Augmenter of liver regeneration (alr) promotes liver outgrowth during zebrafish hepatogenesis

Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.     

Glucocorticoid receptor of frog (Rana esculenta) liver

The presence of a glucocorticoid soluble receptor is demonstrated in frog liver cytosol. The kinetic characterization of frog liver cytosolic receptor for glucocorticoids is reported and its steroid specificity assessed. Results indicate a gross similarity between frog liver and mammalian glucocorticoid receptor, being a major difference the reduced binding capacity.     

Mono(ADP-ribosylation) in rat liver mitochondria

This paper investigates protein mono(ADP-ribosylation) in rat liver mitochondria. In isolated inner mitochondrial membranes, in the presence of both ADP-ribose and NAD+, a protein is mono-(ADP-ribosylated) with high specificity. The reaction apparently consists of enzymatic NAD+ glycohydrolysis and subsequent binding of free ADP-ribose to the acceptor protein. In terms of chemical stability, the resulting bond is unique among the ADP-ribose linkages thus far characterized. Formation of a Schiff base adduct between free ADP-ribose and the acceptor protein is excluded. In intact mitochondria at least three classes of proteins are ADP-ribosylated in vivo. One ADP-ribose-protein linkage is of the carboxylate ester type as indicated by its lability in neutral buffer. Another class of ADP-ribosylated proteins requires hydroxylamine for release of ADP-ribose. The third class is stable in hydroxylamine but labile to alkali, similar to the ADP-ribose-cysteine linkage in transducin formed by pertussis toxin.     

Purification of rat liver S-adenosyl-l-methionine decarboxylase (Short Communication)

The amount of S-adenosyl-l-methionine decarboxylase present in rat liver was enhanced 17-fold by administration of methylglyoxal bis(guanylhydrazone),* a specific inhibitor of the enzyme. The enzyme was purified 1400-fold in 50% yield from such liver extracts by chromatography on columns of the inhibitor bound to Sepharose. The purified enzyme had no spermidine synthetase activity.     

The effect of glutathione monoester (GME) on glutathione (GSH) depleted rat liver

The effect of glutathione monoester (GME) on buthionine sulfoximine (BSO) mediated glutathione (GSH) depletion in rats was studied to understand the defensive role of intraperitoneally supplemented GSH. Administration of glutathione mono ester (GME) (at a dose of 5 mmole/kg body weight, twice a day for 30 days) significantly prevented the buthionine sulfoximine (at a dose of 4 mmole/kg body weight, twice a day for 30 days) induced alterations. This study suggests that glutathione mono ester is hepatoprotective and plays an important role in preventing lipid peroxidation, which leads to cytotoxic effects.     

Biotransformation of (-)-menthone by human liver microsomes

The aim of the current study was to investigate the metabolism of (-)-menthone by liver microsomes of humans. (-)-Menthone (1) was metabolized to (+)-neomenthol (2) (3-reduction) and 7-hydroxymenthone (3) by human liver microsomes. The metabolites formed were analyzed on GC and GC-MS. Kinetic analysis showed that K(m) and V(max) values for the metabolized (-)-menthone to respective (+)-neomenthol and 7-hydroxymenthone by liver microsomes of human sample HG70 were 0.37 mM and 4.91 nmol/min/mg protein and 0.07 mM and 0.71 nmol/min/mg protein.     

Current advances in proteomic analysis of (fatty) liver

In this review, an overview on proteomic studies conducted in livers of farm animals is conducted with a special focus on liver steatosis in waterfowl. Several studies had interest in understanding liver metabolism in dairy cows under various conditions (e.g. fasting) or the evolution of liver proteome during embryonic phases or growing periods in chicken. Those studies provide interesting results leading to a better understanding of the liver metabolism. Liver steatosis development in waterfowl represents a special case and a focus on proteomic studies conducted in these birds will be done. Indeed, recent studies aimed at resolving protein evolution during overfeeding in duck. Proteomic analysis combining two complementary approaches (2-dimensional electrophoresis gels and shot gun strategy) in order to better understand the mechanisms underlying the variability of cooking yield of fatty liver will be presented.     

Biotransformation of (-)-verbenone by human liver microsomes

The biotransformation of (-)-verbenone was investigated with human liver microsomes by using GC-MS. Regioselective biotransformation was observed when (-)-verbenone was incubated with the liver microsomes. (-)-10-Hydroxyverbenone was formed from (-)-verbenone of kinetic analysis showed that the Km and Vmax values for the hydroxylation of (-)-verbenone by liver microsomes from three human samples, HG-70, HG-56 and HG-23, were 1.1 mM and 4.8 nmol/min/nmol P450, 0.6 mM and 2.1 nmol/min/nmol P450, and 2.8 mM and 4.6 nmol/min/nmol P450, respectively.     

Tyrosine aminotransferase activity of frog (Rana esculenta) liver

1. The presence of tyrosine aminotransferase is reported both in particulate and soluble fractions of frog liver. 2. The activity of the soluble enzyme of frog liver was investigated with regard to its dose and time dependence, its substrate specificity and concentration dependence, its thermal sensitivity as well as pH and temperature dependence. 3. It appears that the properties of the soluble tyrosine aminotransferase of frog liver are in close agreement with those reported for the mammalian liver enzyme.     

Effects of clofibrate (alpha-p-chlorophenoxyisobutyryl-ethyl-ester) on male rat liver

In male rats, fed 0.5% clofibrate in their diet for 8 days and 21 days, the ultrastructural morphometric alterations of the hepatocytes were evaluated and compared with the biochemical data. The morphologic alterations of the microbodies were particularly related to the changes of the catalase activity of the liver homogenates. The results showed a marked hypertrophy of the liver and an increase in the volume of the individual hepatocyte. The numerical density and, even more pronounced, the volume density of the microbodies increased excessively during the treatment. The numerical density of the mitochondria decreased markedly after 21 days of administration. The surface of the rough endoplasmic reticulum showed a significant decrease, whereas the surface of the smooth endoplasmic reticulum showed a hypertrophy. The catalase activity of the liver homogenates increased 2-fold after 8 days and remained at this new steady-state after 21 days of treatment. The results suggest that the enzyme content of the microbodies changed after treatment with clofibrate, and support the suggestion that clofibrate may induce the synthesis of a yet unidentified peroxisomal protein.     

Mn(II) binding sites of calf liver arginase

Commercial calf liver arginase was further purified through gel filtration column chromatography. The enzyme is nearly homogeneous in SDS-PAGE; it contains 4 manganese atoms per molecule of enzyme. By dialysis against 1,10-o-phenanthroline at 4°C it is possible to obtain an arginase apo-form containing 2 manganese atoms per molecule of enzyme (apo-2 form) while a treatment of the pur enzyme with o-phenanthroline at 37°C followed by dialysis against 0.1 M NaCl is capable of producing an apoenzyme with only 1 manganese atom per molecule (apo-1 form). The apo-2 and apo-1 arginases retain respectively about 50% and 25% of the full enzymatic activity. NMR titrations of both apo-arginases with increasing concentrations of manganese allowed us to determine the affinity constants for the binding of Mn²⁺ to the protein. It was shown that in this enzyme two manganese atoms are weakly bound, one is more strongly bound and the fourth one is bound so tightly that it is not removed under the experimental conditions used.     

The mouse liver aldehyde oxidase locus (Aox)

Wide variability has been demonstrated in the properties and presumably the genetic constitution of aldehyde oxidases of 30 different strains of inbred mice. Genetic control of aldehyde oxidase (Aox) has been shown to reside in linkage group XIII and to be 9.6±0.4 recombination units from isocitric dehydrogenase (Id-1) and 28.3±3.5 recombination units from dipeptidase-1 (Dip-1). On the basis of these data and a recombination percent of 23.5±3.9 for Id-1 and Dip-1, the following gene order was deduced: Aox-Id-1-Dip-1. Furthermore, aldehyde oxidase activity was shown to be independent of adrenal influence and to have no clear-cut survival value for animals treated with large doses of N ¹-methylnicotinamide.This investigation was supported by USPHS grant AM 05741 and by a grant-in-aid from the American Heart Association.