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Biomedical subjects

R S Holmes

Publications and source records attributed to R S Holmes.

At least 37 records · Page 2Linked to original sources
PubMed1991

Postnatal development of mouse alcohol dehydrogenases: agarose isoelectric focusing analyses of the liver, kidney, stomach and ocular isozymes.

The postnatal development of alcohol dehydrogenase (ADH) isozymes was studied in liver, kidney, stomach and eye tissues of C57BL/6J inbred male mice using agarose isoelectric focusing and histochemical methods. Development profiles were tissue specific, with adult patterns being attained by 30 days in the liver and stomach, and by 42 days in the kidneys. Ocular ADH-C2 increased in activity at the end of the first week, corresponding to the time of eye opening in the neonate.

Age Factors↗
PubMed1991

Aldehyde dehydrogenase (ALDH) isozymes in the gray short-tailed opossum (Monodelphis domestica): tissue and subcellular distribution and biochemical genetics of ALDH3.

Polyacrylamide gel isoelectric focusing (PAGE-IEF), cellulose acetate electrophoresis, and histochemical techniques were used to examine the tissue and subcellular distribution, genetics and biochemical properties of aldehyde dehydrogenase (ALDH) isozymes in a didelphid marsupial, the gray short-tail opossum (Monodelphis domestica). At least 14 zones of activity were resolved by PAGE-IEF and divided into five isozyme groups and three ALDH classes, based upon comparisons with properties previously reported for human, baboon, rat, and mouse ALDHs. Opossum liver ALDHs were distributed among cytosol (ALDHs 1 and 5) and large granular (mitochondrial) fractions (ALDHs 2 and 5). Similarly, kidney ALDHs were distributed between the cytosol (ALDH5) and the mitochondrial fractions (ALDHs 2, 4, and 5), whereas a major isozyme (ALDH3), found in high activity in cornea, esophagus, ear pinna, tail, and stomach extracts, was localized predominantly in the cytosol fraction. Phenotypic variants of the latter enzyme were shown to be inherited in a normal Mendelian fashion, with two alleles at a single locus (ALDH3) showing codominant expression. The data provided evidence for genetic identity of corneal, ear pinna, tail, and stomach ALDH3 and supported biochemical evidence from other mammalian species that this enzyme has a dimeric subunit structure.

Aldehyde Dehydrogenase↗
PubMed1990

Relationships between resistance to cross-linking agents and glutathione metabolism, aldehyde dehydrogenase isozymes and adenovirus replication in human tumour cell lines.

In a panel of 10 human tumour cell lines with no prior exposure to drugs in vitro, resistance to cisplatin correlated with resistance to the nitrogen mustard derivatives Asta Z-7557 (mafosfamide, an activated form of cyclophosphamide), melphalan and chlorambucil. Simultaneous treatment with DL-buthionine-S,R-sulfoximine did not enhance the toxicity of cisplatin or Asta Z-7557, and no correlation was found between drug resistance and cellular levels of metallothioneins (as judged by sensitivity to cadmium chloride), glutathione (GSH), GSH reductase, GSH transferase, or gamma-glutamyltranspeptidase. The two cell lines most resistant to Asta Z-7557 expressed aldehyde dehydrogenase cytosolic isozyme 1, found also in normal ovary, but not isozyme 3. Treatment of resistant cells with cisplatin or Asta Z-7557 inhibited cellular DNA synthesis and replication of adenovirus 5 to a lesser extent than in sensitive cells. The virus could be directly inactivated by both drugs prior to infection, subsequent replication being inhibited to the same extent in sensitive and resistant cells. In contrast to Asta Z-7557 and other DNA damaging agents, cisplatin was much more toxic to adenovirus (D37 0.022-0.048 microM) than to cells (D37 0.25-2.5 microM). The adenovirus 5 mutant Ad5ts125 having a G----A substitution was even more sensitive to cisplatin (D37 7-8 nM) than wild type virus and another mutant. Cisplatin was detoxified less by sonicated resistant resistant cells than sensitive cells, as judged by inactivation of Ad5ts125 added to the reaction mixture. It can be inferred that (i) the major differences in cellular resistance to cisplatin and Asta Z-7557 in the present material did not involve enhanced DNA repair or protection by metallothioneins or GSH, but were associated with the ability to continue cellular and viral DNA synthesis during treatment, (ii) resistance was not associated with less template damage, and (iii) the adenovirus genome may be a suitable probe for predicting tumour resistance to cisplatin and for elucidating the DNA sequence dependence of cisplatin toxicity.

Adenoviruses, Human↗
PubMed1990

Bovine corneal aldehyde dehydrogenase: the major soluble corneal protein with a possible dual protective role for the eye.

Bovine corneal aldehyde dehydrogenase was purified to homogeneity and characterized with aldehyde substrates at pH 7.4. The enzyme was a dimer with a subunit size of 65 kDa. Using kcat/Km values as an indication of substrate efficacy, aldehyde products of lipid peroxidation were recognized as the likely 'natural' substrates. Protein yields from enzyme purification, as well as electrophoretic analyses of crude and purified enzyme preparations, demonstrated that this enzyme is the major soluble protein in bovine cornea, and constitutes around 0.5% wet weight of tissue. A dual role in protecting the eye against UV-B light is proposed--oxidation of aldehydes generated by light induced lipid peroxidation, and the direct absorption of UV-B light by bovine corneal ALDH.

Aldehyde Dehydrogenase↗
PubMed1990

Baboon alcohol dehydrogenase isozymes: purification and properties of liver class I ADH. Moderate alcohol consumption reduces liver class I and class II ADH activities.

The major baboon liver isozyme of alcohol dehydrogenase (ADH-2) has been purified to homogeneity by affinity chromatography, and characterized as a "typical" Class I mammalian ADH isozyme. In contrast to human liver, which possesses three genetic isozymes (alpha 2, beta 2, and gamma 2) and three hybrid isozymes (alpha beta, alpha gamma, and beta gamma) of Class I ADH, baboon liver exhibits activity of a single major Class I isozyme (beta 2). Kinetic analyses, using alcohol substrates of differing chain lengths, and inhibition with pyrazole, support this classification. Agarose-IEF analyses, substrate specificity studies and immunochemical titrations of the major kidney ADH (ADH-1) also support the occurrence of a second Class I isozyme. The baboon was used as a model to study alcohol-induced changes in liver ADH phenotype following moderate and moderately high alcohol consumption. Four male and four female prepubertal baboons were fed nutritionally adequate liquid diets over a 40-week period, including control diets (weeks 1-8, 17-24, 32-40), moderate (12.5 percent of calories) alcohol diet (weeks 9-16), and a moderately high (25 percent of calories) alcohol diet (weeks 25-32). Liver ADH isozyme patterns and Class I ADH activities from biopsy samples, taken every 4 weeks, were monitored during the feeding study. Decreases in both Class I and Class II ADH activities were indicated in most animals, which may reflect adaptive mechanisms in the liver to continuing alcohol metabolism. Moreover, reversible changes were observed, with trends toward a recovery of ADH isozyme activities following return to the control liquid diets. These studies also have particular significance for human biomedical research work on alcohol, since most of the "drinking" population in typical drinking communities are moderate consumers of alcohol. This work, using the baboon as an animal model, has clearly indicated that alcohol consumption at such moderate levels, brings with it adaptations in the level of ADH isozymes in liver, which may play a protective role.

Alcohol Dehydrogenase↗
PubMed1989

Purification and properties of mouse stomach aldehyde dehydrogenase. Evidence for a role in the oxidation of peroxidic and aromatic aldehydes.

The major isozyme of aldehyde dehydrogenase in mouse stomach, AHD-4, has been purified to homogeneity and characterized with a range of aldehyde substrates at pH 7.4. The enzyme was a dimer with a subunit size of 65 kDa. Using V/Km values as an indication of substrate efficacy, aromatic aldehydes were the preferred substrates. The enzyme used either NAD+ or NADP+ as cofactor, but showed a preference for NAD+. AHD-4 showed 'high-Km' properties with respect to acetaldehyde, but differed from the 'high-Km' liver mitochondrial enzyme (AHD-1), in that it was not a semialdehyde dehydrogenase. The enzyme was significantly active towards the peroxidic aldehyde, 4-hydroxynonenal, and may play a role in vivo in the detoxification of aromatic aldehydes and the aldehyde products of lipid peroxidation.

Acetaldehyde↗
PubMed1989

Isoelectric focusing studies of aldehyde dehydrogenases, alcohol dehydrogenases and oxidases from mammalian anterior eye tissues.

1. Isoelectric focusing (IEF) and zymogram methods were used to examine the tissue distribution, multiplicity and substrate specificities of alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) and ocular oxidases (EOXs) from mammalian anterior eye tissues. 2. Baboon, cattle, pig and sheep corneal extracts exhibited high ALDH activities; the corneal ALDHs were distinct from the major liver ALDHs and distinguished by their preference for medium-chain aldehydes. 3. Baboon and pig corneal extracts also showed high ADH activities, by comparison with ovine and bovine samples. Moreover, the ADHs were distinct from the major liver isozymes in pI value and substrate specificity. 4. Mammalian lens extracts exhibited significant ALDH activity of a form corresponding to the major liver cytosolic isozyme. Minor activity of the corneal enzyme was also observed in some species. 5. Lens ADH phenotypes were species-specific, and consisted of either Class II activity (baboon and sheep), Class III ADH activity (pig), or activities of both ADH classes (cattle). 6. Lens extracts also exhibited a complex pattern of ocular oxidase (EOX) activities following IEF. 7. A role in peroxidatic aldehyde detoxification is proposed for these enzymes in anterior eye tissues.

Alcohol Oxidoreductases↗
PubMed1989

Cloning and sequencing of cDNA encoding baboon liver alcohol dehydrogenase: evidence for a common ancestral lineage with the human alcohol dehydrogenase beta subunit and for class I ADH gene duplications predating primate radiation.

The baboon has at least five alcohol dehydrogenases (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and has distinct liver and kidney class I isozymes. A rat liver class I ADH partial cDNA was used to screen a baboon liver cDNA library. A cDNA clone was isolated and sequenced and found to contain the entire coding region for baboon liver ADH, 12 nucleotides of the 5' noncoding region, and 256 nucleotides of the 3' noncoding region. The amino acid sequence deduced from this cDNA most closely resembles that of human liver ADH beta subunit (ADH-beta): 363 of 374 residues were identical. This suggested that baboon liver class I ADH is of the same ancestral lineage as the human ADH-beta. In contrast to human liver, only a single ADH-beta transcript is observed in baboon liver. A comparison of human and baboon ADH 3' noncoding regions suggests that a single nucleotide change in a polyadenylylation signal consensus sequence may, in part, be responsible for the generation of ADH-beta transcripts with variable-length 3' ends in human liver. A nucleotide substitution rate of 0.5 x 10(-9) substitutions per site per year for primate class I ADH genes was deduced from the data, which suggests that the alpha-beta gamma separation of human ADH genes occurred about 60 million years ago, and that primate class I ADH gene duplications predated primate radiation.

Alcohol Dehydrogenase↗
PubMed1988

Genetics of ocular NAD+-dependent alcohol dehydrogenase and aldehyde dehydrogenase in the mouse: evidence for genetic identity with stomach isozymes and localization of Ahd-4 on chromosome 11 near trembler.

Electrophoretic and activity variation of the stomach and ocular isozyme of aldehyde dehydrogenase (designated AHD-4) was observed between C57BL/6J and SWR/J inbred strains of mice. The phenotypes were inherited in a normal mendelian fashion, with two alleles at a single locus (Ahd-4) showing codominant expression. The alleles assorted independently of those at Adh-3 [encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C2)] on chromosome 3. Three chromosome 11 markers, hemoglobin alpha-chain (Hba), trembler (Tr), and rex (Re), were used in backcross analyses which established that Ahd-4 is closely linked to trembler. The distribution patterns for stomach and ocular AHD-4 phenotypes were examined among SWXL recombinant inbred mice, and those for stomach and ocular ADH-C2 among BXD recombinant inbred strains. The data provided evidence for the genetic identity of stomach and ocular ADH-C2 and of stomach and ocular AHD-4.

Alcohol Dehydrogenase↗
PubMed1988

Genetics and development of ocular oxidases in the mouse: evidence for a new locus (Eox-1) closely linked with the aldehyde oxidase loci on chromosome 1.

Isoelectric focusing (IEF) and histochemical techniques were used to examine the genetics, postnatal development and biochemical properties of ocular oxidases (EOXs) among inbred strains of mice. The designation as EOX was made on a provisional basis, since the 'natural' substrate(s) for this enzyme have not been identified. Five major forms were resolved from adult animals, which exhibited high activity in murine lens and low activity in the cornea. An additional ocular oxidase was observed in neonatal animals. Genetic analyses demonstrated that one of these enzymes (EOX-1) is encoded by a locus (Eox-1) which is closely linked with, but distinct from, the aldehyde oxidase (Aox) gene complex on chromosome one of the mouse. These results support the proposal that ocular oxidases are distinct from the major liver AOXs in this organism.

Aldehyde Oxidase↗
PubMed1987

Developmental changes in aldehyde dehydrogenases from mouse tissues.

The postnatal development of aldehyde dehydrogenase (AHD) isozymes from C57BL/6J mouse tissues was examined using agarose-IEF zymogram methods. Mitochondrial isozymes (AHD-1 and AHD-5) were present throughout, increasing to high levels in liver, kidney and stomach by weaning (3 weeks). These activities remained high subsequently, except for kidney AHD-5, which decreased significantly after week 4. The appearance of the cytosolic isozymes was tissue specific and time dependent: liver AHD-2 was undetected until day 21, and increased subsequently; stomach AHD-4 was first observed at day 5, increasing to adult levels by day 21; AHD-6 was active in neonatal kidney and stomach extracts, but was undetected after day 8; and AHD-7 was observed in liver and kidney extracts from day 16. These results supported previous proposals for multiple genes encoding aldehyde dehydrogenases in the mouse, based upon the distinct developmental profiles for the liver, kidney and stomach isozymes.

Aging↗
PubMed1987

Lung carbonyl reductase in the mouse: biochemical and catalytic properties.

Two major forms of aldehyde reductase (AHR) activity were resolved following zone electrophoresis of mouse lung homogenates and distinguished by their differential substrate and inhibitor specificities: alcohol dehydrogenase (ADH) C2 and carbonyl reductase (CBR). CBR was purified to homogeneity by DEAE-cellulose chromatography, affinity chromatography using Blue-sepharose, followed by gel filtration on Sephacryl S-200. The enzyme exhibited a native MW of 122,000, comprising 4 identical subunits. Kinetic and inhibition characteristics resembled those reported by Nakayama and coworkers (1982) for guinea pig lung CBR. Mouse lung CBR exhibited optimal activity at pH 5.0; a preference for NADPH as coenzyme, although reactive with NADH at an order of magnitude higher concentration; poor activity as an ADH, but was strongly inhibited by 4-methyl pyrazole; and was inhibited by quercitin, dithiothreitol and p-OH-mercuribenzoate, but was insensitive to valproate or sorbinil. These properties, coupled with the activity of CBR with a range of aliphatic and aromatic aldehydes, ketones and quinones, distinguish it from other AHRs. The unique localization in lung tissue suggests a possible role for CBR in the detoxification of xenobiotics and of toxic aldehydes derived from lipid peroxidation processes.

Alcohol Oxidoreductases↗
PubMed1987

Genetics of stomach and ocular alcohol dehydrogenase and aldehyde dehydrogenase in the mouse.

Activity variants of the stomach and ocular isozyme of aldehyde dehydrogenase (AHD-4) were observed among inbred strains of mice. The phenotypes were inherited in a normal mendelian fashion, with two alleles showing codominant expression at a single locus (Ahd-4). Linkage data indicated that Ahd-4 is localized on chromosome 11 near Hba (alpha hemoglobin locus), and segregated independently of Adh-3, encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C2).

Alcohol Dehydrogenase↗
PubMed1987

Baboon alcohol dehydrogenase isozymes: phenotypic changes in liver following chronic consumption of alcohol.

According to the nomenclature of Vallee and Bazzone [1983] for mammalian alcohol dehydrogenase (ADH) isozymes, baboon ADHs comprise three major classes of activity, which were distinguished according to the following properties: Class I ADHs. These isozymes exhibited low-Km characteristics with ethanol as substrate, high isoelectric points (8.5-9.3), and sensitivity to 5 mM 4-methyl pyrazole inhibition, and were the major liver (ADH-2) and kidney (ADH-1) isozymes in the baboon. Class II ADHs. These isozymes showed high-Km values for ethanol, neutral isoelectric points (7.7 for the liver ADH-4 [pi-ADH] and 7.2 for the major stomach ADH [ADH-3], respectively), and were insensitive to inhibition with 5 mM 4-methyl pyrazole. Class III ADH. This enzyme was characterized by its inactivity with ethanol as substrate (up to 0.5 M), insensitivity to 4-methyl pyrazole inhibition, preference for medium-chain-length alcohols as substrate (trans-2-hexen-1-ol was routinely used in this study), and an isoelectric point (6.5) similar to that of the human liver chi-ADH (pI 6.4). Major activity variation of the liver pi-ADH (ADH-4) isozyme was observed among the 114 liver samples examined, with 34 percent exhibiting a null (or low-activity) phenotype. An electrophoretic variant phenotype for the major class II stomach isozyme (ADH-3) was also found in the population studied. The baboon was used as a model for studying alcohol-induced changes in liver ADH phenotype following chronic alcohol consumption. Prepuberal male baboons were pair-fed nutritionally adequate liquid diets containing ethanol (50 percent of calories) or isocaloric carbohydrates, and liver ADH isozyme patterns from biopsy samples were monitored for 20 weeks. Dramatic decreases in class II liver ADH activity (ADH-4, or pi-ADH) were observed within 4 weeks after the start of alcohol feeding, and a shift in liver class I isozymes was found during the later stages of alcohol consumption. These changes during chronic alcohol consumption may be adaptations of the liver: these include reduced capacity of the major ADH pathway and increased ethanol oxidation by the microsomal ethanol oxidizing system and possibly by peroxisomal catalase.

Alcohol Dehydrogenase↗