Integrin-linked kinase overexpression and its oncogenic role in promoting tumorigenicity of hepatocellular carcinoma

Background

Integrin-linked kinase (ILK) was first discovered as an integrin β1-subunit binding protein. It localizes at the focal adhesions and is involved in cytoskeleton remodeling. ILK overexpression and its dysregulated signaling cascades have been reported in many human cancers. Aberrant expression of ILK influenced a wide range of signaling pathways and cellular functions. Although ILK has been well characterized in many malignancies, its role in hepatocellular carcinoma (HCC) is still largely unknown.

Methodology/Principal Findings

Quantitative PCR analysis was used to examine ILK mRNA expression in HCC clinical samples. It was shown that ILK was overexpressed in 36.9% (21/57) of HCC tissues when compared to the corresponding non-tumorous livers. The overall ILK expression level was significantly higher in tumorous tissues (P = 0.004), with a significant stepwise increase in expression level along tumor progression from tumor stage I to IV (P = 0.045). ILK knockdown stable clones were established in two HCC cell lines, BEL7402 and HLE, and were subjected to different functional assays. Knockdown of ILK significantly suppressed HCC cell growth, motility and invasion in vitro and inhibited tumorigenicity in vivo. Western blot analysis revealed a reduced phosphorylated-Akt (pAkt) at Serine-473 expression in ILK knockdown stable clones when compared to control clones.

Conclusion/Significance

This study provides evidence about the clinical relevance of ILK in hepatocarcinogenesis. ILK was found to be progressively elevated along HCC progression. Here our findings also provide the first validation about the oncogenic capacity of ILK in vivo by suppressing its expression in HCC cells. The oncogenic role of ILK is implicated to be mediated by Akt pathway.     

Novel adenosine A2A receptor ligands: A synthetic,functional and computational investigation of selected literature adenosine A2A receptor antagonists for extending into extracellular space

Growing evidence has suggested a role in targeting the adenosine A_2A receptor for the treatment of Parkinson’s disease. The literature compounds KW 6002 (2) and ZM 241385 (5) were used as a starting point from which a series of novel ligands targeting the adenosine A_2A receptor were synthesized and tested in a recombinant human adenosine A_2A receptor functional assay. In order to further explore these molecules, we investigated the biological effects of assorted linkers attached to different positions on selected adenosine A_2A receptor antagonists, and assessed their potential binding modes using molecular docking studies. The results suggest that linking from the phenolic oxygen of selected adenosine A_2A receptor antagonists is relatively well tolerated due to the extension towards extracellular space, and leads to the potential of attaching further functionality from this position.     

Electron transfer from Proteus vulgaris to a covalently assembled, single walled carbon nanotube electrode functionalised with osmium bipyridine complex: application to a whole cell biosensor

We report the fabrication and use of electrodes constructed from single walled carbon nanotubes (SWCNTs) chemically assembled on a carbon surface and functionalised with an osmium(II) bipyridine complex (Osbpy). The ability of the electrodes to transduce biologically generated currents from Proteus vulgaris has been established. Our investigations show that there are two contributions to the current: one from electroactive species secreted into solution and another from cell redox sites. The modified electrode can be used to monitor cell metabolism, thereby acting as a whole cell biosensor. The biosensor was used in a 1-h assay to investigate the toxicity of ethanol, sodium azide and the antibiotic ampicillin and gave quantitative data that were closely correlated with standard cell plate viability assays. The results provide proof of principle that the whole cell biosensor could be used for high throughput screening of antimicrobial activity. One of the modified electrodes was used for approximately 1000 measurements over four months demonstrating the robustness of the system.     

Do eel parasites reflect the local crustacean community? A case study from the Rhine River system

In 2003, the parasite fauna of 197 European eels Anguilla anguilla, captured at three different locations (Laufenburg, Karlsruhe and Beneeden Leeuwen) in the River Rhine, was analysed. The eels harboured a total of 18 species, among them the protozoa (Myxidium giardi, Myxobolus kotlani and Trypanosoma granulosum), acanthocephalans (Acanthocephalus anguillae, Acanthocephalus lucii, Echinorhynchus truttae, Pomphorhynchus laevis), nematodes (Paraquimperia tenerrima, Pseudocapillaria tomentosa, Camallanus lacustris, Raphidascaris acus, Spinitectus inermis and Anguillicola crassus), cestodes (Bothriocephalus claviceps and Proteocephalus macrocephalus) and monogeneans (Pseudodactylogyrus sp.). The parasite fauna at the different locations is discussed with respect to the crustacean fauna present at these locations. The investigation shows that changes in the composition of the crustacean fauna, due to the anthropogenic breakdown of a biogeographic barrier, are reflected in the composition of the intestinal eel parasite fauna.     

Cells that produce deleterious autoreactive antibodies are vulnerable to suicide

It is puzzling how autoreactive B cells that escape self-tolerance mechanisms manage to produce Abs that target vital cellular processes without succumbing themselves to the potentially deleterious effects of these proteins. We report that censorship indeed exists at this level: when the Ab synthesis in the cell is up-regulated in IL-6-enriched environments (e.g., adjuvant-primed mouse peritoneum), the cell dies of the increased intracellular binding between the Ab and the cellular autoantigen. In the case in which telomerase is the autoantigen, mouse hybridoma cells synthesizing such an autoantibody, which appeared to grow well in culture, could not grow in syngeneic BALB/c mice to form ascites, but grew nevertheless in athymic siblings. Culture experiments demonstrated that peritoneal cell-derived IL-6 (and accessory factors) affected the growth and functions of the hybridoma cells, including the induction of mitochondria-based apoptosis. Electron microscopy revealed an abundance of Abs in the nuclear chromatin of IL-6-stimulated cells, presumably piggy-backed there by telomerase from the cytosol. This nuclear presence was confirmed by light microscopy analysis of isolated nuclei. In two other cases, hybridoma cells synthesizing an autoantibody to GTP or osteopontin also showed similar growth inhibition in vivo. In all cases, Ab function was crucial to the demise of the cells. Thus, autoreactive cells, which synthesize autoantibodies to certain intracellular Ags, live delicately between life and death depending on the cytokine microenvironment. Paradoxically, IL-6, which is normally growth-potentiating for B cells, is proapoptotic for these cells. The findings reveal potential strategies and targets for immunotherapy.     

Steroid regulation of cell specific secreted phosphoprotein 1 (osteopontin) expression in the pregnant porcine uterus

Secreted phosphoprotein 1 (SPP1, commonly referred to as osteopontin and formerly known as bone sialoprotein 1, early T-lymphocyte activation 1) is an extracellular matrix/adhesion molecule that is upregulated in the pregnant uterus of all mammals examined to date. This study focused on the pig, which has true epitheliochorial placentation and exhibits induction of SPP1 mRNA in luminal epithelium (LE) just before conceptus attachment and in glandular epithelium (GE) after Day 30 of pregnancy. The objective of this study was to determine steroid regulation of SPP1 mRNA and protein in porcine uterine epithelium. To examine the effect of estrogen, cyclic gilts were treated daily (Days 11-14) with 5 mg estradiol benzoate (i.m.) and hysterectomized on Day 15. To evaluate the long-term effect of pseudopregnancy, cyclic gilts were given daily injections (Days 11-15) with steroid as above and hysterectomized on Day 90. In situ hybridization showed high expression of SPP1 mRNA only in LE contiguous with apposing conceptus tissue on Day 15 of pregnancy. In contrast, estrogen injection resulted in moderate but uniform SPP1 mRNA in all LE of Day 15 nonpregnant gilts, with expression maintained through Day 90 of pseudopregnancy. SPP1 mRNA also localized to the GE of Day 90 pseudopregnant gilts, similar to expression in late gestation. Consistent with in situ hybridization results, SPP1 protein localized to the apical surface of LE in all estrogen-treated gilts and in the GE on Day 90 of pseudopregnancy. We conclude that, in pregnant pigs, SPP1 is induced by conceptus estrogen in uterine LE and is regulated in GE in a manner coincident with CL/placental progesterone production.     

Territorial and mating behavior ofXylocopa fimbriata F. andXylocopa gualanensis Cockerell from Costa Rica

The territorial and mating behavior of two Xylocopaspecies X. fimbriataF. and X. gualanensisCockerell from Costa Rica are described. Male territorial activity was common during February through April in the higher areas of a dry forest savanna. Both species maintained territories at the same location within hollows in the foliage of Ardesia revolutaH.B.K., a small evergreen tree which occurs commonly in the dry forest. Xylocopa fimbriatamaintained territories during early morning hours, whereas the territorial period for X. gualanensisoccurred during late afternoon. Males of both species infrequently marked territories. It is suggested that a secretion released from their mesosomal glands is wiped onto their legs during frequent leg-body and leg rubbing which occurs while males hover. The secretion is occasionally applied to marking points on vegetation by their secretion-contaminated legs. Males repeatedly marked select locations when females were near their territories regardless of whether females were searching nearby branches for nesting sites, gathering provisions, or passing through their areas. Some females oriented to and entered certain male territories. An example of female mate choice in selection of male territories is provided. Females flew into male territories from 2–4 cm down-wind of male scent-marked locations. As females approached these locations, males hovered downwind in close proximity to the females' approach path. As females hovered a few centimeters downwind of the scented focal point, males approached from downwind and mounted. The males behavior and the role of scent-marking in the reproductive behavior of lek polygyny is discussed.     

A comparative study of the ground nests of Centris flavifrons and Centris aethiocesta (Hymenoptera: Anthophoridae)

The nests of Centris aethiocesta Snelling and Centris flavifrons (Friese) are described and compared to published accounts of C. flavofasciata. The three are ground nesting solitary bee species in Costa Rica. Centris aethiocesta, like previously published C. flavofasciata, prefers the beach strand, although C. aethiocesta nests in more shaded areas. Centris flavifrons nests are constructed in the dry forest savanna, often in small depressions. All three construct one cell per tunnel, with the tunnel of completed nests filled with soil. Tunnels of completed nests of C. aethiocesta are unbranched; tunnels of C. flavifrons have 6 to 10 side tunnels also filled with soil, while only one side tunnel is published for C. flavofasciata.     

Eggs and clutches of the Spheroolithidae from the Cretaceous Tiantai basin,Zhejiang Province,China

Numerous discoveries in the Tiantai basin of Zhejiang Province, China, enrich our understanding of the parataxonomy, paleobiology and taphonomic histories of fossil eggs from a diverse array of Cretaceous oofamilies. We describe the most abundant of these egg types catalogued in the Zhejiang Museum of Natural History, Spheroolithus cf. zhangtoucaoensis (oofamily Spheroolithidae). Scanning electron microscopy, here utilised for the first time on Spheroolithus eggs from Tiantai, and petrographic microscopy reveal 0.81–1.37-mm thick eggshell composed of a single structural layer of calcite with slightly flaring shell units, irregular pores, horizontal accretion lines and a sweeping extinction pattern under crossed polars. This contradicts previous reports of the presence of two structural layers in Tiantai Spheroolithus. Clutches consist of 2–13 eggs arranged in an irregular, single-layered pattern. As these eggs are among the oldest Asian examples of Spheroolithus currently known, they may shed light on the early evolution of this oogenus. This study establishes the definitive presence of the Spheroolithidae in the Tiantai basin, contra recent reports, and provides the framework for ongoing examination of egg diversity and taphonomy in the Tiantai basin.     

Sediment and eggshell interactions: using abrasion to assess transport in fossil eggshell accumulations

Recent excavation at the Late Cretaceous Egg Mountain locality in Montana revealed abundant Spheroolithus eggshell fragments. The depositional history of the fragments is problematic due to the vertical dispersal of eggshell in homogeneous mudstone. The results of previous studies assessing eggshell transport are based on thin, modern eggshell occurring on well-defined horizons. Therefore, the assessment of depositional history required a different method. Chicken (Gallus gallus domesticus) and ostrich (Struthio camelus) eggshells were placed in a tumbler with water and quartz sand for 168–504 h to simulate transport. The resulting wear on these fragments, revealed under scanning electron microscope, was compared to unabraded eggshell. In addition, the modern eggshell was compared to fossil eggshells from a nesting site, crevasse splay and channel deposits, and Egg Mountain. Fossil eggshells from high-energy paleoenvironments display similar edge rounding to modern eggshell placed in the tumbler. In contrast, eggshell from Egg Mountain lacked edge rounding and resembled fossil eggshell from a nesting site and unabraded modern eggshell, suggesting that the Egg Mountain locality represents a parautochthonous assemblage. This study indicates that sediment interaction leaves distinct patterns of abrasion on eggshell, which may be useful for assessing transport of eggshell at fossil localities.