Biomedical subjects
M McVey
Publications and source records attributed to M McVey.
Using yeast to discover the fountain of youth.
The budding yeast Saccharomyces cerevisiae has long served as a model organism for the study of basic cellular processes. Its short generation time, well-established molecular genetics, and fully sequenced genome have made this organism a favorite of researchers in diverse fields. Much of the information obtained has been shown to apply to higher eukaryotes, including humans. Recently, researchers have begun using yeast to tackle one of the outstanding questions in science: How and why do organisms age? The identification of individual genes in yeast that can affect the aging process itself has elevated this single-celled fungus to full contender status in the aging field. In this Perspective, we present two fundamentally different measures of aging in yeast: replicative life-span and stationary phase survival (chronological life-span). We describe the benefits and limitations of each and present models that attempt to explain these "aging" phenomena. Finally, we present compelling evidence that the use of yeast as a model system will ultimately prove beneficial to the study of human aging.
The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase.
Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.
Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta -opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy.
Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.
The short life span of Saccharomyces cerevisiae sgs1 and srs2 mutants is a composite of normal aging processes and mitotic arrest due to defective recombination.
Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.
Intravenous AMP 579, a novel adenosine A(1)/A(2a) receptor agonist, induces a delayed protection against myocardial infarction in minipig.
The aim of the study was to probe if acute administration of [1S-[1a, 2b,3b, 4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]amino]-3H-imida zo[4,5-b] pyridin-3-yl] cyclopentane carboxamide (AMP 579) could provide a delayed protection against myocardial ischemia-reperfusion injury after 24 h. Anesthetized Yucatan minipigs were given an intravenous (i.v.) loading dose (3 microg/kg) of AMP 579 in 2 min followed by a 68-min infusion (0.3 microg/kg/min) and were allowed to recover. After 24 h, the animals were subjected to an open-chest operation and the left anterior descending coronary artery was occluded for 40 min, followed by 3 h of reperfusion. Results indicated that there were no significant differences in hemodynamic parameters between vehicle- and drug-treated groups either during drug infusion or ischemia-reperfusion. Both groups had similar area at risk (24.9% for vehicle and 25.1% for AMP 579-treated). However, the infarct size was 36.5% of area at risk in vehicle group (n=8) and 12.5% in AMP 579 group (n=8), representing a 66% reduction of infarct size by AMP 579 (p=0.011). This is the first report to demonstrate that in a large animal model, a hemodynamically silent, single i.v. dose of an adenosine receptor agonist can result in a delayed protection against myocardial infarction.
Two classes of sir3 mutants enhance the sir1 mutant mating defect and abolish telomeric silencing in Saccharomyces cerevisiae.
Silent information regulators, or Sir proteins, play distinct roles in chromatin-mediated transcriptional control at the silent mating-type loci, telomeres, and within the rDNA repeats of Saccharomyces cerevisiae. An unusual collection of sir3 mutant alleles was identified in a genetic screen for enhancers of the sir1 mutant mating-defective phenotype. These sir3-eso mutants, like the sir1 mutant, exhibit little or no mating defects alone, but the sir1 sir3-eso double mutants are essentially nonmating. All of the sir3-eso mutants are defective in telomeric silencing. In some mutants, this phenotype is suppressed by tethering Sir1p to telomeres; other mutants are dominant for mating and telomeric silencing defects. Additionally, several sir3-eso mutants are nonmating in combination with the nat1 N-terminal acetyltransferase mutant. The temperature-sensitive allele sir3-8 has an eso phenotype at permissive temperature, yet acts as a null allele at restrictive temperature due to loss of sir3-8 protein. Sequence analysis showed that eight of the nine sir3-eso alleles have mutations within the N-terminal region that is highly similar to the DNA replication initiation protein Orc1p. Together, these data reveal modular domains for Sir3p and further define its function in silencing chromatin.
Sir2: an NAD-dependent histone deacetylase that connects chromatin silencing, metabolism, and aging.
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The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms.
The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.
Adenylyl cyclase, a coincidence detector for nitric oxide.
Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the Vmax of the enzyme without altering the Km for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase.
Does tumor necrosis factor-alpha (TNF-alpha) contribute to myocardial reperfusion injury in anesthetized rats?
1. This study examined the potential role of tumor necrosis factor-alpha (TNF-alpha) in myocardial ischemia-reperfusion injury using an anesthetized rat model of myocardial infarction. 2. The key endpoints were infarct size and serum TNF-alpha levels (measured by a specific ELISA technique). 3. Three groups of rats were studied: vehicle controls (n=6); positive controls for infarct size reduction (ischemic preconditioning; n=6); and a group treated with the selective inhibitor of PDE-IV and TNF-alpha production, rolipram (1 mg/kg i.v. 10-min prereperfusion + 1 microg/kg per minute through 1-hr reperfusion, n=6). 4. Baseline preischemia levels of serum TNF-alpha were low (approximately 0.1 nM) and showed a trend for further reduction in all treatment groups at 1 min and 3 hr into the postischemia reperfusion period. 5. Infarct size (68+/-2% of the ischemic area in controls) was significantly reduced (41% decrease) by preconditioning, but was unchanged in rolipram-treated animals. 6. Collectively, these data argue against an important role for TNF-alpha in lethal reperfusion injury in this rat model of myocardial infarction.
Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. II. Mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied using transient and steady-state kinetics.
The interactions of isoleucyl-tRNA synthetase (IleRS, E) from Staphylococcus aureus with both intermediate analogues and pseudomonic acid (PS-A) have been investigated using transient and steady-state techniques. Non-hydrolyzable analogues of isoleucyl-AMP (I) were simple competitive inhibitors (Ile-ol-AMP, Ki = 50 nM and Ile-NHSO2-AMP, Ki = 1 nM;). PS-A (J) inhibits IleRS via a slow-tight binding competitive mechanism where E.J (Kj = approximately 2 nM), undergoes an isomerization to form a stabilized E*.J complex (K*j = 50 pM). To overcome tight-binding artifacts when K*j << [E], K*j values were estimated from PPi/ATP exchange where [S] >> Km, thus raising K*j,app well above [E]. Using [3H]PS-A, it was confirmed that binding occurs with 1:1 stoichiometry and is reversible. Formation of inhibitor complexes was monitored directly through changes in enzyme tryptophan fluorescence. For Ile-ol-AMP and Ile-NHSO2-AMP, the fluorescence intensity of E.I was identical to that when E.Ile-AMP forms catalytically. Binding of PS-A induced only a small change in IleRS fluorescence that was characterized using transient kinetic competition. SB-205952, a PS-A analogue, produced a 37% quenching of IleRS fluorescence upon binding as a result of radiationless energy transfer. Inhibitor reversal rates were obtained by measuring relaxation between spectroscopically different complexes. Together, these data represent a comprehensive solution to the kinetics of inhibition by these compounds.
Effects of substrate and inhibitor binding on proteolysis of isoleucyl-tRNA synthetase from Staphylococcus aureus.
Binding of ligands to isoleucyl-tRNA synthetase (IleRS; E) from Staphylococcus aureus was investigated through effects on proteolytic digestion. Approximately 50-fold higher concentrations of protease (trypsin or chymotrypsin) were required to inactivate IleRS after incubation with substrates and formation of the E. Ile-AMP intermediate compared with free E. Binding of pseudomonic acid A (PS-A) or isoleucynol adenylate (Ile-ol-AMP) also induced resistance to proteolysis and altered the patterns of IleRS cleavage fragments in an inhibitor-class specific manner. The determinants for PS-A binding were investigated via proteolysis of E.[3H]PS-A. Limited proteolysis of E.[3H]PS-A (excising residues 186-407) could be achieved without significant loss of bound inhibitor, eliminating this region as contributing to inhibitor binding. Assays were developed which allowed IleRS proteolysis to be readily followed using fluorescence polarization. Inhibitor-protected IleRS was labeled with fluorescein isothiocyanate with only a small effect upon catalytic activity (Fl-IleRS). The (pseudo) kinetics of proteolytic cleavage of Fl-IleRS could be measured at low nanomolar Fl-IleRS concentrations in 96/384-well microtiter plates, allowing real-time monitoring of dose-dependent protection from proteolysis. Thus, inhibitor (and substrate) binding could be reproducibly assessed in the absence of measurements of catalytic acitvity. This could potentially form the basis of novel screening assays for ligands to other proteins.
Effect of potent urease inhibitor, fluorofamide, on Helicobacter sp. in vivo and in vitro.
The therapeutic potential of urease inhibition of Helicobacter pylori has been studied by examining the effect of the potent urease inhibitor, fluorofamide (N-(diaminophosphinyl)-4-fluorobenzenamide), on urease activity and bacterial survival in vivo and in vitro. In culture, acid protection in H. pylori was shown to be due to changes in the pH of the medium brought about by the release of ammonia. Both the acid protection and the ammonia release were completely blocked by fluorofamide at low doses (ED50 = approximately 100 nM). However, fluorofamide was unstable under acidic conditions (T1/2 = 5.7 min at pH 2). Despite this, fluorofamide was the best available compound to test in vivo. In ferrets naturally infected with H. mustelae, a single dose (50 mg/kg, per os) of fluorofamide completely inhibited bacterial urease. In repeat dosing studies, fluorofamide (50 mg/kg per os, three times a day) was compared with the Helicobacter triple therapy regime (amoxycillin, metronidazole, and bismuth subcitrate). Fluorofamide failed to eradicate the H. mustelae infection, compared to 80% eradication with triple therapy. However, histological samples showed a profound reduction in bacterial numbers following fluorofamide treatment. A combination of fluorofamide and amoxycillin was dosed to ferrets (seven days of treatment with 50 mg/kg fluorofamide plus 10 mg/kg amoxycillin per os twice a day); however, this failed to eradicate the infection, despite there being a reduction in bacterial numbers in 3/5 ferrets after 21 days after dosing stopped. It was concluded that urease inhibitors (either alone or in combination with antibiotics) are unlikely to have therapeutic potential for Helicobacter pylori infections. This is probably because, in vivo, some bacteria (perhaps dormant forms) are not entirely dependent upon urease for survival. However, given the acid instability of fluorofamide, the possibility that more stable urease inhibitors might have therapeutic potential, cannot be excluded.
Cardioprotective effects of the novel adenosine A1/A2 receptor agonist AMP 579 in a porcine model of myocardial infarction.
This study examined the cardioprotective effects and pharmacology of the novel adenosine A1/A2 receptor agonist ([1S-[1a,2b,3b, 4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]amino]-3H-imida zo[4,5-b] pyridyl-3-yl] cyclopentane carboxamide) (AMP 579), in a model of myocardial infarction. Experiments were performed in pentobarbital-anesthetized pigs in which myocardial infarction was induced by a 40-min occlusion of the left anterior descending coronary artery, followed by 3 hr of reperfusion. This procedure resulted in approximately 20% of the left ventricle being made ischemic in all test groups. In untreated animals, an infarct size equal to 56 +/- 5% of the ischemic area was observed. Preconditioning, with two cycles of 5 min of ischemia followed by 10-min reperfusion, resulted in a 70% reduction in infarct size (17 +/- 5%) relative to risk area. Administration of AMP 579 30 min before ischemia (3 microg/kg i.v. followed by 0.3 microg/kg/min i.v. through 1 hr of reperfusion) did not change blood pressure, HR or coronary blood flow but resulted in marked cardioprotection: a 98% reduction in infarct size (1 +/- 1%) relative to risk area. Moreover, whereas approximately 90% of control pigs suffered ventricular fibrillation during ischemia, no fibrillation was observed in animals treated with AMP 579. Further experiments determined the effects of AMP 579 when administered 30 min after the onset of myocardial ischemia, 10 min before reperfusion. Two doses were studied: a low hemodynamically silent dose (3 microg/kg + 0.3 microg/kg/min through 1 hr of reperfusion) and a 10-fold higher dose that did cause reductions in blood pressure and HR. Both doses of AMP 579 produced a comparable cardioprotective effect, reducing infarct size to approximately 50% of that observed in control animals. The cardioprotective effect of AMP 579 was a consequence of adenosine receptor stimulation, because it was completely inhibited by pretreatment with the specific adenosine receptor antagonist CGS 15943 (1 mg/kg i.v.). However, the selective A1 receptor agonist GR 79236 (3 microg/kg + 0.3 microg/kg/min i.v.) did not reduce infarct size, which suggests that under these experimental conditions, stimulation of adenosine A2 receptors is important for the cardioprotective effect of AMP 579. The adenosine-regulating agent acadesine (5 mg/kg + 0.5 mg/kg/min i.v.) also failed to reduce infarct size. In conclusion, the novel adenosine A1/A2 receptor agonist AMP 579 produces marked cardioprotection whether administered before myocardial ischemia or reperfusion. Cardioprotection is not dependent on changes in afterload or myocardial oxygen demand and is a consequence of adenosine receptor stimulation. The pharmacological profile of AMP 579 in this model is consistent with its potential utility in the treatment of acute myocardial infarction.
A comparative analysis of the neuroprotective properties of competitive and uncompetitive N-methyl-D-aspartate receptor antagonists in vivo: implications for the process of excitotoxic degeneration and its therapy.
Injection of the N-methyl-D-aspartate receptor agonist, quinolinic acid, into the rat striatum in vivo results in the degeneration of cholinergic and GABAergic neurons, as determined seven days later using the marker enzymes, choline acetyltransferase and glutamate decarboxylase, respectively. Such damage was dose-dependently prevented by CGP 37849 or MK-801 (competitive and uncompetitive N-methyl-D-aspartate receptor antagonists, respectively) administered systemically or intrastriatally at the same time as quinolinic acid. The neuroprotective activity of CGP 37849 was associated with the D-enantiomer, CGP 40116 (ED50 7.5 mg/kg i.p.), which was approximately 1.5-fold and 3.5-fold more potent than the related compounds, D-CPPene and CGS 19755, respectively. CGP 37849 was a weaker neuroprotectant than MK-801 (ED50 0.8 mg/kg i.p) when administered systemically, but was dramatically more potent following coinjection with quinolinic acid (ED50's 0.2 and 117 nmol, respectively). When injected intrastriatally 0.5-2 h post-quinolinic acid, CGP 37849 was protective over the entire period studied, whereas MK-801 was less effective at all post-quinolinic acid injection times. The finding that CGP 37849 is neuroprotective when administered intrastriatally 1-2 h post-quinolinic acid supports the hypothesis that a period exists following excitotoxic insult in which neurons are not committed to die, and can be rescued by blockade of ongoing N-methyl-D-aspartate receptor activation. Competition studies indicated that, when coinjected with 100-400 nmol quinolinic acid into the striatum, CGP 37849 exhibited kinetics predicted of a competitive N-methyl-D-aspartate receptor antagonist (declining neuroprotective potency with increasing doses of agonist), whereas MK-801 displayed a complex picture, with weak protective activity at low doses of quinolinic acid. Following systemic administration, neither antagonist was markedly affected by the dose of excitotoxin. When given i.p. at up to 6 h post-quinolinic acid, CGP 37849 and MK-801 showed essentially identical profiles of post-insult protection; degeneration of cholinergic neurons was reduced significantly throughout the entire post-insult period, whereas GABAergic neurons were protected only when drugs were administered 2 h or earlier post-quinolinic acid. The data indicate that competitive and uncompetitive N-methyl-D-aspartate receptor antagonists are effective neuroprotectants in vivo, and that parameters such as drug lipophilicity or mechanism of action at the receptor do not impinge upon their properties as systemically active cerebroprotectants.
Total-body protein turnover in parenterally fed neonates: effects of energy source studied by using [15N]glycine and [1-13C]leucine.
The effects of nonprotein energy source (ie, glucose only vs glucose and lipid) on nitrogen retention and total-body protein turnover were studied in 20 parenterally fed newborn infants. All infants received approximately 3 g amino acids and 80-90 kcal.kg body wt.d. Total-body protein synthesis was estimated by using three constant-infusion, end-product methods: enrichment of urinary urea and ammonia in response to a [15N]glycine label and exhaled carbon dioxide enrichment in response to a [1-13C]leucine label. No differences were seen in nitrogen retention between the two energy sources. The estimate of total-body protein turnover obtained from the 13C label was similar to that obtained with the [15N]urea label. No differences in turnover rates were observed between the two diet groups. Use of the glucose-plus-lipid fuel system enhanced energy storage and the reutilization of amino acid for protein synthesis.
A comparison of the estimates of whole-body protein turnover in parenterally fed neonates obtained using three different end products.
Protein turnover rates in neonates have been calculated largely by measuring urinary [15N]urea enrichment following administration of [15N]glycine. Although ammonia has been increasingly recognized as an end product of nitrogen metabolism, in neonates it yields a different estimate of protein turnover than does urea. Comparisons of ammonia and urea end products in parenterally fed neonates have not previously been reported. A third and independent way of estimating protein turnover, developed for adults, is to use breath 13CO2 as an end product following administration of [1-13C]leucine. We therefore carried out simultaneous measurements of protein turnover in 10 parenterally fed neonates, using the three end products. The infants were clinically stable, weighed 2.6 +/- 0.2 kg, and received 3.1 +/- 0.2 g.kg-1.d-1 of amino acid, 2.2 +/- 0.1 g.kg-1.d-1 of lipids, and an energy intake of 90 +/- 4 kcal.kg-1.d-1 (1 kcal = 4.186 kJ). The turnover estimates derived from the 13CO2 and [15N]urea end products were very similar. The [15N]ammonia end product produced values approximately 66% (p less than 0.01) of the other two. We conclude that the ammonia and urea end products probably originate in different precursor pools. The similarity of the urea and breath carbon dioxide results helps validate the use of the urea end product in studying the nitrogen metabolism of parenterally fed neonates. Ideally in future studies two or more end products should be used, since they provide information about different aspects of the neonates' protein metabolism.