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Biomedical subjects

E M Ellis

Publications and source records attributed to E M Ellis.

At least 19 recordsLinked to original sources
PubMed2001

Expression of rat aldehyde reductase AKR7A1: influence of age and sex and tissue-specific inducibility.

The regulation of the aldo-keto reductase AKR7A1 was examined in the livers of male and female rats during development by using Western blots, and its contribution to carbonyl metabolism was assessed by using enzyme assays. Hepatic levels of AKR7A1 are low in fetal rats and rise to a peak at around 6 weeks of age in animals of both sexes. Higher levels of the enzyme are found in adult male rat liver than in adult female rat liver. The reductase, therefore, appears to be subject to sex-specific regulation. The effect of growth hormone in mediating this difference in expression was examined by using hypophysectomized animals whose serum growth hormone levels had been feminized by continuous administration. Results demonstrate that such treatment leads to a reduction in AKR7A1 expression. AKR7A1 was found to be constitutively expressed in rat tissues such as liver, kidney, small intestine, and testis, but it was not detected in nasal mucosa, skeletal muscle, heart, adrenal gland, brain, or spleen. However, AKR7A1 was inducible by the synthetic antioxidant ethoxyquin in liver, kidney, and small intestine, but not in the other tissues examined. These results show that levels of this important detoxication enzyme vary considerably according to age and sex and that dietary antioxidants can also influence its level in several tissues.

Age Factors↗
PubMed2001

Three aldo-keto reductases of the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an industrially important yeast, which is also used extensively as a model eukaryote. The S. cerevisiae genome has been sequenced in its entirety and therefore represents an ideal organism in which to carry out functional analysis of genes. We have identified several open reading frames in the S. cerevisiae genome which show significant similarity to members of the aldo-keto reductase superfamily. The physiological roles of these gene products have not been previously determined, but their similarity to other enzymes suggests they may perform roles in carbohydrate metabolism and detoxification pathways. Cloning and expression of three of these enzymes has allowed their substrate specificities to be determined. Expression profiling and gene disruption analysis will allow potential roles for these enzymes within the cell to be examined.

Alcohol Oxidoreductases↗
PubMed2000

Purification from rat liver of a novel constitutively expressed member of the aldo-keto reductase 7 family that is widely distributed in extrahepatic tissues.

Antiserum raised against human aflatoxin B(1) aldehyde reductase 1 (hAFAR1) has been used to identify a previously unrecognized rat aldo-keto reductase (AKR). This novel enzyme is designated rat aflatoxin B(1) aldehyde reductase 2 (rAFAR2) and it characteristically migrates faster during SDS/PAGE than does the archetypal ethoxyquin-inducible rAFAR protein (now called rAFAR1). Significantly, rAFAR2 is essentially unreactive with polyclonal antibodies raised against rAFAR1. Besides its distinct electrophoretic and immunochemical properties, rAFAR2 appears to be regulated differently from rAFAR1 as it is expressed in most rat tissues and does not appear to be induced by ethoxyquin. Multiple forms of rAFAR2 have been identified. Anion-exchange chromatography on Q-Sepharose, followed by adsorption chromatography on columns of Matrex Orange A and Cibacron Blue, have been employed to purify rAFAR2 from rat liver cytosol. The Q-Sepharose chromatography step resulted in the resolution of rAFAR2 into three peaks of AKR activity, two of which were purified and shown to be capable of catalysing the reduction of 2-carboxybenzaldehyde, succinic semialdehyde, 4-nitrobenzaldehyde and 9,10-phenathrenequinone. The two most highly purified rAFAR2-containing preparations eluted from the Cibacron Blue column were 91 and 98% homogeneous. Analysis of these by SDS/PAGE indicated that the least anionic (peak CBA5) comprised a polypeptide of 37.0 kDa, whereas the most anionic (peak CBA6) contained two closely migrating polypeptides of 36.8 and 37.0 kDa; by contrast, in the present study, rAFAR1 was estimated by SDS/PAGE to be composed of 38.0 kDa subunits. Final purification of the 37 kDa polypeptide in CBA5 and CBA6 was accomplished by reversed-phase HPLC. Partial proteolysis of the two preparations of the 37 kDa polypeptide with Staphylococcus aureus V8 protease yielded fragments of identical size, suggesting that they represent the product of a single gene. Furthermore, the peptide maps from CBA5 and CBA6 differed substantially from that yielded by rAFAR1, indicating that they are genetically distinct from the inducible reductase. A peptide generated by CNBr digestion of the 37 kDa polypeptide from CBA6 was shown by Edman degradation to share 88% sequence identity with residues Tyr(168)-Leu(183) of rAFAR1. This provides evidence that the rat protein identified by its cross-reactivity with anti-hAFAR1 serum is an additional member of the AKR7 family.

Aldehyde Reductase↗
PubMed2000

Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver.

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.

Aflatoxin B1↗
PubMed1999

Cellular response to cancer chemopreventive agents: contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress.

Cancer chemopreventive agents can act by inhibiting either the acquisition of mutations or the neoplastic processes that occur subsequent to mutagenesis. Compounds that reduce the rate at which mutations arise, referred to as blocking agents, exert their effects largely through their ability to induce the expression of antioxidant and detoxification proteins. This is achieved by the transcriptional activation of a small number of genes that are co-regulated through the presence of an antioxidant responsive element (ARE) in their promoters. Blocking agents can cause gene induction by producing oxidative and/or chemical stress within the cell and, as the inducible proteins act to ameliorate the metabolic insult, the process represents a form of adaptive response. The transcription factors which mediate this response through the ARE are members of the basic leucine zipper superfamily. The mechanism whereby cells sense and respond to the chemical signal(s) generated by chemopreventive blocking agents is discussed.

Adaptation, Physiological↗
PubMed1998

Growth hormone- and testosterone-dependent regulation of glutathione transferase subunit A5 in rat liver.

The class Alpha glutathione S-transferase (GST) subunit A5 is expressed in the livers of young male and female rats. After sexual maturation, this protein is no longer detectable in the livers of male rats, but is still expressed in female rats. We have previously demonstrated that the sexually dimorphic secretion of growth hormone regulates the levels of certain class Mu GSTs in rat liver, and this study was designed to investigate the hormonal regulation of GSTA5. Control and hypophysectomized rats of both sexes were used to study the role of growth hormone in the regulation of hepatic GSTA5; and the influence of testosterone on the expression of this same subunit was investigated in intact females and castrated males. Liver cytosols were subjected to SDS/PAGE and immunoblotting using antibodies directed towards rat (r)GSTA5, and to affinity purification on glutathione-Sepharose followed by reverse-phase HPLC in order to quantify the relative levels of rGSTA1, A2, A3, A4, M1 and M2 subunits. These analyses revealed that the expression of rGSTA5 is, indeed, regulated by both growth hormone and testosterone.

Animals↗
PubMed1998

Regulation of rat glutathione S-transferase A5 by cancer chemopreventive agents: mechanisms of inducible resistance to aflatoxin B1.

The rat can be protected against aflatoxin B1 (AFB1) hepatocarcinogenesis by being fed on a diet containing the synthetic antioxidant ethoxyquin. Evidence suggests that chemoprotection against AFB1 is due to increased detoxification of the mycotoxin by one or more inducible drug-metabolising enzymes. The glutathione S-transferase (GST) isoenzymes in rat liver that contribute to ethoxyquin-induced chemoprotection against AFB1 have been identified by protein purification. This approach resulted in the isolation of several heterodimeric class alpha GST, all of which contained the A5 subunit and possessed at least 50-fold greater activity towards AFB1-8,9-epoxide than previously studied transferases. Molecular cloning and heterologous expression of rat GSTA5-5 has led to the demonstration that it exhibits substantially greater activity for AFB1-8,9-epoxide than other rat transferases. The A5 homodimer can also catalyse the conjugation of glutathione with other epoxides, such as trans-stilbene oxide and 1,2-epoxy-3-(4'-nitrophenoxy)propane, and possesses high catalytic activity for the reactive aldehyde 4-hydroxynonenal. Western blotting has shown that the A5 subunit is not only induced by ethoxyquin but that it is also induced by other cancer chemopreventive agents, such as butylated hydroxyanisole, oltipraz, benzyl isothiocyanate, indole-3-carbinol and coumarin. In addition to GSTA5, we have identified a novel aflatoxin-aldehyde reductase (AFAR) that is similarly induced by ethoxyquin. However, immunoblotting has shown that GSTA5 and AFAR are not always co-ordinately regulated by chemoprotectors. In order to gain a better understanding of the mechanisms responsible for the induction of GSTA5 protein, the GSTA5 gene has been cloned. It was isolated on two overlapping bacteriophage lambda clones and found to be approximately 12 kb in length. The transcriptional start site of GSTA5 has been identified 228 bp upstream from the ATG translational initiation codon. Computer-assisted analysis of the upstream sequence has indicated the presence of a putative antioxidant responsive element (located between -421 and -429 bp) which may be responsible for the induction of GSTA5 by chemopreventive agents.

Aflatoxin B1↗
PubMed1997

Protection conferred by selenium deficiency against aflatoxin B1 in the rat is associated with the hepatic expression of an aldo-keto reductase and a glutathione S-transferase subunit that metabolize the mycotoxin.

Fischer 344 rats fed on a diet that is deficient in selenium are more resistant to the hepatocarcinogen aflatoxin B1 (AFB1) than those fed on a selenium-sufficient diet. Hepatic cytosol from either selenium-deficient Fischer 344 rats or Hooded Lister rats possesses a marked increase in both reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB(1)-8,9-epoxide than hepatic cytosol from selenium-sufficient rats. The elevation in hepatic AFB1-aldehyde reductase (AFAR) activity in selenium-deficient animals is accompanied by an increase of 11- and 15-fold in the levels of AFAR protein in liver cytosol from Fischer 344 and Hooded Lister rats, respectively. The amount of AFAR protein in selenium-sufficient and -deficient Fischer rats was modulated by treatment with N-acetylcysteine; this antioxidant reduced basal expression of AFAR but did not modulate the relative overexpression of AFAR during selenium deficiency. The enhanced capacity to conjugate glutathione with AFB(1)-8,9-epoxide in selenium-deficient livers from Fischer 344 and Hooded Lister rats is associated with a 5- and 7-fold increase, respectively, in the hepatic levels of the AFB1-metabolizing alpha-class GSTA5 subunit. The elevated levels of AFAR and GSTA5 protein in the selenium-deficient animals coincided with increases in the steady-state levels of their mRNAs. In selenium-deficient Fischer 344 rats, AFAR and GSTA5 were both found to be expressed throughout the centrilobular and midzonal areas of the liver lobule but were essentially absent from periportal hepatocytes. The effect of selenium insufficiency is pleiotropic, and it was also noted that the theta-class GSTT1 is overexpressed 3- and 10-fold in livers of selenium-deficient Hooded Lister and Fischer 344 rats. Inasmuch as GSTT1 is responsible for the metabolic activation of dihaloalkanes, selenium deficiency may increase the susceptibility of rats to mutagens such as dichloromethane.

Acetylcysteine↗
PubMed1996

Regulation of carbonyl-reducing enzymes in rat liver by chemoprotectors.

Feeding rats on diets containing the synthetic antioxidants ethoxyquin, butylated hydroxyanisole, and oltipraz results in 15-, 9-, and 6-fold increases, respectively, in the hepatic levels of aflatoxin B1-dialdehyde reductase (AFAR) protein. By contrast, treatment of rats with either of the inducing agents phenobarbital or 3-methylcholanthrene results in an approximate increase of only 1.4-fold in the amount of AFAR in rat liver. Northern blotting has shown that these increases in levels of hepatic AFAR protein are accompanied by corresponding increases in AFAR mRNA. Immunodepletion of AFAR from rat liver extracts has revealed that AFAR makes a considerable contribution to carbonyl metabolism in livers from animals treated with synthetic antioxidants and that it is the major reductase that can utilize aflatoxin B1-dialdehyde as a substrate. The immunodepletion experiments also revealed the presence of at least one other inducible carbonyl-reducing enzyme that, like AFAR, can metabolize 9,10-phenanthraquinone. Carbonyl-reducing activity from rat liver has been resolved into six enzyme-containing peaks by anion-exchange chromatography on Q-Sepharose. This method has been used to show that, in addition to AFAR, two other rat liver carbonyl-reducing enzymes are induced by ethoxyquin, and that these are distinct from NAD(P)H: quinone oxidoreductase. Collectively, these data show that synthetic antioxidants can influence substantially the capacity of rat liver to metabolize reactive carbonyl-containing compounds.

Aldehyde Reductase↗
PubMed1996

Regulation of glutathione S-transferases and aldehyde reductase by chemoprotectors: studies of mechanisms responsible for inducible resistance to aflatoxin B1.

A number of xenobiotics, including the synthetic antioxidant ethoxyquin, inhibit aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat. Two detoxification enzymes that mediate ethoxyquin-induced chemoprotection against AFB1 have been identified by protein purification: a glutathione S-transferase (GST) Yc2 subunit with at least 100-fold greater activity towards AFB1-8,9-epoxide than previously studied transferases, and a unique aldehyde reductase with activity towards the dialdehydic form of AFB1-8,9-dihydrodiol. Molecular cloning has revealed that the Yc2 subunit is a class alpha GST and that the aflatoxin-metabolizing aldehyde reductase (AFAR) is a distant member of the aldo-keto reductase superfamily. Enzyme assay and western blotting have shown that many chemoprotectors, such as ethoxyquin, butylated hydroxyanisole, butylated hydroxytoluene, oltipraz and indole-3-carbinol, that inhibit AFB1-mediated hepatocarcinogenesis induce both GST Yc2 and AFAR. However, western blotting suggests that these enzymes are not always coordinately regulated, as treatment with phenobarbital and beta-naphthoflavone results in differences in the relative increase in hepatic GST Yc2 and AFAR. These findings indicate that GST Yc2 and AFAR represent important resistance mechanisms against AFB1 in the rat. This conclusion is supported by the observation that GST Yc2 and AFAR are overexpressed in rat liver preneoplastic nodules, which display pleiotropic drug resistance.

Aflatoxin B1↗
PubMed1995

Substrate specificity of an aflatoxin-metabolizing aldehyde reductase.

The enzyme from rat liver that reduces aflatoxin B1-dialdehyde exhibits a unique catalytic specificity distinct from that of other aldo-keto reductases. This enzyme, designated AFAR, displays high activity towards dicarbonyl-containing compounds with ketone groups on adjacent carbon atoms; 9,10-phenanthrenequinone, acenaphthenequinone and camphorquinone were found to be good substrates. Although AFAR can also reduce aromatic and aliphatic aldehydes such as succinic semialdehyde, it is inactive with glucose, galactose and xylose. The enzyme also exhibits low activity towards alpha,beta-unsaturated carbonyl-containing compounds. Determination of the apparent Km reveals that AFAR has highest affinity for 9,10-phenanthrenequinone and succinic semialdehyde, and low affinity for glyoxal and DL-glyceraldehyde.

Aldehyde Reductase↗
PubMed1993

An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a subfamily of aldo-keto reductases.

Protection of liver against the toxic and carcinogenic effects of aflatoxin B1 (AFB1) can be achieved through the induction of detoxification enzymes by chemoprotectors such as the phenolic antioxidant ethoxyquin. We have cloned and sequenced a cDNA encoding an aldehyde reductase (AFB1-AR), which is expressed in rat liver in response to dietary ethoxyquin. Expression of the cDNA in Escherichia coli and purification of the recombinant enzyme reveals that the protein exhibits aldehyde reductase activity and is capable of converting the protein-binding dialdehyde form of AFB1-dihydrodiol to the nonbinding dialcohol metabolite. We show that the mRNA encoding this enzyme is markedly elevated in the liver of rats fed an ethoxyquin-containing diet, correlating with acquisition of resistance to AFB1. AFB1-AR represents the only carcinogen-metabolizing aldehyde reductase identified to date that is induced by a chemoprotector. Alignment of the amino acid sequence of AFB1-AR with other known and putative aldehyde reductases shows that it defines a subfamily within the aldo-keto reductase superfamily.

Aflatoxin B1↗
PubMed1993

The Saccharomyces cerevisiae MTS1 gene encodes a putative RNA-binding protein involved in mitochondrial protein targeting.

Most proteins present in the mitochondrion are nuclear encoded, and are directed to the organelle by virtue of a targeting sequence at the N terminus of the precursor protein. Mitochondrial (mt) protein targeting appears to require several accessory proteins that recognise mt precursors both in the cytoplasm and at the mt surface. We describe here the use of yeast genetics to identify a protein that is required for mt protein targeting. Two yeast mutants (mts1 and mts2) were isolated as extragenic suppressors of a known targeting defect in the presequence of the beta-subunit of ATP synthase. We have cloned and sequenced the wild-type allele of one of these genes (MTS1) and shown that it encodes a member of a family of RNA-binding proteins that is essential for growth.

Amino Acid Sequence↗
PubMed1991

Watchers in the night: an anthropological look at sleep disorders.

An anthropological point of view is introduced to explain the origin of sleep disorders. Four groups of sufferers are discussed: (1) Those that fail to maintain primary relationships; (2) those that feel increased vulnerability during the night; (3) those who have failed to separate or individuate; and (4) those who show a higher arousability than sound sleepers. A case example is offered for each group. A description of sleeping arrangements of the Kung San people of the Kalahari desert; speculations of the need for arousability in primitive society to prevent predators from attacking serve to bolster the view point. A theory that suggests that sleep disorders in children are caused by Western sleeping arrangements, encouraging mother and child to sleep apart, is challenged.

Adolescent↗
PubMed1990

Adult agoraphobia and childhood separation anxiety: using children's literature to understand the link.

Separation-anxiety stories are frequent in children's literature and have eight characteristic elements. Peter Rabbit is used as an early and very simple example. A case study follows which illustrates the fear of separating from the family in an adult agoraphobic. The Wizard of Oz is presented as a more expanded version of a separation-anxiety story. A case study follows which illustrates a woman's difficulty with maturity and separation. A review of the literature is made in which studies are cited which indicate a history of childhood-separation problems in adult agoraphobics. Studies also show that panic disorder in these patients is often precipitated by losses and separations. Studies also show that agoraphobics may have poor problem-solving skills. Children's stories may serve as a guide to children toward mastery of separation fear. A case study follows of a man who developed a panic disorder while watching E.T. E.T. is presented as a separation-anxiety story for the late 1900s. The paper concludes with a brief review of children's separation-anxiety stories and how they reflect broad cultural changes.

Adult↗
PubMed1987

OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium.

The tppB locus of Salmonella typhimurium encodes the anaerobically-induced tripeptide permease. We have demonstrated that expression of tppB requires the function of the ompR and envZ gene products, originally identified as positive regulatory proteins required for the osmotic regulation of porin expression. Significantly, tppB expression is not osmotically regulated. We have also identified three additional genes whose expression depends on OmpR. Thus OmpR and EnvZ serve a more general regulatory role than has previously been supposed. This study provides the first detailed genetic analysis of the ompB locus of S. typhimurium.

Amino Acid Sequence↗