Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.     

不明原因肝功能异常患者的临床诊断思维

目的：建立不明原因肝功能异常患者的临床诊断思维以提高疑难肝病的诊治水平。方法：回顾性分析我院收治的4例不明原因肝功能异常患者的临床资料及诊治经过,并复习相关文献。结果：导致肝功能异常的病因虽极为复杂但通过详细询问病史,进行细致全面的体格检查以及必要的实验室和辅助检查,慎重采取诊断性治疗措施,提高少见病例对诊断影响的认识,绝大多数的病因可以查明。结论：不明原因肝功能异常患者的临床表现多样,病因复杂,建立相应的临床诊断思维可减少误诊和漏诊。     

Veliparib overcomes multidrug resistance in liver cancer cells

Overexpression of ATP-binding cassette (ABC) transporter is one of the most important factors taking responsibility for the progress of multidrug resistance (MDR) in multiple cancers. In this study, we investigated that veliparib, a PARP inhibitor which is in clinical development, could overcome ABCB1-mediated MDR in liver cancer cells. Veliparib could significantly enhance the cytotoxic effects of a series of conventional chemotherapeutic drugs in ABCB1-overexpression liver cancer cells. Mechanism study showed that veliparib could significantly enhance the accumulation of doxorubicin in ABCB1-overexpression liver cancer cells, without down-regulating the expression level of ABCB1. Finally, veliparib could significantly inhibit the ATPase activity of ABCB1 transporter. This study could provide information that combine veliparib with other chemotherapeutic drugs may benefit liver cancer patients.     

FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.     

Wnt/β-Catenin Signaling Protects Mouse Liver against Oxidative Stress-induced Apoptosis through the Inhibition of Forkhead Transcription Factor FoxO3

Numerous liver diseases are associated with extensive oxidative tissue damage. It is well established that Wnt/β-catenin signaling directs multiple hepatocellular processes, including development, proliferation, regeneration, nutrient homeostasis, and carcinogenesis. It remains unexplored whether Wnt/β-catenin signaling provides hepatocyte protection against hepatotoxin-induced apoptosis. Conditional, liver-specific β-catenin knockdown (KD) mice and their wild-type littermates were challenged by feeding with a hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce chronic oxidative liver injury. Following the DDC diet, mice with β-catenin-deficient hepatocytes demonstrate increased liver injury, indicating an important role of β-catenin signaling for liver protection against oxidative stress. This finding was further confirmed in AML12 hepatocytes with β-catenin signaling manipulation in vitro using paraquat, a known oxidative stress inducer. Immunofluorescence staining revealed an intense nuclear FoxO3 staining in β-catenin-deficient livers, suggesting active FoxO3 signaling in response to DDC-induced liver injury when compared with wild-type controls. Consistently, FoxO3 target genes p27 and Bim were significantly induced in β-catenin KD livers. Conversely, SGK1, a β-catenin target gene, was significantly impaired in β-catenin KD hepatocytes that failed to inactivate FoxO3. Furthermore, shRNA-mediated deletion of FoxO3 increased hepatocyte resistance to oxidative stress-induced apoptosis, confirming a proapoptotic role of FoxO3 in the stressed liver. Our findings suggest that Wnt/β-catenin signaling is required for hepatocyte protection against oxidative stress-induced apoptosis. The inhibition of FoxO through its phosphorylation by β-catenin-induced SGK1 expression reduces the apoptotic function of FoxO3, resulting in increased hepatocyte survival. These findings have relevance for future therapies directed at hepatocyte protection, regeneration, and anti-cancer treatment.