Insoluble uricase in liver peroxisomes of Old World monkeys

1. Subcellular localization form and properties of liver uricase of Macaca fascicularis were examined. 2. Uricase was present as the insoluble form in the peroxisomal core. 3. Evidence was obtained to show that the peroxisomal core is uricase itself. 4. The number and mol. wts of the subunits of the enzyme were identical to those of rat liver uricase. 5. The same results were also obtained for liver uricase of Macaca fuscata.     

Several enzymic properties of the DNA-polymerase alpha complexes with antibodies

A highly specific rabbit antiserum against DNA polymerase alpha from regenerating rat liver (antigen AG 1) and an antiserum against the preparation of the enzyme proteolytic fragments possessing catalytic activity (antigen AG 2) were obtained. The enzyme neutralization test revealed that antibodies against AG 2 inhibit the DNA polymerase activity in a much stronger degree, than those against AG 1. Data from a kinetic analysis of the enzyme complexed with the antibodies against AG 1 suggest that the catalytic and binding sites for dNTP and free Mg2+ are altered. The value of apparent Km for activated DNA is unchanged in the DNA polymerase complexes with antibodies both against AG 1 and AG 2.     

Serological survey of a captive macaque colony in China for antibodies to simian type D retroviruses.

Sera from 510 macaques consisting of Macaca mulatta, Macaca assamensis, Macaca fascicularis, Macaca nemestrina, and Macaca arctoides were investigated for antibodies to simian AIDS type D retrovirus (SRV) by ELISA and Western blot with viral antigens purified from supernatants of SRV-1 infected cell cultures. Of these monkeys, 104 were seropositive by ELISA; only 23 were confirmed by Western blot. The true positive reaction to SRV was found in 15 of 463 (3.2%) M. mulatta and eight of eleven (72.7%) M. assamensis.     

Role of nitric oxide in the expression of hepatic vascular stress genes in response to sepsis

This study examined the role of nitric oxide (NO) on the expression of the hepatic vasoregulatory gene during polymicrobial sepsis. Aminoguanidine (AG, 100 mg/kg) or Nomega-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg) was injected intraperitoneally at 0, 3, 6, 10, and 20 h after a cecal ligation and puncture (CLP). The heart rate increased 24 h after the CLP, and this increase was attenuated by L-NAME and further attenuated by AG. The mean arterial pressure in the CLP animals did not change significantly 24 h after the onset of sepsis but was increased after the L-NAME injection. Sepsis increased the serum aminotransferase levels, which were attenuated by AG but augmented by L-NAME. CLP increased the mRNA level of the ET-1 and ETB receptors in the liver. This increase was prevented by AG but augmented by L-NAME. The level of iNOS and HO-1 mRNA expression were increased by CLP, which was prevented by both AG and L-NAME. The level of TNF-alpha and COX-2 mRNA expression increased after CLP, and was attenuated by AG. These results show that iNOS and eNOS are regulated differently in sepsis. While eNOS appears to have a protective role in liver microcirculation, the strong upregulation of iNOS might contribute to a microvascular dysfunction and hepatic injury.     

Acetyl glutamate--a model of signals for intracellular proteolysis

The proteolysis at neutral pH of mitochondria from liver and brain is more marked in isolated preparations than "in vivo" indicating activation of proteases or inactivation of repressors during isolation. Acetyl glutamate (AG), found in liver mitochondria of ureotelic animals, plays a crucial role as activator of carbamylphosphate synthetase. Since AG levels change under a number of conditions, we checked for an AG deacylase in mitochondria, for otherwise AG must be exported and destroyed by cytosol deacylases. We noted on incubation of mitochondrial extracts with AG an increase in trichloracetic acid-soluble ninhydrin-reacting material but not in acetate liberation, indicating activation of proteases. This was checked with 14C-labelled mitochondria. Under certain conditions AG and other acyl aminoacids stimulate approximately 5 to 20% the proteolysis with rat live and with brain mitochondria.     

Amarogentin,a secoiridoid glycoside,activates AMP- activated protein kinase (AMPK) to exert beneficial vasculo-metabolic effects

IntroductionAMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined.MethodsMolecular docking of Gentiana lutea constituents was performed with crystal structure of human α₂β₁γ₁ trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG) to α₂ subunit was confirmed through isothermal titration calorimetry (ITC) and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining.ResultsAG interacts with the α₂ subunit of AMPK and activates the trimeric kinase with an EC₅₀ value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC) and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice.ConclusionIn conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK.General significanceAmarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.     

Antioxidative and anti-diabetic effects of amaranth (Amaranthus esculantus) in streptozotocin-induced diabetic rats

The anti-diabetic and antioxidative effect of amaranth grain (AG) and its oil fraction (AO) was studied in streptozotocin-induced diabetic rats. Male Sprague-Dawley rats were divided into four groups after induction of STZ-diabetes: normal control; diabetic control; diabetic-AG supplement (500 g kg(-1) diet); diabetic-AO supplement (100 g kg(-1) diet) and fed experimental diets for 3 weeks. Serum glucose, insulin, activities of serum marker enzymes of liver function and liver cytosolic antioxidant enzymes were measured. The AG and AO supplement significantly decreased the serum glucose and increased serum insulin level in diabetic rats. Serum concentration of liver function marker enzymes, GOT and GPT, were also normalized by AG and AO treatment in diabetic rats. Liver cytosolic SOD and GSH-reductase activities were significantly increased, and catalase, peroxidase and GSH-Px activities were decreased in diabetic rats. AG and AO supplement reverted the antioxidant enzyme activities to near normal values. Hepatic lipid peroxide product was significantly higher, and GSH content was decreased in diabetic rats. However, AG and AO supplement normalized these values. Our data suggest that AG and AO supplement, as an antioxidant therapy, may be beneficial for correcting hyperglycaemia and preventing diabetic complications.     

The effect of aminoguanidine against cholestatic liver injury in rats

We investigated the protective role of aminoguanidine (AG) in rat liver injury induced by chronic biliary obstruction. Secondary biliary cirrhosis was induced by bile duct ligation for 14 days. Swiss albino rats were divided into three groups: Common bile duct ligated (CBDL) rats; Group A, CBDL rats treated with AG as Group B and simple laparotomy group known as the Sham group; Group C. Group B received 200 mg/kg of AG intraperitoneally daily throughout 14 days. The present data showed decreased gama glutamyl transferase (GGT), aspartate aminotransferase (AST), bilirubin and alanine aminotransferase (ALT) levels in the AG treated rats, when compared with CBDL rats (p < 0.05). In the AG treated rats, tissue levels of malondialdehyde (MDA) were significantly lower than that in CBDL rats (p < 0.001). Although the levels of glutathione (GSH) in AG treated rats were higher and myeloperoxidase (MPO) were lower than that in CBDL rats, the difference was not statistically significant (p > 0.05). The levels of interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were significantly lower and although the levels of interleukin-6 (IL-6) were lower in AG treated rats than that in CBDL rats, the difference was not statistically significant. Administration of AG in the rats with biliary obstruction resulted in inhibition of ductular proliferation and portal inflammation. The present study demonstrates that intraperitoneal administration of AG in CBDL rats maintains antioxidant defenses, reduces liver oxidative and cytokine damage and ductular proliferation and portal inflammation. This effect of AG may be useful in the preservation of liver injury in cholestasis.     

Mouse AKR1E1 is an ortholog of pig liver NADPH dependent 1,5-anhydro-D-fructose reductase

In many organisms, glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is reduced to 1,5-anhydro-D-glucitol (AG). AF reductase, which catalyzes the latter reaction, was purified from pig liver, but mouse ortholog has not yet been reported. In the database, aldo-keto reductase family 1, member E1 (AKR1E1) showed highest homology to pig enzyme. We confirmed that cloned AKR1E1 is mouse ortholog based on enzymatic properties of purified recombinant protein.     

Effects of protein malnutrition on IL-6-mediated signaling in the liver and the systemic acute-phase response in rats

This study examines the effects of malnutrition on IL-6 signaling pathways of rats fed 2% vs. 20% casein diets for 14 days. Effects of malnutrition on the abundance and IL-6-stimulated phosphorylation of signaling proteins in the JAK-STAT and MAP kinase pathways were examined in the liver. Changes of the acute-phase response as reflected by serum alpha(1)-acid glycoprotein (AG), TNF-alpha (TNF), and IL-1beta (IL-1) were compared in the two dietary groups at 0, 4, 8, 16, and 24 h after IL-6 administration. Under basal conditions, the abundance of the IL-6 receptor, gp130, JAK1, STAT1, and STAT3 proteins and levels of phosphorylation of ERK1/2 and p38 were significantly increased in the liver in the 2% casein group compared with the 20% casein group. With IL-6 stimulation, the increased phosphorylation per unit of protein of these signaling proteins was not different in the liver between the two groups. Before IL-6 stimulation, serum levels of TNF, IL-1, IL-6, and AG were significantly higher in the 2% casein group than in the 20% casein group. After bolus injection of IL-6, changes in IL-1 and AG were similar in the two dietary groups, although a slight decline in AG level was noted after 8 h of IL-6 administration in the 2% protein group. These data demonstrate that protein malnutrition produces changes in inflammation-related proteins characteristic of a low-grade systemic inflammatory response and, thus, can serve as an inflammatory stimulus. The capacity for response to IL-6 is preserved, suggesting adaptive preservation of acute-phase responsiveness during malnutrition.     

Association of genetic variation in co-stimulatory molecule genes with outcome of liver transplant in Iranian patients

CD28, cytotoxic T-lymphocyte associated antigen 4 (CTLA4), inducible costimulator (ICOS) and programmed cell death 1 are closely-linked genes located on chromosome 2q and encode co-stimulatory molecules, which are T-cell activity regulators. The principal assignment of T-cell mediated immune response in allograft rejection is an interesting topic of multiple studies. Although the variation in these genes may influence the graft survival and the amount of immunosuppression needed, the studies so far have been restricted solely to the CTLA4 gene. In 145 patients who underwent liver allograft transplantation, 10 single nucleotide polymorphisms of CD28, CTLA4, ICOS, and PD.1 genes were defined. To distinguish the polymorphisms of all 10 SNPs, PCR-RFLP method was used and according to the standard criteria, acute rejection episodes were determined. CTLA4-1661, AA genotype was significantly more frequent in the patients with acute rejection and AG genotype was significantly more frequent in the patients without rejection. Frequencies of CTLA4+49 AG A allele and CTLA4-1661AG A allele were significantly higher than those of CTLA4+49 AG and CTLA4-1661AG, G allele in the patients with acute rejection. ICOS+693, GG genotype and G allele were significantly less frequent in the patients with acute rejection and CD28 CT genotype was significantly more in patients with acute rejection. The present results demonstrate that potentially functional genetic variation in T-cell co-stimulatory molecules including ICOS, CTLA4 and CD28 can influence liver transplant outcome.     

RsaI polymorphism of the ERβ gene in women with endometriosis

We examined the frequency of RsaI polymorphism of the ERβ gene in 54 patients diagnosed with endometriosis and 46 controls. Peripheral blood was collected from women undergoing laparoscopy with a confirmed diagnosis of endometriosis. Polymorphisms of the ERβ gene and p53 were assessed by PCR and analyzed on 2% agarose gel stained with ethidium bromide. The AG polymorphism genotype frequency in patients with endometriosis was 59.3%, with 40.7% GG. In the control group, the frequency of AG was 6.5%, with 93.5% GG. The frequency of heterozygous AG was nine times higher in patients with endometriosis than in the control group (P < 0.0001).     

Rosmarinic acid,a major polyphenolic component of Perilla frutescens,reduces lipopolysaccharide (LPS)-induced liver injury in D-galactosamine (D-GalN)-sensitized mice

The protective activity of rosmarinic acid from Perilla frutescens on liver injury induced by LPS in D-GalN-sensitized mice was examined. We also investigated the effects of antitumor necrosis factor-alpha antibody (anti-TNF), superoxide dismutase (SOD), and aminoguanidine (AG) on this model in order to elucidate the mechanism of rosmarinic acid protection. Perilla extract (PE) and rosmarinic acid (RA) treatments significantly reduced the elevation of plasma asparatate aminotransferase levels, as well as anti-TNF and SOD treatment, compared with controls, but this reduction was not seen in the AG group. These results were confirmed by histological examination using hematoxylin-eosin and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Increases in tumor necrosis factor-alpha (TNF-alpha) mRNA expression in liver and in plasma TNF-alpha levels, which were observed in control mice, were not significantly reduced by PE or RA. PE and RA treatments also did not significantly diminish iNOS mRNA expression or plasma nitrate/nitrite levels. Nitrotyrosine and N(epsilon)-(hexanonyl)lysine (HEL) production, the residue of oxidative stress, was observed in livers from controls, but not in those mice pretreated with PE or RA. These results suggest that the liver protection of RA is due to the scavenging or reducing activities-superoxide or peroxynitirite rather than to inhibition of TNF-alpha production.     

Aromatization of norethindrone to ethynylestradiol in human adult liver [Poster 41]

Homogenates of human adult liver are capable of aromatizing norethindrone (17-ethynyl-19-nortestosterone) to ethynylestradiol (17-ethynylestradiol). The evidence of ethynylestradiol formation was obtained using a Bio-Rad AG1-X2 column, thin-layer chromatography and co-crystallization.     

Cross-links of quadruplex structures from human telomeric DNA by dinuclear platinum complexes show the flexibility of both structures

The folding of AG(3)(T(2)AG(3))(3) was investigated in the presence of Na(+) or K(+) ions, by using the dinuclear platinum complexes [{trans-PtCl(NH(3))(2)}(2)H(2)N(CH(2))(n)NH(2)]Cl(2) (n = 2 or 6). AG(3)(T(2)AG(3))(3) has been previously found to adopt two different quadruplex structures: the antiparallel one in a solution containing Na(+) and the parallel one in a K(+)-containing crystal. The two structures are strikingly distinct and are not expected to form the same platinum cross-links. Therefore, characterization of the cross-links formed with platinum complexes in solution allowed the predominant conformation(s) to be identified. The bases coordinating the platinum atoms were identified by chemical and 3'-exonuclease digestions. The observed cross-links showed that the parallel structure exists in solution whatever the cation and confirmed the existence of the antiparallel structure in the presence of both cations as previously reported from cross-linking experiments of AG(3)(T(2)AG(3))(3) by mononuclear platinum complexes. Furthermore, the major platinum cross-links were unexpectedly formed between two guanines belonging to the same G-quartet. Their formation was rationalized using molecular dynamics simulations in implicit solvent of the two quadruplex structures. It was shown that they were flexible, allowing some guanines to leave reversibly the top G-quartet and thus rendering their N(7) atom accessible to platinum complexes. Our results also suggest that the human telomere sequence could be a target for such platinum complexes.     

Differential metabolism of diradyl glycerol molecular subclasses and molecular species by rabbit brain diglyceride kinase

Elevations in the mass of ether-linked diglycerides (i.e. 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) and 1-O-alkyl-2-acyl-sn-glycerol (Alkyl AG)) during cellular activation are prolonged in comparison to their 1,2-diacyl-sn-glycerol (DAG) counterparts. Since the metabolic removal of DAG is determined, in large part, by the rate of its phosphorylation by diglyceride kinase, we quantified differences in the activity of diglyceride kinase utilizing individual subclasses of diradyl glycerols as substrate. Rabbit brain microsomal diglyceride kinase activity was over 30-fold greater utilizing DAG as substrate (25.8 nmol.mg-1.min-1) in comparison to AAG (0.8 nmol.mg-1.min-1). No alterations in the affinity of microsomal diglyceride kinase for ATP were present (Km approximately 0.5 mM) utilizing each diradyl glycerol subclass. Similar subclass specificities for diglyceride kinase (i.e. DAG greater than Alkyl AG much greater than AAG) were present in brain and liver cytosol as well as in liver microsomes utilizing multiple assay conditions. In sharp contrast, Escherichia coli diglyceride kinase phosphorylated DAG, Alkyl AG, or AAG diradyl glycerol molecular subclasses at identical rates. Furthermore, although DAG was rapidly hydrolyzed by diglyceride lipase, catabolism of AAG or Alkyl AG by plasmalogenase, alkyl ether hydrolase, or diglyceride/monoglyceride lipase was undetectable. Collectively, these results demonstrate the importance of the differential catabolism of each diradyl glycerol molecular subclass as a primary determinant of their biologic half-lives. Since individual subclasses of diglycerides have distinct physical properties and physiologic functions, these results underscore the importance of lipid subclass specific metabolism in tailoring individual cellular responses during activation.     

Aromatization of norethindrone to ethynylestradiol in human adult liver

Homogenates of human adult liver are capable of aromatizing norethindrone (17 alpha-ethynyl-19-nortestosterone) to ethynylestradiol (17 alpha-ethynylestradiol). The evidence of ethynylestradiol formation was obtained using a Bio-Rad AG1-X2 column, thin layer chromatographies and co-crystallization. Neither acid nor base was used in any step in product identification.     

Sex differences in the accumulation of estrogen receptors in nuclei of liver cells and increase of angiotensinogen concentration in the blood of rats after administration of low doses of synthetic estrogens

The significance of sex differences in the level of estrogen receptors (ER) in hepatocytes for direct effects of estrogens in male and female rat livers was investigated. 4-5-fold increase in ER level and 20-30%-elevation in plasma angiotensinogen (AG) occurred after a single injection of 0.5 microgram of hexestrol (HE) in female and gonadectomized male rats. In male liver, where the cytosol ER content is two fold lower than that in female rats, nuclear ER level was shown to be very low and unchanged after HE injection, plasma AG also did not change. Injection of 0.5 microgram of ethinylestradiol produced similar effect. Injection of a greater dose of estrogen caused an enhancement in plasma AG level in males. The existence of sex differences in hepatic ER level seems to cause in some conditions different response of metabolic processes in male and female rat liver after estrogenization.     

αA-Crystallin-Derived Mini-Chaperone Modulates Stability and Function of Cataract Causing αAG98R-Crystallin

Background

A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37°C. Our previous studies have shown that αA-crystallin–derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin–derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin.

Methodology/Principal Findings

Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA–αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin.

Conclusion/Significance

These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant αA-crystallins and preserve the chaperone function.     

Hydranencephaly in two rhesus monkeys (Macaca mulatta)

Spontaneously occurring hydranencephaly was diagnosed at necropsy and confirmed histologically in two stillborn fetuses that were delivered from young female rhesus macaques (Macaca mulatta).