression using iterative mega-prime PCR.

Mammalian mitochondria possess their own multicopy genome, mtDNA. Although much is known about mtDNA replication and transcription, our knowledge of the mechanisms governing mt-RNA processing, stability and translation remains rudimentary. We have taken a step towards addressing these issues by altering the luciferase reporter gene to accommodate the variation in mitochondrial codon recognition. 19 essential substitutions have been generated by an iterative mega-primer PCR technique. To mimic mt-mRNA species and to optimise intramitochondrial translation, further engineering has produced a template which, when transcribed in vitro, generates an RNA species with only two nucleotides upstream from the initiation codon, an absence of a 3' untranslated region and a polyadenylated tail of 40 residues. It is intended that mt-luciferase (mt-luc) RNA will be an excellent reporter for revealing cis-acting elements essential for in organello RNA processing, maturation and expression. Additionally, the mt-luc gene can be readily incorporated into any novel mitochondrial transducing vectors to assess intra-organellar transcription and translation.

DNA Primers↗
PubMed1999

Demonstration of rapid onset vascular endothelial dysfunction after hyperhomocysteinemia: an effect reversible with vitamin C therapy.

BACKGROUND: Hyperhomocysteinemia is a major and independent risk factor for vascular disease. The mechanisms by which homocysteine promotes atherosclerosis are not well understood. We hypothesized that elevated homocysteine concentrations are associated with rapid onset endothelial dysfunction, which is mediated through oxidant stress mechanisms and can be inhibited by the antioxidant vitamin C. METHODS AND RESULTS: We studied 17 healthy volunteers (10 male and 7 female) aged 33 (range 21 to 59) years. Brachial artery diameter responses to hyperemic flow (endothelium dependent), and glyceryltrinitrate (GTN, endothelium independent) were measured with high resolution ultrasound at 0 hours (fasting), 2 hours, and 4 hours after (1) oral methionine (L-methionine 100 mg/kg), (2) oral methionine preceded by vitamin C (1g/day, for 1 week), and (3) placebo, on separate days and in random order. Plasma homocysteine increased (0 hours, 12.8+/-1.4; 2 hours, 25.4+/-2.5; and 4 hours, 31. 2+/-3.1 micromol/l, P<0.001), and flow-mediated dilatation fell (0 hours, 4.3+/-0.7; 2 hours, 1.1+/-0.9; and 4 hours, -0.7+/-0.8%) after oral L-methionine. There was an inverse linear relationship between homocysteine concentration and flow-mediated dilatation (P<0. 001). Pretreatment with vitamin C did not affect the rise in homocysteine concentrations after methionine (0 hours, 13.6+/-1.6; 2 hours, 28.3+/-2.9; and 4 hours, 33.8+/-3.7 micromol/l, P=0.27), but did ameliorate the reduction in flow-mediated dilatation (0 hours, 4. 0+/-1.0; 2 hours, 3.5+/-1.2 and 4 hours, 2.8+/-0.7%, P=0.02). GTN-induced endothelium independent brachial artery dilatation was not affected after methionine or methionine preceded by vitamin C. CONCLUSIONS: We conclude that an elevation in homocysteine concentration is associated with an acute impairment of vascular endothelial function that can be prevented by pretreatment with vitamin C in healthy subjects. Our results support the hypothesis that the adverse effects of homocysteine on vascular endothelial cells are mediated through oxidative stress mechanisms.

Adult↗
PubMed1999

Secondary structure mapping of an RNA ligand that has high affinity for the MetJ repressor protein and interference modification analysis of the protein-RNA complex.

The secondary structure of an RNA aptamer, which has a high affinity for the Escherichia coli MetJ repressor protein, has been mapped using ribonucleases and with diethyl pyrocarbonate. The RNA ligand is composed of a stem-loop with a highly structured internal loop. Interference modification showed that the bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ. Most of the terminal stem-loop could be removed with little effect on the binding. Ethylation interference suggests that none of the phosphate groups are absolutely essential for tight binding. The data suggest that the MetJ binding site on the aptamer is distinct from that of the natural DNA target, the 8-base pair Met box.

Bacterial Proteins↗
PubMed1999

Molecular characterization of the guinea pig cytomegalovirus UL83 (pp65) protein homolog.

The tegument phosphoproteins of human cytomegalovirus (HCMV) elicit cytotoxic T-lymphocyte (CTL) responses and are hence candidates for subunit vaccine development. Little is known, however, about the tegument proteins of nonhuman cytomegaloviruses, such as guinea pig CMV (GPCMV). DNA sequence analysis of the Eco R I "C" fragment of the GPCMV genome identified an open reading frame (ORF) which is colinear with that of the HCMV tegument phosphoprotein, UL83 (pp65). This ORF was found to have identity to HCMV UL83 and was predicted to encode a 565-amino-acid (aa) protein with a molecular mass of 62.3 kDa. Transcriptional analyses revealed that a GPCMV UL83 probe hybridized with both 2.2 kb and 4.2 kb mRNA species at 48 h post-infection (p.i.); synthesis of these messages was blocked by phosphonoacetic acid (PAA), defining these as "late" gene transcripts. In vitro translation of the UL83 ORF in reticulocyte lysate resulted in synthesis of a 65 kDa protein. Immunofluorescence experiments revealed that the putative GPCMV UL83 homolog exhibited a predominantly nuclear localization pattern. Polyclonal antisera were raised against a UL83/glutathione-S-transferase (GST) fusion protein. This antibody identified a 70-kDa virion-associated protein, the putative GPCMV UL83 homolog, in immunoblot and radioimmunoprecipitation experiments. Labeling experiments with 32P-orthophosphate indicated that the GPCMV UL83 protein is phosphorylated. Western blot analysis of glycerol tartrate gradient-purified virions and dense bodies confirmed that the putative GPCMV UL83 homolog was a constituent of both fractions.

Amino Acid Sequence↗