Metamorphosis of the malaria parasite in the liver is associated with organelle clearance

Malaria parasites encounter diverse conditions as they cycle between their vertebrate host and mosquito vector. Within these distinct environments, the parasite undergoes drastic transformations, changing both its morphology and metabolism. Plasmodium species that infect mammals must first take up residence in the liver before initiating red blood cell infection. Following penetration into hepatocytes, the parasite converts from an invasion-competent, motile, elongated sporozoite to a metabolically active, round trophozoite. Relatively little is known about the cellular events involved in sporozoite metamorphosis. Our data uncover the early cellular events associated with these transformations. We illustrate that the beginning of metamorphosis is marked by the disruption of the membrane cytoskeleton beneath the plasma membrane, which results in a protruding area around the nucleus. As this bulbous region expands, the two distal ends of the sporozoite gradually retract and disappear, leading to cell sphericalization. This shape change is associated with major interior renovations and clearance of superfluous organelles, e.g. micronemes involved in invasion. The membrane cytoskeleton is reorganized into dense lamellar arrays within the cytoplasm and is partially expulsed by converting parasites. Simultaneously, micronemes are compartmentalized into large exocytic vesicles and are then discharged into the environment. At the completion of metamorphosis, the parasites only retain organelles necessary for replication. These observations lay the groundwork for further investigations on the developmental pathways implicated in the metamorphosis of the malaria parasite.

Identification of deregulated miRNAs and their targets in hepatitis B virus-associated hepatocellular carcinoma

AIM： TO identify the differentially expressed miRNAs and their targets in hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC）. METHODS： Six hundred and sixty seven human miRNAs were quantitatively analyzed by Taqman lowdensity miRNA array （TLDA） in HBV-HCC tissues. Gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analyses were used to analyze the significant function and pathway of the differentially expressed miRNAs in HBV-HCC. TargetScan software was used to predict the targets of deregulated miRNAs. Western blotting and luciferase assay were performed to verify the targets of these miRNAs.RESULTS： Ten up-regulated miRNAs （miR-217, miR- 518b, miR-517c, miR-520g, miR-519a, miR-522, miR- 518e, miR-525-3p, miR-512-3p, and miR-518a-3p） and 11 down-regulated miRNAs （miR-138, miR-214, miR-214#, miR-199a-5p, miR-433, miR-511, miR-592, miR-483-3p, miR-483-5p, miR-708 and miR-1275） were identified by Taqman miRNAs array and confirmed quantitatively by reverse transcription polymerase chain reaction in HCC and adjacent non-tumor tissues. GO and KEGG pathway analysis revealed that ＂regulation of actin cytoskeleton＂ and ＂pathway in cancer＂ are most likely to play critical roles in HCC tumorigenesis. MiR- 519a and ribosomal protein S6 kinase polypeptide 3 （RPS6KA3） were predicted as the most significant can-didates by miRNA-mRNA network. In addition, cyclin D3 （CCND3） and clathrin heavy chain （CHC）, usually up-regulated in HCC tissues, were validated as the di- rect target of miR-138 and miR-199a-5p, respectively.

Hepatocyte cytoskeleton during ischemia and reperfusion -influence of ANP-mediated p38 MAPK activation

AIM： To determine functional consequences of this activation, whereby we focused on a potential regulation of the hepatocyte cytoskeleton during ischernia and reperfusion. METHODS： For in vivo experiments, animals received ANP （5 μg/kg） intravenously. In a different experimental setting, isolated rat livers were perfused with KH-buffer ＋ANP （200 nmol/L）＋SB203580 （2 μmol/L）. Livers were then kept under ischernic conditions for 24 h, and either transplanted or reperfused. Actin, Hsp27, and phosphorylated Hsp27 were determined by Western blotting, p38 MAPK activity by in vitro phosphorylation assay. F-actin distribution was determined by confocal microscopy. RESULTS： We first confirmed that ANP preconditioning leads to an activation of p38 MAPK and observed alterations of the cytoskeleton in hepatocytes of ANP- preconditioned organs. ANP induced an increase of hepatic F-actin after ischemia, which could be prevented by the p38 MAPK inhibitor SB203580 but had no effect on bile flow. After ischemia untreated livers showed a translocation of Hsp27 towards the cytoskeleton and an increase in total Hsp27, whereas ANP preconditioning prohibited translocation but caused an augmentation of Hsp27 phosphorylation. This effect is also mediated via p38 MAPK, since it was abrogated by the p38 MAPK inhibitor SB203580. CONCLUSION： This study reveals that ANP-mediated p38 MAPK activation leads to changes in hepatocyte cytoskeleton involving an elevation of phosphorylated Hsp27 and thereby for the first time shows functional consequences of ANP-induced hepatic p38 MAPK activation.

Hepatoma cell line HepG2.2.15 demonstrates distinct biological features compared with parental HepG2

AIM:To investigate the biological features of hepatitis B virus(HBV)-transfected HepG2.2.15 cells. METHODS:The cell ultrastructure,cell cycle and apoptosis,and the abilities of proliferation and invasion of HBV-transfected HepG2.2.15 and the parent HepG2 cells were examined by electron microscopy,flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trans-well assay.Oncogenicity of the two cell lines was compared via subcutaneous injection and orthotopic injection or implantation in nude mice,and the pathological analysis of tumor formation was performed.Two cytoskeletal proteins were detected by Western blotting. RESULTS:Compared with HepG2 cells,HepG2.2.15 cells showed organelle degeneration and filopodia disappearance under electron microscope.HepG2.2.15 cells proliferated and migrated slowly in vitro,and hardly formed tumor and lung metastasis in nude mice.Flow cytometry showed that the majority of HepG2.2.15 cells were arrested in G1 phase,and apoptosis was minor in both cell lines.Furthermore,the levels of cytoskeletal proteins F-actin and Ezrin were decreased in HepG2.2.15 cells. CONCLUSION:HepG2.2.15 cells demonstrated a lower proliferation and invasion ability than the HepG2 cells due to HBV transfection.