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Biomedical subjects

C M Grant

Publications and source records attributed to C M Grant.

At least 19 recordsLinked to original sources
PubMed2001

Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions.

Sulphydryl groups (-SH) play a remarkably broad range of roles in the cell, and the redox status of cysteine residues can affect both the structure and the function of numerous enzymes, receptors and transcription factors. The intracellular milieu is usually a reducing environment as a result of high concentrations of the low-molecular-weight thiol glutathione (GSH). However, reactive oxygen species (ROS), which are the products of normal aerobic metabolism, as well as naturally occurring free radical-generating compounds, can alter this redox balance. A number of cellular factors have been implicated in the regulation of redox homeostasis, including the glutathione/glutaredoxin and thioredoxin systems. Glutaredoxins and thioredoxins are ubiquitous small heat-stable oxidoreductases that have proposed functions in many cellular processes, including deoxyribonucleotide synthesis, repair of oxidatively damaged proteins, protein folding and sulphur metabolism. This review describes recent findings in the lower eukaryote Saccharomyces cerevisiae that are leading to a better understanding of their role in redox homeostasis in eukaryotic cell metabolism.

Gene Expression Regulation, Fungal↗
PubMed2001

The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain.

A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain. This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome. The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation. The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound. GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells. This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth. This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress. Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis. These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress. Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant. Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.

Anaerobiosis↗
PubMed2001

Using tests of false belief with children with autism: how valid and reliable are they?

Twenty-two children with autism were given four tests of false belief understanding: the Sally-Anne task, two variants of the deceptive box task, and the three boxes task. The overall consistency of the children's performance was high, 77 percent of the participants either passing or failing all of the tasks. The convergent validity (across-task consistency) of the deceptive box and the three boxes paradigms was high, and the convergent validity of the three boxes and Sally-Anne tasks was also acceptable. However, a weaker level of convergent validity was found for the deceptive box and Sally-Anne tasks, suggesting that these paradigms test slightly different aspects of cognition. The reliability (within-child consistency) of the children's performances across two versions of the deceptive box task was high. These findings are discussed in terms of their practical implications for practitioners and researchers.

Adolescent↗
PubMed2000

Differential regulation of glutaredoxin gene expression in response to stress conditions in the yeast Saccharomyces cerevisiae.

Glutaredoxins are small heat-stable proteins that are active as glutathione-dependent oxidoreductases and are encoded by two genes, designated GRX1 and GRX2, in the yeast Saccharomyces cerevisiae. We report here that the expression of both genes is induced in response to various stress conditions including oxidative, osmotic, and heat stress and in response to stationary phase growth and growth on non-fermentable carbon sources. Furthermore, both genes are activated by the high-osmolarity glycerol pathway and negatively regulated by the Ras-protein kinase A pathway via stress-responsive STRE elements. GRX1 contains a single STRE element and is induced to significantly higher levels compared to GRX2 following heat and osmotic shock. GRX2 contains two STRE elements, and is rapidly induced in response to reactive oxygen species and upon entry into stationary phase growth. Thus, these data support the idea that the two glutaredoxin isoforms in yeast play distinct roles during normal cellular growth and in response to stress conditions.

DNA-Binding Proteins↗
PubMed2000

A single glutaredoxin or thioredoxin gene is essential for viability in the yeast Saccharomyces cerevisiae.

Glutaredoxins and thioredoxins are small heat-stable oxidoreductases that have been conserved throughout evolution. The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2). We report here that the quadruple trx1 trx2 grx1 grx2 mutant is inviable and that either a single glutaredoxin or a single thioredoxin (i.e. grx1 grx2 trx1, grx1 grx2 trx2, grx1 trx1 trx2, grx2 trx1 trx2) is essential for viability. Loss of both thioredoxins has been reported previously to lead to methionine auxotrophy consistent with thioredoxins being the sole reductants for 3'-phosphoadenosine 5'-phosphosulphate reductase (PAPS) in yeast. However, we present evidence for the existence of a novel yeast hydrogen donor for PAPS reductase, as strains lacking both thioredoxins assimilated sulphate under conditions that minimized the generation of reactive oxygen species (low aeration and absence of functional mitochondria). In addition, the assimilation of [35S]-sulphate was approximately 60-fold higher in the trx1 trx2 grx1 and trx1 trx2 grx2 mutants compared with the trx1 trx2 mutant. Furthermore, in contrast to the trx1 trx2 mutant, the trx1 trx2 grx2 mutant grew on minimal agar plates, and the trx1 trx2 grx1 mutant grew on minimal agar plates under anaerobic conditions. We propose a model in which the novel reductase activity normally functions in the repair of oxidant-mediated protein damage but, under conditions that minimize the generation of reactive oxygen species, it can serve as a hydrogen donor for PAPS reductase.

Culture Media↗
PubMed2000

The effects of breed and level of nutrition on whole-body and muscle protein metabolism in pure-bred Aberdeen angus and Charolais beef steers.

Eighteen pure-bred steers (live weight 350 kg) from each of two breeds, Aberdeen Angus (AA) and Charolais (CH), were split into three equal groups (six animals each) and offered three planes of nutrition during a 20-week period. The same ration formulation was offered to all animals with amounts adjusted at 3-week intervals to give predicted average weight gains of either 1.0 kg/d (M/M group) or 1.4 kg/d (H/H group). The remaining group (M/H) were offered the same amount of ration as the M/M group until 10 weeks before slaughter when the ration was increased to H. Data on animal performance, carcass characteristics and fibre-type composition in skeletal muscle are presented elsewhere (Maltin et al. 2000; Sinclair et al. 2000). On three occasions (17, 10 and 2 weeks before slaughter) the animals were transferred to metabolism stalls for 1 week, during which total urine collection for quantification of Ntau-methylhistidine (Ntau-MeH) elimination was performed for 4 d. On the last day, animals were infused for 11 h with [2H5]phenylalanine with frequent blood sampling (to allow determination of whole-body phenylalanine flux) followed by biopsies from m. longissimus lumborum and m. vastus lateralis to determine the fractional synthesis rate of mixed muscle protein. For both breeds, the absolute amount of Ntau-MeH eliminated increased with animal age or weight (P < 0.001) and was significantly greater for CH steers, at all intake comparisons, than for AA (P < 0.001). Estimates of fractional muscle breakdown rate (FBR; calculated from Ntau-MeH elimination and based on skeletal muscle as a fixed fraction of live weight) showed an age (or weight) decline for M/M and H/H groups of both breeds (P < 0.001). FBR was greater for the H/H group (P = 0.044). The M/H group also showed a lower FBR for the first two measurement periods (both at M intake) but increased when intake was raised to H. When allowance was made for differences in lean content (calculated from fat scores and eye muscle area in carcasses at the end of period 3), there were significant differences in muscle FBR with intake (P = 0.012) but not between breed. Whole-body protein flux (WBPF; g/d) based on plasma phenylalanine kinetics increased with age or weight (P < 0.001) and was similar between breeds. The WBPF was lower for M/M compared with H/H (P < 0.001) based on either total or per kg live weight0.75. Muscle protein fractional synthesis rate (FSR) declined with age for both breeds and tended to be higher at H/H compared with M intakes (intake x period effects, P < 0.05). Changing intake from M to H caused a significant increase (P < 0.001) in FSR. The FSR values for AA were significantly greater than for CH at comparable ages (P = 0. 044). Although FSR and FBR responded to nutrition, these changes in protein metabolism were not reflected in differences in meat eating quality (Sinclair et al. 2000).

Age Factors↗
PubMed1999

Redox control of exofacial protein thiols/disulfides by protein disulfide isomerase.

Protein disulfide isomerase (PDI) facilitates proper folding and disulfide bonding of nascent proteins in the endoplasmic reticulum and is secreted by cells and associates with the cell surface. We examined the consequence of over- or underexpression of PDI in HT1080 fibrosarcoma cells for the redox state of cell-surface protein thiols/disulfides. Overexpression of PDI resulted in 3.6-4. 2-fold enhanced secretion of PDI and 1.5-1.7-fold increase in surface-bound PDI. Antisense-mediated underexpression of PDI caused 38-53% decreased secretion and 10-33% decrease in surface-bound PDI. Using 5,5'-dithio-bis(2-nitrobenzoic acid) to measure surface protein thiols, a 41-50% increase in surface thiols was observed in PDI-overexpressing cells, whereas a 29-33% decrease was observed in underexpressing cells. Surface thiol content was strongly correlated with cellular (r = 0.998) and secreted (r = 0.969) PDI levels. The pattern of exofacial protein thiols was examined by labeling with the membrane-impermeable thiol reactive compound, 3-(N-maleimidylpropionyl)biocytin. Fourteen identifiable proteins on HT1080 cells were labeled with 3-(N-maleimidylpropionyl)biocytin. The intensity of labeling of 11 proteins was increased with overexpression of PDI, whereas the intensity of labeling of 3 of the 11 proteins was clearly decreased with underexpression of PDI. These findings indicated that secreted PDI was controlling the redox state of existing exofacial protein thiols or reactive disulfide bonds.

Base Sequence↗
PubMed1999

Cervical screening interval: costing the options in one health authority.

BACKGROUND: This is a study of the costs of the cervical screening programme in one health authority with a mixed three and five year, and thus inequitable, cervical screening interval. The costs of three year and five yearly screening are compared, and considered in terms of likely numbers of averted cases of and deaths from cervical cancer. METHODS: The study uses an activity-based costing procedure to calculate the component and total costs of the cervical screening programme. RESULTS: The main costs of the cervical screening programme are the costs of taking and processing smears. In 1994-1995 the total cost of a three year recall policy was 768 570 pound silver per 100000 eligible women and that of a five year recall policy was 476768 pound silver per 100000 eligible women. Best estimates of the numbers of cases of and deaths from invasive cervical cancer averted by three over five yearly screening are 1.4 and 0.7 per 100000 eligible women, respectively. Because of uncertainty regarding colposcopy costs a sensitivity analysis was carried out, giving a range of cost differences between three and five yearly screening of 278477 pound silver and 351 768 pound silver. CONCLUSIONS: The health service costs of three yearly screening are considerably greater than those of five yearly screening. Despite this, a significant proportion of smear-takers are screening more frequently than five yearly, with implications for anxiety of screened women, as well as health service costs.

Adult↗
PubMed1999

Differential protein S-thiolation of glyceraldehyde-3-phosphate dehydrogenase isoenzymes influences sensitivity to oxidative stress.

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, in which protein -SH groups form mixed disulfides with low-molecular-weight thiols such as glutathione. We report here the identification of glyceraldehyde-3-phosphate dehydrogenase as the major target of protein S-thiolation following treatment with hydrogen peroxide in the yeast Saccharomyces cerevisiae. Our studies reveal that this process is tightly regulated, since, surprisingly, despite a high degree of sequence homology (98% similarity and 96% identity), the Tdh3 but not the Tdh2 isoenzyme was S-thiolated. The glyceraldehyde-3-phosphate dehydrogenase enzyme activity of both the Tdh2 and Tdh3 isoenzymes was decreased following exposure to H2O2, but only Tdh3 activity was restored within a 2-h recovery period. This indicates that the inhibition of the S-thiolated Tdh3 polypeptide was readily reversible. Moreover, mutants lacking TDH3 were sensitive to a challenge with a lethal dose of H2O2, indicating that the S-thiolated Tdh3 polypeptide is required for survival during conditions of oxidative stress. In contrast, a requirement for the nonthiolated Tdh2 polypeptide was found during exposure to continuous low levels of oxidants, conditions where the Tdh3 polypeptide would be S-thiolated and hence inactivated. We propose a model in which both enzymes are required during conditions of oxidative stress but play complementary roles depending on their ability to undergo S-thiolation.

Dose-Response Relationship, Drug↗
PubMed1998

Glutathione and catalase provide overlapping defenses for protection against hydrogen peroxide in the yeast Saccharomyces cerevisiae.

Glutathione (GSH) is an abundant and ubiquitous low-molecular-weight thiol which has proposed roles in many cellular processes including protection against the deleterious effects of reactive oxygen species. Our experiments have addressed the role of GSH in protection against hydrogen peroxide in the yeast Saccharomyces cerevisiae, and have shown that GSH and catalase provide overlapping defense systems. GSH appears to be the primary antioxidant for protection against hydrogen peroxide since mutants lacking GSH (gsh1) or glutathione reductase (glr1) are sensitive, whereas, strains lacking catalase A (cta1) or catalase T (ctt1) are unaffected in resistance to this oxidant. Furthermore, following treatment with hydrogen peroxide, the levels of oxidized, protein-bound and extracellular GSH were all increased at the expense of intracellular GSH. However, there are two lines of evidence that indicate catalases are required in the absence of GSH; firstly, strains that lack both catalase A and T accumulate increased levels of oxidized glutathione following treatment with hydrogen peroxide; and secondly, deletion of catalase genes exacerbates the hydrogen peroxide sensitivity of glr1 and gsh1 mutants.

Antioxidants↗
PubMed1998

The cytoplasmic Cu,Zn superoxide dismutase of saccharomyces cerevisiae is required for resistance to freeze-thaw stress. Generation of free radicals during freezing and thawing.

The involvement of oxidative stress in freeze-thaw injury to yeast cells was analyzed using mutants defective in a range of antioxidant functions, including Cu,Zn superoxide dismutase (encoded by SOD1), Mn superoxide dismutase (SOD2), catalase A, catalase T, glutathione reductase, gamma-glutamylcysteine synthetase and Yap1 transcription factor. Only those affecting superoxide dismutases showed decreased freeze-thaw tolerance, with the sod1 mutant and the sod1 sod2 double mutant being most affected. This indicated that superoxide anions were formed during freezing and thawing. This was confirmed since the sod1 mutant could be made more resistant by treatment with the superoxide anion scavenger MnCl2, or by freezing in the absence of oxygen, or by the generation of a rho0 petite. Increased expression of SOD2 conferred freeze-thaw tolerance on the sod1 mutant indicating the ability of the mitochondrial superoxide dismutase to compensate for the lack of the cytoplasmic enzyme. Free radicals generated as a result of freezing and thawing were detected in cells directly using electron paramagnetic resonance spectroscopy with either alpha-phenyl-N-tert-butylnitrone or 5, 5-dimethyl-1-pyrroline-N-oxide as spin trap. Highest levels were formed in the sod1 and sod1 sod2 mutant strains, but lower levels were detected in the wild type. The results show that oxidative stress causes major injury to cells during aerobic freezing and thawing and that this is mainly initiated in the cytoplasm by an oxidative burst of superoxide radicals formed from oxygen and electrons leaked from the mitochondrial electron transport chain.

Copper↗
PubMed1998

Involvement of the Saccharomyces cerevisiae UTH1 gene in the oxidative-stress response.

The UTH1 gene was identified by screening a Saccharomyces cerevisiae promoter-probe gene bank for oxidative stress-responsive genes. Transcription of UTH1 was decreased by the superoxide anion and increased by hydrogen peroxide. Deletion of UTH1 did not affect the growth of grande cells, however in a rho0 background it caused retarded growth. The uth1 mutant showed increased resistance to peroxides and, in contrast, was sensitive to superoxide or the thiol oxidant diamide. Furthermore, the mutant exhibited increased survival under starvation conditions, with elevated levels of dormant cells in starved cell cultures. A multicopy plasmid containing the first half of the ORF could confer increased resistance to superoxide and increased sensitivity to peroxides/diamide/starvation on wild-type cells. The same plasmid in the uth1 background caused a highly increased mortality.

Amino Acid Sequence↗
PubMed1998

The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species.

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40-52% identity and 61-76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using beta-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins.

Amino Acid Sequence↗
PubMed1998

Toxicity of linoleic acid hydroperoxide to Saccharomyces cerevisiae: involvement of a respiration-related process for maximal sensitivity and adaptive response.

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type Saccharomyces cerevisiae at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (gsh1 and gsh2), glutathione reductase (glr1), respiratory competence ([rho0] petite), and yAP-1p-mediated expression (yap1) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [rho0] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the yap1 and glr1 mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 microg ml-1). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37 degrees C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.

Adaptation, Physiological↗
PubMed1997

Mitochondrial function is required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae.

Yeast strains that lack mitochondrial function are sensitive to oxidative stress caused by reactive oxygen species (ROS). Specifically, rho0 mutants that lack mitochondrial DNA, and strains deleted for the nuclear genes COX6 and COQ3 that are required for function of the respiratory electron transport chain, were sensitive to H2O2. In addition, treatment with mitochondrial inhibitors including antimycin A, oligomycin, potassium cyanide and sodium azide increased sensitivity to H2O2. The mechanism does not appear to depend on the antioxidant status of the cell since respiratory-deficient strains were able to mount an inducible adaptive response to H2O2. We suggest that the oxidant sensitivity is due to a defect in an energy-requiring process that is needed for detoxification of ROS or for the repair of oxidatively damaged molecules.

Electron Transport↗
PubMed1997

Identification of a Saccharomyces cerevisiae mitochondrial-DNA which can act as a promoter tightly regulated by carbon source when placed in the nucleus.

Screening of a promoter probe gene bank for DNA sequences that could act as inducible promoters following growth on non-fermentable carbon sources led to the identification of the mitochondrially encoded cytochrome oxidase subunit 1 gene (COX1) as an active sequence. Carbon-source regulation of this promoter was confirmed by a beta-galactosidase assay which showed a 31- and 180-fold induction of expression after growth on ethanol or lactate-based media respectively. Two elements matching the CCAAT-binding-factor motif, which is involved in activating transcription on non-fermentable carbon sources, were identified in the putative promoter. Expression was found to be reduced to low levels in otherwise isogenic hap3 and hap4 mutant strains. Thus, this mitochondrial DNA when placed in the nucleus can act as a promoter that is subject to strict carbon-source regulation. These observations are discussed both with respect to the origin of the S. cerevisiae COX1 gene in particular and with respect to the origin of introns in general.

Blotting, Southern↗
PubMed1997

Stationary-phase regulation of the Saccharomyces cerevisiae SOD2 gene is dependent on additive effects of HAP2/3/4/5- and STRE-binding elements.

SOD2, encoding manganese superoxide dismutase (MnSOD), is essential for stationary-phase survival of yeast cells. In addition, stationary-phase cells are more resistant to oxidative stress than exponential-phase cells. The use of a SOD2::lacZ fusion construct in this study shows that transcription of SOD2 increases 6.5-fold as cells enter stationary phase in rich, glucose medium. The increase in SOD2 expression appears to be due to two phenomena-the switch to a non-fermentable carbon source and nutrient limitation. Analysis of SOD2 transcription in mutant Saccharomyces cerevisiae strains showed that the gene was negatively regulated by intracellular cAMP levels which decrease as cells enter stationary phase. Mutation of 'stress-responsive' (STRE) elements in the SOD2 promoter which respond to cAMP levels resulted in the loss of cAMP-dependent expression but only partially reduced the increase in expression as cells entered stationary phase. A putative Yap1p-binding site was found to be inactive and mutation of YAP1 had no effect on the STRE-mediated expression. To fully eliminate the stationary-phase response, it was necessary to mutate a HAP2/3/4/5 complex binding site in addition to the STRE elements. It is postulated that the effects of the STRE sites and the HAP2/3/4/5 complex binding site are additive.

Bacterial Proteins↗