Loss of the orphan nuclear receptor SHP is more pronounced in fibrolamellar carcinoma than in typical hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a major problem in oncology. The molecular mechanisms which underlie its pathogenesis are poorly understood. Recently the Small Heterodimer Partner (SHP), an orphan nuclear receptor, was suggested to be involved as a tumor suppressor in hepatocellular carcinoma development. To date, there are no such studies regarding fibrolamellar carcinoma, a less common variant of HCC, which usually affects young people and displays distinct morphological features. The aim of our project was to evaluate the SHP levels in typical and fibrolamellar hepatocellular carcinoma with respect to the levels of one of the cell cycle regulators, cyclin D1. We assessed the immunoreactivity levels of SHP and cyclin D1 in 48 typical hepatocellular carcinomas, 9 tumors representing the fibrolamellar variant, 29 non malignant liver tissues and 7 macroregenerative nodules. We detected significantly lower SHP immunoreactivity in hepatocellular carcinoma when compared to non malignant liver tissue. Moreover, we found that SHP immunoreactivity is reduced in fibrolamellar carcinoma when compared to typical hepatocellular carcinoma. We also found that SHP is more commonly lost in HCC which arises in the liver with steatosis. The comparison between the cyclin D1 and SHP expression revealed the negative correlation between these proteins in the high grade HCC. Our results indicate that the impact of loss of SHP protein may be even more pronounced in fibrolamellar carcinoma than in a typical form of HCC. Further investigation of mechanisms through which the loss of SHP function may influence HCC formation may provide important information in order to design more effective HCC therapy.     

siRNA carriers based on carbosilane dendrimers affect zeta potential and size of phospholipid vesicles

One of the major limitations in gene therapy is an inability of naked siRNA to passively diffuse through negatively charged cell membranes. Therefore, the siRNA transport into a cell requires efficient carriers. In this work we analyzed the charge-dependent interaction of the complexes of cationic carbosilane dendrimers (CBD) and anti-HIV siRNA (dendriplexes) with the model membranes - large unilamellar vesicles (LUV). We used the second generation of branched with CBD carbon-silicon bonds (CBD-CS) which are water-stable and that of oxygen-silicon bonds (CBD-OS) which are slowly hydrolyzed in aqueous solutions. The LUVs were composed of zwitterionic dimyristoylphosphatidylcholine (DMPC), negatively charged dipalmitoylphosphatidylglycerol (DPPG) and their mixture (DMPC/DPPG, molar ratio 7:3). The interaction of dendriplexes with LUVs affected both zeta potential and size of the vesicles. The changes of these values were larger for the negatively charged LUV. CBD-CS resulted in the decrease of zeta potential values to more negative ones, whereas an opposite effect took place for CBD-OS suggesting a different kind of interaction between LUVs and the dendriplexes. The results indicate that both CBD-CS and CBD-OS can be used for transport of siRNA into the cells. However, CBD-CS are preferred due to a better stability in water and improved bioavailability of siRNA on their surface.     

Investigation of a wide spectrum of inherited metabolic disorders by 13C NMR spectroscopy

High-resolution (1)H NMR spectroscopy of body fluids has proved to be very useful in diagnostics of inherited metabolic diseases, whereas (13)C NMR remains almost unexploited. In this paper the application of (13)C NMR spectroscopy of fivefold concentrated urine samples for diagnosis of selected metabolic diseases is reported. Various marker metabolites were identified in test urine samples from 33 patients suffering from 10 different diseases, providing information which could be crucial for their diagnoses. Spectra were accumulated for 2 h or overnight when using spectrometers operating at 9.4 or 4.7 T magnetic fields, respectively. Interpretation of the measurement results was based on a comparison of the peak positions in the measured spectrum with reference data. The paper contains a table with (13)C NMR chemical shifts of 73 standard compounds. The method can be applied individually or as an auxiliary technique to (1)H NMR or any other analytical method.     

An involvement of polokinases in control of progress of the cell-cycle--the mechanism of transient translocation and formation of an activated protein-protein complexes during mitosis

Polokinases are a subfamily of the mitotic serine/threonine kinases involved in coordination of a run of mitosis of eukaryotic cells. The main polo-like-kinase 1p (PLK1) is a passenger protein transiently localized to centrosomes, kinetochores and central spindle during mitosis and is required for bi-orientation of the normal metaphase spindle. Its activity is regulated at the level of protein stability and by action of upstream kinases, so that it peaks in metaphase and drops as cells exit mitosis. Regulation of location and activity of Plk1p is bi-phasic: the COOH terminal polo box domain binds to an array of mitotic phosphoproteins and followed by an allosteric conformation is activated to phosphorylate many its substrates. These mode of action involves polokinases into critical transitions of the cell cycle phases, and in control at some checkpoints of this cycle.     

Structure and serological properties of the O-antigen of two clinical Proteus mirabilis strains classified into a new Proteus O77 serogroup

Two Proteus mirabilis strains, 3 B-m and 3 B-k, were isolated from urine and faeces of a hospitalized patient from Lodz, Poland. It was suggested that one strain originated from the other, and the presence of the bacilli in the patient's urinary tract was most probably a consequence of autoinfection. The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of P. mirabilis 3 B-m and studied by sugar analysis and nuclear magnetic resonance spectroscopy, including two-dimensional rotating frame Overhause effect spectroscopy (ROESY) and 1H,13C heteronuclear single quantum coherence (HSQC) experiments. The following structure of the linear trisaccharide-repeating unit of the O-polysaccharide was established:-->2)-beta-D-Glcp-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where 6dTal2Ac stands for 2-O-acetyl-6-deoxy-L-talose. It resembles the structure of the O-polysaccharide of Proteus penneri O66, which includes additional lateral residues of 2,3-diacetamido-2,3,6-trideoxy-L-mannose. The lipopolysaccharides from two P. mirabilis strains studied were serologically identical to each other but not to that from any of the existing 76 Proteus O-serogroups. Therefore, the strains were classified into a new O77 serogroup specially created in the genus Proteus. Serological studies using Western blot and enzyme-linked immunosorbent assay with intact and adsorbed O-antisera showed that the P. mirabilis O77 antigen is related to Proteus vulgaris O2 and P. penneri O68 antigens, and a putative disaccharide epitope responsible for the cross-reactivity was revealed.     

Statins impair antitumor effects of rituximab by inducing conformational changes of CD20

Background

Rituximab is used in the treatment of CD20⁺ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas.

Methods and Findings

Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a ⁵¹Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.

Conclusions

Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.     

Efficiency of nitrogen and phosphorus utilization in progenies of factorial crosses between European and exotic cultivars of spring barley (Hordeum vulgare L.)

Previous observations suggested that some landraces, primitive cultivars and other barleys originating from the harsh conditions of the Middle East, North Africa and Tibet might serve as interesting sources of adaptation to low-input agriculture. This opportunity was verified in field experiments performed under reduced rates of nitrogen (N) and phosphorus (P) fertilization. Genetic variation in major components of the efficiency of N and P utilization was examined among F₂ and F₃ generations of crosses between four European female and seven exotic male lines of spring barley. Preponderance of general combining ability effects was found for most efficiency components, suggesting that the characters are mainly controlled by additive gene action. Non-additive effects appeared to be less or non-significant. Under reduced fertilization, hybrid progenies of the Syrian cv. M.Dingo/D.Alla 106 and the Moroccan cv. Moroc 9–75 exhibit an enhanced yielding and the highest capacity to utilize N and P efficiently in grain mass formation. The results proved that the exotic barleys appear to be particularly useful for breeding programs aiming at improved barley adaptation to less favourable fertilization regimes.     

Biodistribution and Efficacy Studies of the Proteasome Inhibitor BSc2118 in a Mouse Melanoma Model

Inhibition of the proteasome offers many therapeutic possibilities in inflammation as well as in neoplastic diseases. However, clinical use of proteasome inhibitors is limited by the development of resistance or severe side effects. In our study we characterized the anti-tumor properties of the novel proteasome inhibitor BSc2118. The sensitivity of tumor lines to BSc2118 was analyzed in comparison to bortezomib using crystal violet staining in order to assess cell viability. The In Vivo distribution of BSc2118 in mouse tissues was tracked by a fluorescent-modified form of BSc2118 (BSc2118-FL) and visualized by confocal microscopy. Inhibition of the 20S proteasome was monitored both in cultured cell lines and in mice, respectively. Finally, safety and efficacy of BSc2118 was evaluated in a mouse melanoma model. BSc2118 inhibits proliferation of different tumor cell lines with a similar potency as compared with bortezomib. Systemic administration of BSc2118 in mice is well tolerated, even when given in a dose of 60 mg/kg body weight. After systemic injection of BSc2118 or bortezomib similar proteasome inhibition patterns are observed within the murine organs. Detection of BSc2118-FL revealed correlation of distribution pattern of BSc2118 with inhibition of proteasomal activity in cells or mouse tissues. Finally, administration of BSc2118 in a mouse melanoma model shows significant local anti-tumor effects. Concluding, BSc2118 represents a novel low-toxic agent that might be alternatively used for known proteasome inhibitors in anti-cancer treatment.