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M D Boleda

Publications and source records attributed to M D Boleda.

10 recordsLinked to original sources
PubMed1996

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).

DNA↗
PubMed1993

Physiological substrates for rat alcohol dehydrogenase classes: aldehydes of lipid peroxidation, omega-hydroxyfatty acids, and retinoids.

Alcohol dehydrogenase classes exhibit important differences in both substrate specificity and tissue distribution which suggest distinct physiological functions. We have studied the kinetic constants at pH 7.5 of the rat alcohol dehydrogenase classes, purified from liver (classes I and III) and from stomach (class IV), with three groups of relevant physiological compounds: cytotoxic aldehydes generated in lipid peroxidation, omega-hydroxyfatty acids, and retinoids. Classes I and IV actively reduce 4-hydroxynonenal, 2-hexenal, and hexanal, which are toxic compounds known to be produced in significant amounts during lipid peroxidation. Class III shows poor activity with these aldehydes. Class IV exhibits the best kcat/Km values (2150 mM-1 x min-1 for 4-hydroxynonenal), which suggest a role for this enzyme in the elimination of the cytotoxic aldehydes in tissues that are susceptible to lipid peroxidation, such as skin, cornea, and mucosa of the respiratory and digestive tracts, where class IV is localized. The three classes are very active with omega-hydroxyfatty acids, suggesting that all of them are involved in the physiological oxidation of these compounds in the rat tissues. The kinetic constants support that oxidation of omega-hydroxyfatty acids is a physiological function for class III, in addition to its role as formaldehyde dehydrogenase. Finally, classes I and IV are active in retinol oxidation and retinal reduction. Class IV may play a crucial role in the generation of retinoic acid in epithelia, where this compound is involved in development and cell differentiation. In conclusion, alcohol dehydrogenase is an enzyme with multiple metabolic roles, and the different substrate specificity and tissue localization for each class provide organs and tissues with distinct physiological functions.

Alcohol Dehydrogenase↗
PubMed1992

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.

Alcohol Dehydrogenase↗
PubMed1992

Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus.

Alcohol dehydrogenase (ADH), the major enzyme catalyzing the biological oxidation of ethanol in mammals, includes four classes with very different capacities for ethanol oxidation. Class III ADH is present in all the tissues and is well conserved throughout evolution. This enzyme has a low activity with ethanol, is specific for the glutathione-dependent oxidation of formaldehyde, and is therefore a formaldehyde dehydrogenase (FALDH). Until now there have been few and conflicting studies concerning its intracellular distribution, which is important for the understanding of its role in cell function. In the present work we used biochemical and immunocytochemical methods to assess the distribution of FALDH in rat hepatocytes and astroglial cells. With the glutathione-dependent formaldehyde dehydrogenase assay, we found the highest activity in the cytosol of hepatocytes and brain cells (12 and 2.6 mU/mg protein, respectively), but nuclei also exhibited significant activity (1.16 and 2.1 mU/mg protein, respectively). The immunocytochemical results showed the presence of FALDH binding sites in both the cytoplasm and the nucleus of the different cell types studied. Whereas no specific gold particle labeling was seen associated with any cytoplasmic component, in the nucleus the particles were found mainly over condensed chromatin and interchromatin regions. Finally, the gold particle density over both the nucleus and cytoplasm was greater in differentiated than in proliferating astrocytes in primary culture. In contrast, class I ADH, primarily responsible for ethanol metabolism, was found only in the cytoplasm of hepatocytes. We propose that one of the functions of FALDH is to protect cell structures, including DNA, from the toxic effects of endogenous formaldehyde, which is an intermediate in many metabolic process.

Aldehyde Oxidoreductases↗
PubMed1990

RNP, Sm and SS-B antigens from calf thymus: molecular stability upon enzymatic digestion.

RNP, Sm and SS-B nuclear antigens from calf thymus were studied with respect to the size distribution on sucrose gradients as well as to the molecular integrity and related structural changes when they were subjected to enzymatic digestions under different conditions. Making a difference with RNP particles, the Sm size distribution is concentration dependent, a property in accordance with the complexity of the Sm particles in comparison with the RNPs. The use of combined effects of temperature, endogenous proteases and RNase A, allowed us to gain insight into the limits of stability of the three antigenic particles. Following treatments in the absence of RNAse A, the degradation products (32-38 Kd molecular weight) of the 70 Kd RNP polypeptide remain stable and associated with other molecules within the RNP particle. It was also found that the phosphate groups of the SS-B protein moiety are only accessible to alkaline phosphatase if the RNA of the SS-B particle is degraded by the action of RNAse A.

Animals↗
PubMed1989

Role of extrahepatic alcohol dehydrogenase in rat ethanol metabolism.

Rat alcohol dehydrogenase exhibits three isoenzymes with very different capacities of ethanol oxidation and with characteristic distribution in tissues. ADH-1 (class II isoenzyme, Km = 5 M) is especially concentrated in the most external organs: auditive, bucal, and nasal mucoses, cornea, esophagus, stomach, rectum, penis, and vagina. ADH-2 (class III isoenzyme) is present in all organs but has a poor activity with ethanol. ADH-3 (class I isoenzyme, Km = 1.4 mM) is the main liver isoenzyme, also present in lung, intestine, kidney, and sexual organs. At 33 mM ethanol and pH 7.5, total hepatic activity (3.5 +/- 0.6 units) represents 90% of the whole activity in the male rat, while the remaining 10% is distributed in many organs. The skin is the extrahepatic organ with the highest total activity (88 +/- 15 mU) followed by testis and small intestine. ADH-3 accounts for 96% of total activity (90% hepatic and 6% extrahepatic) and ADH-1 contributes with 4% (extrahepatic). However, in conditions that may be found in the digestive tract mucose after ethanol ingestion (pH 7.5, 1 M ethanol), stomach and small intestine activities represent 10% of the liver activity at 33 mM ethanol. Therefore, oral administration of ethanol will result in a higher contribution of the extrahepatic activity than will intravenous or intraperitoneal administration, because of the great ADH-1 content of the digestive tract. On the other hand, pyrazole inhibition constants at pH 7.5 for ADH-1 (33 mM) and ADH-3 (4.2 microM) are much higher than those at pH 10.0 (0.56 mM and 0.4 microM) and indicate that at the usual concentration of inhibitor only ADH-3 activity will be effectively suppressed. ADH-1 will be, therefore, responsible in part for the residual ethanol oxidation activity in pyrazole-treated rats.

Alcohol Dehydrogenase↗
PubMed1987

Mammalian alcohol dehydrogenase: characteristics of class III isoenzymes.

The kinetic characteristics of alcohol dehydrogenase class III have been studied using class III isoenzymes purified from human liver (X-ADH) and rat liver (ADH-2). Our results confirm that long chain primary alcohols and aldehydes are the best substrates, although some aromatic compounds can also be actively metabolized. Kinetic analysis suggests an ordered bibi mechanism for X-ADH. Ethanol can be oxidized by class III isoenzymes at high substrate concentration, but with a very slow rate. Thus, their contribution to physiological ethanol elimination is probably insignificant. The general properties of the class III isoenzymes isolated from different mammals by ourselves and other authors are discussed.

Alcohol Dehydrogenase↗