STUDIES ON THE PROPERTIES OF RETINAL ALCOHOL DEHYDROGENASE FROM THE RAT

An NAD-dependent alcohol dehydrogenase (alcohol:NAD oxidoreductase; EC 1.1.1.1) has been isolated and partially purified from the retinal cytosol of the rat. Its substrate specificity and sensitivity to inhibitors of hepatic alcohol dehydrogenase have been investigated. Ethanol, 1-propanol and 1-butanol served as substrates for this enzyme but the K m values were more than 100-fold higher than those reported for hepatic alcohol dehydrogenase. Methanol and retinol were unreactive with this alcohol dehydrogenase. Inhibition by pyrazole was observed but the K t was about 100-fold higher than the value observed for hepatic alcohol dehydrogenase. n -Butyraldoxime inhibited retinal alcohol dehydrogenase with a K t of 2 ΜM, a value which approximates its K t for hepatic alcohol dehydrogenase. 1, 10-Phenanthroline was ineffective as an inhibitor. Oxidation of retinol was observed in retinal homogenates in the presence of NADP but no inhibition was observed with ethanol, methanol or pyrazole. We conclude that oxidation of retinol is not catalysed by soluble retinal alcohol dehydrogenase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65178/1/j.1471-4159.1971.tb00195.x.pd

A comparison of rat and human liver formaldehyde dehydrogenase

- An NAD- and GSH-dependent formaldehyde dehydrogenase (formaldehyde: NAD+ oxidoreductase, EC 1.2.1.1) was purified from rat and human liver, and the properties of these enzymes were compared. The GSH requirement of the enzyme obtained from both species could not be replaced by dithiothreitol, CoA or cysteine, and NADP could not substitute for NAD. The pH optimum, and the Km of formaldehyde and NAD+, were similar for both rat and human liver formaldehyde dehydrogenase. By employing inhibitors such as folic acid and 1,10-phenanthroline, several qualitative differences between rat and human liver formaldehyde dehydrogenase have been detected.- The molecular weight of purified human formaldehyde dehydrogenase was estimated at 90000. However, the molecular weight of human formaldehyde dehydrogenase appeared to be greater than 250000 when it was determined in preparations which also contained alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and aldehyde dehydrogenase (aldehyde: NAD+ oxidoreductase, EC 1.2.1.3). These data suggest that formaldehyde dehydrogenase exists in a complex with other proteins or in a polymeric form until the ultimate steps in purification.- The capacity for NAD-linked formaldehyde oxidation was greater in human liver than in rat liver. The possible implications of this are discussed in regard to the unique susceptibility of man to methanol poisoning.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33505/1/0000002.pd

Requirement of NOX2 and Reactive Oxygen Species for Efficient RIG-I-Mediated Antiviral Response through Regulation of MAVS Expression

The innate immune response is essential to the host defense against viruses, through restriction of virus replication and coordination of the adaptive immune response. Induction of antiviral genes is a tightly regulated process initiated mainly through sensing of invading virus nucleic acids in the cytoplasm by RIG-I like helicases, RIG-I or Mda5, which transmit the signal through a common mitochondria-associated adaptor, MAVS. Although major breakthroughs have recently been made, much remains unknown about the mechanisms that translate virus recognition into antiviral genes expression. Beside the reputed detrimental role, reactive oxygen species (ROS) act as modulators of cellular signaling and gene regulation. NADPH oxidase (NOX) enzymes are a main source of deliberate cellular ROS production. Here, we found that NOX2 and ROS are required for the host cell to trigger an efficient RIG-I-mediated IRF-3 activation and downstream antiviral IFNβ and IFIT1 gene expression. Additionally, we provide evidence that NOX2 is critical for the expression of the central mitochondria-associated adaptor MAVS. Taken together these data reveal a new facet to the regulation of the innate host defense against viruses through the identification of an unrecognized role of NOX2 and ROS

Intermediate filament cytoskeleton of the liver in health and disease

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising ~70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation