Deficiency in augmenter of liver regeneration accelerates liver fibrosis by promoting migration of hepatic stellate cell

Background

Augmenter of liver regeneration (ALR) protects liver from various injuries, however, the association of ALR with liver fibrosis, particularly its effect on hepatic stellate cells (HSC), remains unclear. In this study, we investigated the impact of ALR on the activation of HSC, a pivotal event in occurrence of liver fibrosis.

Regulation of polyamine synthesis in human hepatocytes by hepatotrophic factor augmenter of liver regeneration

Different stages of liver regeneration are regulated by a variety of factors such as the liver growth associated protein ALR, augmenter of liver regeneration. Furthermore, small molecules like polyamines were proven to be essential for hepatic growth and regeneration. Therefore, using primary human hepatocytes in vitro we investigated the effect of ALR on the biosynthesis of polyamines. We demonstrated by HPLC analysis that recombinant ALR enhanced intracellular hepatic putrescine, spermidine, and spermine levels within 9-12h. The activation of polyamine biosynthesis was dose dependent with putrescine showing the strongest increase. Additionally, ALR treatment induced mRNA expression of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase, both key enzymes of polyamine biosynthesis. Further, ALR induced c-myc mRNA expression, a regulator of ODC expression, and therefore we assume that ALR exerts its liver regeneration augmenting effects through stimulation of its signalling pathway leading in part to enhanced polyamine synthesis.     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

肝再生增强因子研究进展

肝再生增强因子是新近克隆的蛋白质因子,能特异地刺激肝源细胞的增殖,并对ＣＣl4所引起的急性肝衰竭有效治作用。本文综述了肝再生增强因子的发现、基因克隆及组织分布等。目前已开始了该因子的基因工程产品研制,它有望成为一种治疗肝病的新药。     

Augmenter of liver regeneration: Essential for growth and beyond

Liver regeneration is a well-orchestrated process that is triggered by tissue loss due to trauma or surgical resection and by hepatocellular death induced by toxins or viral infections. Due to the central role of the liver for body homeostasis, intensive research was conducted to identify factors that might contribute to hepatic growth and regeneration. Using a model of partial hepatectomy several factors including cytokines and growth factors that regulate this process were discovered. Among them, a protein was identified to specifically support liver regeneration and therefore was named ALR (Augmenter of Liver Regeneration). ALR protein is encoded by GFER (growth factor erv1-like) gene and can be regulated by various stimuli. ALR is expressed in different tissues in three isoforms which are associated with multiple functions: The long forms of ALR were found in the inner-mitochondrial space (IMS) and the cytosol. Mitochondrial ALR (23 kDa) was shown to cooperate with Mia40 to insure adequate protein folding during import into IMS. On the other hand short form ALR, located mainly in the cytosol, was attributed with anti-apoptotic and anti-oxidative properties as well as its inflammation and metabolism modulating effects. Although a considerable amount of work has been devoted to summarizing the knowledge on ALR, an investigation of ALR expression in different organs (location, subcellular localization) as well as delineation between the isoforms and function of ALR is still missing. This review provides a comprehensive evaluation of ALR structure and expression of different ALR isoforms. Furthermore, we highlight the functional role of endogenously expressed and exogenously applied ALR, as well as an analysis of the clinical importance of ALR, with emphasis on liver disease and in vivo models, as well as the consequences of mutations in the GFER gene.     

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl4-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl₄ in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl₄-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.     

Measurement of oxidative stress in human liver by EPR spin-probe technique

A method for the measurement of reactive oxygen species (ROS) in human hepatic tissue has been developed. The method is based on the EPR detection of the nitroxide radical produced by reaction of the hydroxylamine spin-probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate with ROS generated under pseudo-physiologic conditions in fine needle biopsies of healthy (10 controls) and diseased (22 patients) human liver. Measures of malonaldehyde in 9 liver biopsies (3 controls and 6 patients) have also been obtained by high pressure liquid chromatography and values parallel those obtained by the spin-probe technique. The amount of ROS found in healthy human liver (median = 1.8 × 10^-11 mol/mg) was significantly lower than values found in liver affected by hepatitis B (median = 5.8 × 10^-10 mol/mg; p < 0.02) or by hepatitis C (median = 2.7 × 10^-9 mol/mg; p < 0.003) as well as compared to some other non-viral liver diseases (NVLD): autoimmune hepatitis, primary biliary cirrhosis, primary schlerosing cholangitis (median = 9.8 × 10^-9 mol/mg; p < 0.005). NVLD also showed significantly higher ROS levels compared to hepatitis B (p < 0.04) and hepatitis C (p < 0.04).

The mechanism, potentiality and limitations of our method are discussed.     

肝再生增强因子超家族研究进展

从断奶大鼠的肝脏中纯化得到了肝刺激物质（ＨＳＳ）的有效成份,即肝再生增强因子（ＡＬＲ）的蛋白质,酶解并对其多肽末端测序,据此推导出简并核苷酸序列,合成探针,对大鼠肝脏来源的ｃＤＮＡ文库进行筛选,首选获得了大鼠ＡＬＲ的ｃＤＮＡ克隆,随后又分别克隆了人和小鼠的ＡＬＲ的ｃＤＮＡ。与此同时,从酵母细胞中克隆了与线粒体氧化－－磷酸化功能密切相关的ＥＲＶ１基因,然后克隆了人的ＥＲＶ１同源基因,从功能上证实人     

The crystal structure of augmenter of liver regeneration: A mammalian FAD-dependent sulfhydryl oxidase

The crystal structure of recombinant rat augmenter of liver regeneration (ALRp) has been determined to 1.8 A. The protein is a homodimer, stabilized by extensive noncovalent interactions and a network of hydrogen bonds, and possesses a noncovalently bound FAD in a motif previously found only in the related protein ERV2p. ALRp functions in vitro as a disulfide oxidase using dithiothreitol as reductant. Reduction of the flavin by DTT occurs under aerobic conditions resulting in a spectrum characteristic of a neutral semiquinone. This semiquinone is stable and is only fully reduced by addition of dithionite. Mutation of either of two cysteine residues that are located adjacent to the FAD results in inactivation of the oxidase activity. A comparison of ALRp with ERV2p is made that reveals a number of significant structural differences, which are related to the in vivo functions of these two proteins. Possible physiological roles of ALR are examined and a hypothesis that it may serve multiple roles is proposed.     

重组[B18Ile]人胰岛素的鉴定和特征

突变体「Ｂ１８Ｉｌｅ」猪胰岛素前体经分离纯化,转肽,得到重组「Ｂ１８Ｉｌｅ」人胰素「Ｂ１８Ｉｌｅ」人胰岛素能结晶,其与受体的结合能力为猪胰岛素的８２％,保留了与猪胰岛素基本相同的体内活力,从本文结果和分析表明Ｂ１８Ｖａｌ可能不是胰岛素表现生物功能所必需的。     

Efficacy of sauchinone as a novel AMPK-activating lignan for preventing iron-induced oxidative stress and liver injury

Iron-overload disorders cause hepatocyte injury and inflammation by oxidative stress, possibly leading to liver fibrosis and hepatocellular carcinoma. This study investigated the efficacy of sauchinone, a bioactive lignan, in preventing iron-induced liver injury and explored the mechanism of sauchinone's activity. To create iron overload, mice were injected with phenylhydrazine, and the effects on hepatic iron and histopathology were assessed. Phenylhydrazine treatment promoted liver iron accumulation and ferritin expression, causing hepatocyte death and increased plasma arachidonic acid (AA). Sauchinone attenuated liver injury (EC₅₀ = 10 mg/kg) and activated AMPK in mice. Treatment of hepatocytes with iron and AA simulated iron overload conditions: iron + AA synergistically amplified cytotoxicity, increasing H₂O₂ and the mitochondrial permeability transition. Sauchinone protected hepatocytes from iron + AA-induced cytotoxicity, preventing the induction of mitochondrial dysfunction and apoptosis (EC₅₀ = 1 μM), similar to the result using metformin. Sauchinone treatment activated LKB1, which led to AMPK activation: these events contributed to cell survival. Evidence of cytoprotection by LKB1 and AMPK activation was revealed in the reversal of sauchinone's restoration of the mitochondrial membrane potential by either dominant negative mutant AMPKα or chemical inhibitor. In conclusion, sauchinone protects the liver from toxicity induced by iron accumulation, and sauchinone's effects may be mediated by LKB1-dependent AMPK activation.     

Carotenoids modulate the trade-off between egg production and resistance to oxidative stress in zebra finches

The allocation of resources to reproduction and survival is a central question of studies of life history evolution. Usually, increased allocation to current reproduction is paid in terms of reduced future reproduction and/or decreased survival. However, the proximal mechanisms underlying the cost of reproduction are poorly understood. Recently, it has been shown that increased susceptibility to oxidative stress might be one of such proximate links between reproduction and self-maintenance. Organisms possess a range of antioxidant defenses, including endogenously produced molecules (e.g., enzymes) and compounds ingested with food (e.g., carotenoids). If reproductive effort increases the production of reactive oxygen species, the availability of antioxidant defenses may partly or fully counteract the free-radical damages. One could, therefore, expect that the trade-off between reproduction and oxidative stress is modulated by the availability of antioxidant defenses. We tested this hypothesis in zebra finches. We manipulated reproductive effort by either allowing or preventing pairs to breed. Within each breeding or non-breeding group, the availability of antioxidant compounds was manipulated by supplementing or not supplementing the drinking water with carotenoids. We found that although birds in the breeding and non-breeding groups did not differ in their resistance to oxidative stress (the breakdown of red blood cells submitted to a controlled free-radical attack), one aspect of breeding effort (i.e., the number of eggs laid by birds in both breeding and non-breeding groups) was negatively correlated with resistance to oxidative stress only in birds that did not benefit from a carotenoid-supplemented diet. This result therefore suggests that carotenoid availability can modulate the trade-off between reproduction and resistance to oxidative stress.     

Mitochondria as a primary target for vascular hypoperfusion and oxidative stress in Alzheimer's disease

It has been widely accepted that vascular hypoperfusion induces oxidative stress and the outcome of this misbalance is brain energy failure. This abnormality leads to neuronal death which manifests as cognitive impairment and the development of brain pathology as in Alzheimer's disease (AD). It has been demonstrated that the AD brain is characterized by impairments in energy metabolism. We theorize that hypoperfusion induced mitochondrial failure plays a key role in the generation of reactive oxygen species, resulting in oxidative damage to brain cellular compartments, especially in the vascular endothelium and in selective population of neurons with high metabolic activity in the AD brain. All of these abnormalities have been found to occur before classic AD pathology inducing neuronal degeneration and amyloid deposition during the progression of AD. Therefore, expanding investigations into both the mechanisms behind amyloid beta (Abeta) deposition and the possible accelerating effects of environmental factors such as chronic hypoxia/reperfusion may open a new avenue for effective treatments of AD. Future studies examining the importance of mitochondrial pathobiology in brain cellular compartments provide insight not only into the better understanding of the neurodegenerative and/or cerebrovascular disease but also provide targets for treating these conditions.     

Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

Background/Aim

Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells.

Methods

Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [³H]-thymidine assay.

Results

The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1).

Conclusion

rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.     

Engineered ZnO and TiO(2) nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli

Extensive use of engineered nanoparticle (ENP)-based consumer products and their release into the environment have raised a global concern pertaining to their adverse effects on human and environmental health. The safe production and use of ENPs requires improvement in our understanding of environmental impact and possible ecotoxicity. This study explores the toxicity mechanism of ZnO and TiO(2) ENPs in a gram-negative bacterium, Escherichia coli. Internalization and uniform distribution of characterized bare ENPs in the nano range without agglomeration was observed in E. coli by electron microscopy and flow cytometry. Our data showed a statistically significant concentration-dependent decrease in E. coli cell viability by both conventional plate count method and flow cytometric live-dead discrimination assay. Significant (p<0.05) DNA damage in E. coli cells was also observed after ENP treatment. Glutathione depletion with a concomitant increase in hydroperoxide ions, malondialdehyde levels, reactive oxygen species, and lactate dehydrogenase activity demonstrates that ZnO and TiO(2) ENPs induce oxidative stress leading to genotoxicity and cytotoxicity in E. coli. Our study substantiates the need for reassessment of the safety/toxicity of metal oxide ENPs.     

Monoamine oxidase-A is an important source of oxidative stress and promotes cardiac dysfunction,apoptosis, and fibrosis in diabetic cardiomyopathy

Oxidative stress is closely associated with the pathophysiology of diabetic cardiomyopathy (DCM). The mitochondrial flavoenzyme monoamine oxidase A (MAO-A) is an important source of oxidative stress in the myocardium. We sought to determine whether MAO-A plays a major role in modulating DCM. Diabetes was induced in Wistar rats by single intraperitoneal injection of streptozotocin (STZ). To investigate the role of MAO-A in the development of pathophysiological features of DCM, hyperglycemic and age-matched control rats were treated with or without the MAO-A-specific inhibitor clorgyline (CLG) at 1 mg/kg/day for 8 weeks. Diabetes upregulated MAO-A activity; elevated markers of oxidative stress such as cardiac lipid peroxidation, superoxide dismutase activity, and UCP3 protein expression; enhanced apoptotic cell death; and increased fibrosis. All these parameters were significantly attenuated by CLG treatment. In addition, treatment with CLG substantially prevented diabetes-induced cardiac contractile dysfunction as evidenced by decreased QRS, QT, and corrected QT intervals, measured by ECG, and LV systolic and LV end-diastolic pressure measured by microtip pressure transducer. These beneficial effects of CLG were seen despite the persistent hyperglycemic and hyperlipidemic environments in STZ-induced experimental diabetes. In summary, this study provides strong evidence that MAO-A is an important source of oxidative stress in the heart and that MAO-A-derived reactive oxygen species contribute to DCM.