Medium- and short-chain dehydrogenase/reductase gene and protein families : Medium-chain and short-chain dehydrogenases/reductases in retinoid metabolism.

Retinoic acid (RA), the most active retinoid, is synthesized in two steps from retinol. The first step, oxidation of retinol to retinaldehyde, is catalyzed by cytosolic alcohol dehydrogenases (ADHs) of the medium-chain dehydrogenase/reductase (MDR) superfamily and microsomal retinol dehydrogenases (RDHs) of the short-chain dehydrogenase/reductase (SDR) superfamily. The second step, oxidation of retinaldehyde to RA, is catalyzed by several aldehyde dehydrogenases. ADH1 and ADH2 are the major MDR enzymes in liver retinol detoxification, while ADH3 (less active) and ADH4 (most active) participate in RA generation in tissues. Several NAD(+)- and NADP(+)-dependent SDRs are retinoid active. Their in vivo contribution has been demonstrated in the visual cycle (RDH5, RDH12), adult retinoid homeostasis (RDH1) and embryogenesis (RDH10). K(m) values for most retinoid-active ADHs and RDHs are close to 1 microM or lower, suggesting that they participate physiologically in retinol/retinaldehyde interconversion. Probably none of these enzymes uses retinoids bound to cellular retinol-binding protein, but only free retinoids. The large number of enzymes involved in the two directions of this step, also including aldo-keto reductases, suggests that retinaldehyde levels are strictly regulated.

Functional impact of treatment with ranibizumab under a reactive strategy in patients with neovascular age-related macular degeneration. / Impacto funcional del tratamiento con ranibizumab bajo una estrategia reactiva en pacientes con degeneración macular asociada a la edad exudativa neovascular.

OBJECTIVE: To analyse the functional recovery using a pro re nata (PRN) dosing strategy with intravitreal injections of ranibizumab for patients with neovascular age-related macular degeneration (AMD). MATERIAL AND METHODS: An observational, retrospective, single-centre study, was conducted on patients with neovascular AMD managed with a PRN strategy with ranibizumab, and were followed-up for a minimum of 18 months. Sociodemographic and clinical data were collected from medical records. The percentage of visual acuity (VA) recovered after losing 5 or more letters was calculated taking into account the previous visit, as well as considering the best VA recorded prior to the retreament. RESULTS: The analysis included 128 patients. The mean (SD) follow-up period was 18.9 (2.3) months. The mean (SD) elapsed days between onset of symptoms and diagnosis, and between prescription and administration of treatment was 50.2 (57.4) and 10.9 (16.0), respectively. Only 108 patients were prescribed ranibizumab after losing 5 or more letters of VA. The mean (SD) VA recovery compared to the previous VA was 70.3% (114.4). On the other hand, the mean (SD) VA recovery when considering the best VA registered before the retreatment was 43.5% (112.9), with 59.4% of re-treatments having a VA recovery below 75%, and with 11.7% not presenting any VA recovery. CONCLUSIONS: A PRN dosing strategy with intravitreal ranibizumab for neovascular AMD may not be efficient in preserving and/or recovering VA in the long-term, due to a cumulative irreversible VA loss.

PARP-2 sustains erythropoiesis in mice by limiting replicative stress in erythroid progenitors.

Erythropoiesis is a tightly regulated process in which multipotential hematopoietic stem cells produce mature red blood cells. Here we show that deletion of poly(ADP-ribose) polymerase-2 (PARP-2) in mice leads to chronic anemia at steady state, despite increased erythropoietin plasma levels, a phenomenon not observed in mice lacking PARP-1. Loss of PARP-2 causes shortened lifespan of erythrocytes and impaired differentiation of erythroid progenitors. In erythroblasts, PARP-2 deficiency triggers replicative stress, as indicated by the presence of micronuclei, the accumulation of γ-H2AX (phospho-histone H2AX) in S-phase cells and constitutive CHK1 and replication protein A phosphorylation. Transcriptome analyses revealed the activation of the p53-dependent DNA-damage response pathways in PARP-2-deficient cells, culminating in the upregulation of cell-cycle and cell death regulators, concomitant with G2/M arrest and apoptosis. Strikingly, while loss of the proapoptotic p53 target gene Puma restored hematocrit levels in the PARP-2-deficient mice, loss of the cell-cycle regulator and CDK inhibitor p21 leads to perinatal death by exacerbating impaired fetal liver erythropoiesis in PARP-2-deficient embryos. Although the anemia displayed by PARP-2-deficient mice is compatible with life, mice die rapidly when exposed to stress-induced enhanced hemolysis. Our results pinpoint an essential role for PARP-2 in erythropoiesis by limiting replicative stress that becomes essential in the absence of p21 and in the context of enhanced hemolysis, highlighting the potential effect that might arise from the design and use of PARP inhibitors that specifically inactivate PARP proteins.

Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco.

A synthetic gene based on the primary sequence of the mature spruce budworm antifreeze protein (sbwAFP) was constructed by primer overlap extension. The amino acid codons were chosen to mimic those of a highly expressed tobacco nuclear gene. A DNA sequence encoding the amino-terminal leader sequence from the tobacco pathogen related protein 1b (PR), which targets the protein to the apoplastic space, was fused in frame to the synthetic sbwAFP gene. This fusion was placed downstream of the cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator in a T-DNA binary vector. Transgenic tobacco lines transcribing PR-sbwAFP were selected by RT-PCR. The apoplastic protein fractions of sbwAFP expressing tobacco lines exhibited enhanced antifreeze activity as demonstrated by the ability to inhibit ice re-crystallization and increased thermal hysteresis.

Molecular modelling of human gastric alcohol dehydrogenase (class IV) and substrate docking: differences towards the classical liver enzyme (class I).

A three-dimensional model of the human class IV alcohol dehydrogenase has been calculated based upon the X-ray structure of the class I enzyme. As judged from the model, the substrate-binding site is wider than in class I, compatible with the differences in substrate specificities and the large difference in Km value for ethanol. Substrate docking performed for the class I structure and the class IV model show all-trans-retinol and 11-cis-retinol to bind better to the class IV enzyme. The calculations also indicate that 16-hydroxyhexadecanoic acid binds in a different manner for the two enzyme classes. A simulation of coenzyme-binding indicates that the adenine ring of the coenzyme might be differently bound in class IV than in class I, decreasing the interactions with Asp-223 which is compatible with the higher k(cat) values for class IV.

Retinoids, omega-hydroxyfatty acids and cytotoxic aldehydes as physiological substrates, and H2-receptor antagonists as pharmacological inhibitors, of human class IV alcohol dehydrogenase.

Kinetic constants of human class IV alcohol dehydrogenase (sigmasigma-ADH) support a role of the enzyme in retinoid metabolism, fatty acid omega-oxidation, and elimination of cytotoxic aldehydes produced by lipid peroxidation. Class IV is the human ADH form most efficient in the reduction of 4-hydroxynonenal (k(cat)/Km: 39,500 mM(-1) min(-1)). Class IV shows high activity with all-trans-retinol and 9-cis-retinol, while 13-cis-retinol is not a substrate but an inhibitor. Both all-trans-retinoic and 13-cis-retinoic acids are potent competitive inhibitors of retinol oxidation (Ki: 3-10 microM) which can be a basis for the regulation of the retinoic acid generation and of the pharmacological actions of the 13-cis-isomer. The inhibition of class IV retinol oxidation by ethanol (Ki: 6-10 mM) may be the origin of toxic and teratogenic effects of ethanol. H2-receptor antagonists are poor inhibitors of human and rat classes I and IV (Ki > 0.3 mM) suggesting a small interference in ethanol metabolism at the pharmacological doses of these common drugs.

Alcohol dehydrogenase of human and rat blood vessels. Role in ethanol metabolism.

Alcohol dehydrogenase (ADH) activity has been detected in all arteries and veins examined from humans and rat. In distinct human autopsy vessels, activity values range from 0.9 +/- 0.2 to 9.9 +/- 7.7 mU/mg. Distribution of the activity in human aorta was: intima (23.5%), media (74%) and adventia (2.5%). In most of the samples the beta1 beta1 isozyme of class I ADH was the only form responsible for the ADH activity. Class IV ADH (sigma sigma-ADH) was present in three of the 28 individuals examined. The rat blood vessels showed class IV, but not class I, ADH localized in endothelium and media. The physiological role of vascular ADH is probably related to retinoid metabolism and elimination of lipid peroxidation aldehydes. A contribution to human ethanol metabolism is supported by the significant amount of low-Km activity and the extension of the vascular system.

DNA footprint enhancement using tandem binding sites.

A concatenated DNA fragment containing a five-repeat binding site was used for DNase I footprinting. Under the same conditions, the tandem repeat assay greatly enhanced the DNA footprint as compared with a native DNA sequence with only one binding site. This technique provides an approach for improving poor DNA footprints.

Experimental design: a useful tool for PCR Optimization.

Because of complex interactions among the components of PCR and the wide and increasing variety of applications in which this technique is used, optimization is necessary for every reaction. Here we describe the use of experimental design techniques (2k fractional factorial design and central composite design) to attain easier, quicker and cheaper PCR optimization of DNA from blood spots. By determining the factors affecting the product yield first (factors screening), the quantity of template DNA needed for PCR and the Mg2+ concentration are easily optimized (factors optimization).

Alcohol dehydrogenase isoenzymes in rat development. Effect of maternal ethanol consumption.

The alcohol dehydrogenase (ADH) isoenzymes (alcohol:NAD oxidoreductase, EC 1.1.1.1) of classes I, III and IV were investigated by activity and starch gel electrophoresis analyses during rat ontogeny. Class I was studied in the liver, class III in the brain and class IV in the stomach and eyes. Classes I and IV exhibited very low activity during the fetal period, reaching 12% and 3%, respectively, of the adult value at birth. Class III was relatively more active in the fetus, with 38% of the adult activity at birth. In the three cases, activity increased after birth and adult values were found around day 20 (classes I and III), day 39 (stomach class IV) and after day 91 (eye class IV). The very low activity of the isoenzymes responsible for ethanol oxidation, i.e. liver class I and stomach class IV, in the fetus demonstrates that metabolism of ethanol during gestation is essentially performed by the maternal tissues. Development of ADH isoenzymes were also studied in the offspring of rats exposed to an alcoholic liquid diet. Activities of liver class I and stomach class IV were severely reduced: they were only 30% and 50%, respectively, of the control values. In contrast, eye class IV activity did not change and brain class III showed a 30% increase. Moreover, the concentration of liver soluble protein exhibited a 1.3-1.5-fold increase with respect to control animals. The effects on activities and liver protein were more pronounced in the adult than in the perinatal period, and they seem irreversible since normal values were not recovered after 6 weeks of feeding with a non-alcoholic diet. The low activities of the alcohol-oxidizing isoenzymes indicate tht maternal ethanol consumption results in an impaired ethanol metabolism of the offspring.

Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies.

Kinetic effects of a single-amino acid mutation in a highly variable loop (residues 114-120) of class IV ADH.

Class IV alcohol dehydrogenase shows a deletion at position 117 with respect to class I enzymes, which typically have a Gly residue. In class I structures, Gly117 is part of a loop (residues 114-120) that is highly variable within the alcohol dehydrogenase family. A mutant human class IV enzyme was engineered in which a Gly residue was inserted at position 117 (G117ins). Its kinetic properties, regarding ethanol and primary aliphatic alcohols, secondary alcohols and pH profiles, were determined and compared with the results obtained in previous studies in which the size of the 114-120 loop was modified. For the enzymes considered, a smaller loop was associated with a lower catalytic efficiency towards short-chain alcohols (ethanol and propanol) and secondary alcohols, as well as with a higher K(m) for ethanol at pH 7.5 than at pH 10.0. The effect can be rationalized in terms of a more open, solvent-accessible active site in class IV alcohol dehydrogenase, which disfavors productive binding of ethanol and short-chain alcohols, specially at physiological pH.

Properties of rat retina alcohol dehydrogenase.

The rat eye fraction, including retina, pigment epithelium and choroid, contains an alcohol dehydrogenase (ADH) isoenzyme that is not present in rat liver. Starch gel electrophoresis of retina ADH shows an anodic band that can be visualized by activity staining, using either ethanol or pentanol as substrates. Ethanol is a poor substrate (Km: 336 mM, at pH 10.0) for the purified retina ADH which prefers long chain, 2-unsaturated and aromatic alcohols. The enzyme has a pH optimum of 10.0 for ethanol oxidation and it is inhibited by 4-methylpyrazole (KI: 10 microM). Electrophoretic and kinetic properties clearly differentiate the retina ADH from the hepatic cathodic ADH isoenzymes and from an anodic chi-ADH-like form that we have also detected in rat liver. At the pH and ethanol concentrations found "in vivo," retina ADH can oxidize ethanol to an appreciable extent. The subsequent production of acetaldehyde and redox change may be responsible for visual disorders during alcohol intoxication.

Site directed mutagenesis to probe for active site components of liver mitochondrial aldehyde dehydrogenase.

Mutational analysis allowed us to rule out an essential role for the histidine residues and for serine 74 in mammalian aldehyde dehydrogenase. The later though, was found to be important in coenzyme interaction. The function of the serine could not be replaced by threonine or by cysteine. The absolute requirement for cysteine 302 and for glutamate 268 was verified using mutational analysis. The fact that these two residues are completed conserved among all aldehyde dehydrogenases is consistent with their being essential in the catalytic process.