bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of a novel DNA decoy that inhibits the oncogenic β-catenin/T-cell factor pathway
Authors: Seki Yosuke , Yamamoto Hirofumi , Yee Ngan Chew , Yasui Masayoshi , Tomita Naruya , Kitani Kotaro , Takemasa Ichiro , Ikeda Masataka , Sekimoto Mitsugu , Matsuura Nariaki , Albanese Chris , Kaneda Yasufumi , Pestell Richard G. , Monden Morito
Published: April 2006

The oncogenic β-catenin/T-cell factor (TCF) signal is a common trigger inducing expressions of various cancer-related genes and is activated in various types of human malignancy. The aim of this study was to create an effective double-stranded DNA decoy that would interfere with endogenous TCF hyperactivity in tumor cells. We first established the TCF-activated model using nontumor human embryonic kidney 293 (HEK293) cells by introducing a β-catenin cDNA. Based on a consensus TCF-binding sequence in the cyclin D1 and c-myc promoters, several double-stranded oligodeoxynucleotides were designed and tested for their ability to inhibit TCF activity in the HEK293 model. Among them, the 18-mer oligodeoxynucleotide stably formed double-stranded DNA and efficiently inhibited TCF activity. FITC-labeled oligodeoxynucleotide was efficiently incorporated into the nucleus at 6 hours and remained within cells for up to 72 to 96 hours. When compared with scrambled oligodeoxynucleotide, we found that the 18-mer TCF decoy significantly inhibited TCF activity and promoter activities of the downstream target genes, such as cyclin D1, c-myc, and matrix metalloproteinase 7 in HCT116 colon cancer cells. Reverse transcription-PCR assays indicated that mRNA expression of these genes decreased with treatment of the TCF decoy. Proliferation assay showed that the TCF decoy significantly inhibited growth of HCT116 tumor cells, but not of nontumor HEK293 cells. Our data provide evidence that the TCF decoy reduced both TCF activity and transcriptional activation of downstream target genes. Thus, this TCF decoy is potentially an efficient and nontoxic molecular targeting therapy for controlling malignant properties of cancer cells. [Mol Cancer Ther 2006;5(4):985–94]
Diarylureas are small-molecule inhibitors of insulin-like growth factor I receptor signaling and breast cancer cell growth
Authors: Gable Karissa L. , Maddux Betty A. , Penaranda Cristina , Zavodovskaya Marianna , Campbell Michael J. , Lobo Margaret , Robinson Louise , Schow Steven , Kerner John A. , Goldfine Ira D. , Youngren Jack F.
Published: April 2006

In breast and certain other cancers, receptor tyrosine kinases, including the insulin-like growth factor I receptor (IGF-IR), play an important role in promoting the oncogenic process. The IGF-IR is therefore an important target for developing new anti–breast cancer therapies. An initial screening of a chemical library against the IGF-IR in breast cancer cells identified a diaryl urea compound as a potent inhibitor of IGF-IR signaling. This class of compounds has not been studied as inhibitors of the IGF-IR. We studied the effectiveness of one diaryl urea compound, PQ401, at antagonizing IGF-IR signaling and inhibiting breast cancer cell growth in culture and in vivo. PQ401 inhibited autophosphorylation of the IGF-IR in cultured human MCF-7 cells with an IC50 of 12 μmol/L and autophosphorylation of the isolated kinase domain of the IGF-IR with an IC50 <1 μmol/L. In addition, PQ401 inhibited the growth of cultured breast cancer cells in serum at 10 μmol/L. PQ401 was even more effective at inhibiting IGF-I-stimulated growth of MCF-7 cells (IC50, 6 μmol/L). Treatment of MCF-7 cells with PQ401 was associated with a decrease in IGF-I-mediated signaling through the Akt antiapoptotic pathway. Twenty-four hours of treatment with 15 μmol/L PQ401 induced caspase-mediated apoptosis. In vivo, treatment with PQ401 (i.p. injection thrice a week) reduced the growth rate of MCNeuA cells implanted into mice. These studies indicate that diaryl urea compounds are potential new agents to test in the treatment of breast and other IGF-I-sensitive cancers. [Mol Cancer Ther 2006;5(4):1079–86]
Preclinical activity of ABT-869, a multitargeted receptor tyrosine kinase inhibitor
Authors: Albert Daniel H. , Tapang Paul , Magoc Terrance J. , Pease Lori J. , Reuter David R. , Wei Ru-Qi , Li Junling , Guo Jun , Bousquet Peter F. , Ghoreishi-Haack Nayereh S. , Wang Baole , Bukofzer Gail T. , Wang Yi-Chun , Stavropoulos Jason A. , Hartandi Kresna , Niquette Amanda L. , Soni Nirupama , Johnson Eric F. , McCall J. Owen , Bouska Jennifer J. , Luo Yanping , Donawho Cherrie K. , Dai Yujia , Marcotte Patrick A. , Glaser Keith B. , Michaelides Michael R. , Davidsen Steven K.
Published: April 2006

ABT-869 is a structurally novel, receptor tyrosine kinase (RTK) inhibitor that is a potent inhibitor of members of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor families (e.g., KDR IC50 = 4 nmol/L) but has much less activity (IC50s > 1 μmol/L) against unrelated RTKs, soluble tyrosine kinases, or serine/threonine kinases. The inhibition profile of ABT-869 is evident in cellular assays of RTK phosphorylation (IC50 = 2, 4, and 7 nmol/L for PDGFR-β, KDR, and CSF-1R, respectively) and VEGF-stimulated proliferation (IC50 = 0.2 nmol/L for human endothelial cells). ABT-869 is not a general antiproliferative agent because, in most cancer cells, >1,000-fold higher concentrations of ABT-869 are required for inhibition of proliferation. However, ABT-869 exhibits potent antiproliferative and apoptotic effects on cancer cells whose proliferation is dependent on mutant kinases, such as FLT3. In vivo ABT-869 is effective orally in the mechanism-based murine models of VEGF-induced uterine edema (ED50 = 0.5 mg/kg) and corneal angiogenesis (>50% inhibition, 15 mg/kg). In tumor growth studies, ABT-869 exhibits efficacy in human fibrosarcoma and breast, colon, and small cell lung carcinoma xenograft models (ED50 = 1.5–5 mg/kg, twice daily) and is also effective (>50% inhibition) in orthotopic breast and glioma models. Reduction in tumor size and tumor regression was observed in epidermoid carcinoma and leukemia xenograft models, respectively. In combination, ABT-869 produced at least additive effects when given with cytotoxic therapies. Based on pharmacokinetic analysis from tumor growth studies, efficacy correlated more strongly with time over a threshold value (cellular KDR IC50 corrected for plasma protein binding = 0.08 μg/mL, ≥7 hours) than with plasma area under the curve or Cmax. These results support clinical assessment of ABT-869 as a therapeutic agent for cancer. [Mol Cancer Ther 2006;5(4):995–1006]
The dietary flavonoid apigenin sensitizes malignant tumor cells to tumor necrosis factor–related apoptosis-inducing ligand
Authors: Horinaka Mano , Yoshida Tatsushi , Shiraishi Takumi , Nakata Susumu , Wakada Miki , Sakai Toshiyuki
Published: April 2006

Dietary flavonoid apigenin is expected to have preventive and therapeutic potential against malignant tumors. In this report, we show for the first time that apigenin markedly induces the expression of death receptor 5 (DR5) and synergistically acts with exogenous soluble recombinant human tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) to induce apoptosis in malignant tumor cells. TRAIL is a promising candidate for cancer therapeutics due to its ability to selectively induce apoptosis in cancer cells. The combined use of apigenin and TRAIL at suboptimal concentrations induces Bcl-2-interacting domain cleavage and the activation of caspases-8, -10, -9, and -3. Furthermore, human recombinant DR5/Fc chimera protein and caspase inhibitors dramatically inhibit apoptosis induced by the combination of apigenin and TRAIL. On the other hand, apigenin-mediated induction of DR5 expression is not observed in normal human peripheral blood mononuclear cells. Moreover, apigenin does not sensitize normal human peripheral blood mononuclear cells to TRAIL-induced apoptosis. These results suggest that this combined treatment with apigenin and TRAIL might be promising as a new therapy against malignant tumors. [Mol Cancer Ther 2006;5(4):945–51]
Potent activation of mitochondria-mediated apoptosis and arrest in S and M phases of cancer cells by a broccoli sprout extract
Authors: Tang Li , Zhang Yuesheng , Jobson Hillary E. , Li Jun , Stephenson Katherine K. , Wade Kristina L. , Fahey Jed W.
Published: April 2006

We have previously shown that broccoli sprouts are a rich source of chemopreventive isothiocyanates, which potently induce carcinogen-detoxifying enzymes and inhibit the development of mammary and skin tumors in rodents. However, the principal isothiocyanate present in broccoli sprout extracts, sulforaphane, not only induces carcinogen-detoxifying enzymes but also activates apoptosis and blocks cell cycle progression. In this article, we show that an aqueous extract of broccoli sprouts potently inhibits the growth of human bladder carcinoma cells in culture and that this inhibition is almost exclusively due to the isothiocyanates. Isothiocyanates are present in broccoli sprouts as their glucosinolate precursors and blocking their conversion to isothiocyanates abolishes the antiproliferative activity of the extract. Moreover, the potency of isothiocyanates in the extract in inhibiting cancer cell growth was almost identical to that of synthetic sulforaphane, as judged by their IC50 values (6.6 versus 6.8 μmol/L), suggesting that other isothiocyanates in the extract may be biologically similar to sulforaphane and that nonisothiocyanate substances in the extract may not interfere with the antiproliferative activity of the isothiocyanates. Further study showed that the isothiocyanate extract of broccoli sprouts activated the mitochondria-mediated apoptosis pathway and halted cells in S and M phases. Cell cycle arrest was associated with down-regulation of Cdc25C and disruption of mitotic spindles. These data show that broccoli sprout isothiocyanate extract is a highly promising substance for cancer prevention/treatment and that its antiproliferative activity is exclusively derived from isothiocyanates. [Mol Cancer Ther 2006;5(4):935–44]
Cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal HER2/neuexpressing tumors
Authors: Helguera Gustavo , Rodríguez José A. , Penichet Manuel L.
Published: April 2006

We have previously generated antihuman HER2/neu–humanized IgG3 fused to interleukin-2 (IL-2), IL-12, or granulocyte macrophage colony-stimulating factor (GM-CSF) [monofunctional fusion proteins (mono-AbFP)] or fused to IL-2 and IL-12 or IL-12 and GM-CSF [bifunctional fusion proteins (bi-AbFP)]. These AbFPs retained cytokine and antigen-binding activities. We have now further characterized the AbFPs and determined the heparin-binding activity of the fused cytokines, their ability to trigger IFN-γ secretion and natural killer (NK) activation, and their direct antitumor efficacy. Flow cytometry revealed heparin-binding activity in the AbFPs containing IL-12 and IL-2, although this activity seems to be decreased in the bi-AbFPs. However, both bi-AbFPs retained the capacity to stimulate IL-12-dependent IFN-γ secretion in the NK cell line KY-1, and IL-12/IL-2 bi-AbFP induced NK activity in splenocytes. The antitumor effectiveness of bi-AbFPs and mono-AbFP combinations was studied in mice challenged i.p. with three different human HER2/neu murine syngeneic models (D2F2/E2, CT26-HER2/neu, and MC38-HER2/neu). Although a significant variability in the profile of antitumor response was observed in the different tumor models, the combination of IL-12 and GM-CSF mono-AbFPs protected 100% of D2F2/E2-challenged and 75% of CT26-HER2/neu–challenged mice. In contrast, bi-AbFPs protected less than the combination of mono-AbFPs and, in some models, even less than mono-AbFPs alone. However, in all cases, most of long-term survivors showed protection after s.c. rechallenge with the tumors and later with the parental tumors not expressing HER2/neu. These results show that, although the pattern of protection is tumor model dependent, treatments with AbFPs can effectively generate high levels of protection against peritoneal tumors expressing HER2/neu, which may be relevant in patients with primary or metastatic peritoneal carcinomatosis that may be observed in ovarian, colon, stomach, bladder, lung, and breast cancers. [Mol Cancer Ther 2006;5(4):1029–40]
Integrin-linked kinase functions as a downstream mediator of endothelin-1 to promote invasive behavior in ovarian carcinoma
Authors: Rosanò Laura , Spinella Francesca , Di Castro Valeriana , Dedhar Shoukat , Nicotra Maria Rita , Natali Pier Giorgio , Bagnato Anna
Published: April 2006

The endothelin-1 (ET-1) axis represents a novel target in several malignancies, including ovarian carcinoma. Upon being activated, the endothelin A receptor (ETAR) mediates multiple tumor-promoting activities, including mitogenesis, escape from apoptosis, angiogenesis, metastasis-related protease activation, epithelial-mesenchymal transition, and invasion. Integrin-linked kinase (ILK) is a multidomain focal adhesion protein that conveys intracellular signaling elicited by β1-integrin and growth factor receptors. In this study, we investigate whether the signaling triggered by ETAR leading to an aggressive phenotype is mediated by an ILK-dependent mechanism. In HEY and OVCA 433 ovarian carcinoma cell lines, activation of ETAR by ET-1 enhances the expression of α2β1 and α3β1 integrins. ILK activity increases as ovarian cancer cells adhere to type I collagen through β1 integrin signaling, and do so to a greater extent on ET-1 stimulation. ET-1 increases ILK mRNA and protein expression and activity in a time- and concentration-dependent manner. An ILK small-molecule inhibitor (KP-392) or transfection with a dominant-negative ILK mutant effectively blocks the phosphorylation of downstream signals, Akt and glycogen synthase kinase-3β. The blockade of ET-1/ETAR-induced ILK activity results in an inhibition of matrix metalloproteinase activation as well as of cell motility and invasiveness in a phosphoinositide 3 kinase–dependent manner. In ovarian carcinoma xenografts, ABT-627, a specific ETAR antagonist, suppresses ILK expression, Akt and glycogen synthase kinase-3β phosphorylation, and tumor growth. These data show that ILK functions as a downstream mediator of the ET-1/ETAR axis to potentiate aggressive cellular behavior. Thus, the ILK-related signaling cascade can be efficiently targeted by pharmacologic blockade of ETAR. [Mol Cancer Ther 2006;5(4):833–42]
Efficacy of a nitric oxide–releasing nonsteroidal anti-inflammatory drug and cytotoxic drugs in human colon cancer cell linesin vitroand xenografts
Authors: Leonetti Carlo , Scarsella Marco , Zupi Gabriella , Zoli Wainer , Amadori Dino , Medri Laura , Fabbri Francesco , Rosetti Marco , Ulivi Paola , Cecconetto Lorenzo , Bolla Manlio , Tesei Anna
Published: April 2006

We previously showed that NCX 4040 inhibits in vitro and in vivo tumor growth and induces apoptosis in human colon cancer cell lines. On the basis of these results, NCX 4040 antitumor activity in combination with 5-fluorouracil (5-FU) or oxaliplatin was evaluated in vitro and in vivo in human colon cancer models. The cytotoxicity of different NCX 4040 and 5-FU or oxaliplatin combination schemes was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr, and LRWZ) by the sulforhodamine B assay, and apoptosis was assessed by flow cytometry. NCX 4040 and 5-FU combination was always additive in vitro regardless of the scheme used. Sequential NCX 4040→oxaliplatin treatment produced a strong synergism in three cell lines, with a ratio index ranging from 3.7 to 4. The synergistic effect was accompanied by apoptosis induction (up to 40%). In the in vivo experiments, xenografted mice were treated with the sequential combination of NCX 4040 and oxaliplatin, and apoptosis was evaluated immunohistochemically in excised tumors. Furthermore, in WiDr xenografts, this sequence caused a significantly higher reduction (∼60%) in tumor growth compared with single-drug treatments and produced extensive apoptotic cell death (15.3%), significantly higher (P < 0.01) than that observed in untreated tumors (2.7%) or in tumors treated with NCX 4040 (5.1%) or oxaliplatin (5.7%) alone. These data show that NCX 4040 sensitizes colon cancer cell lines to the effect of antitumor drugs and suggests that their combination could be useful for the clinical management of colon cancer. [Mol Cancer Ther 2006;5(4):919–26]
Promise of vitamin D analogues in the treatment of hyperproliferative conditions
Authors: Masuda Sonoko , Jones Glenville
Published: April 2006

1α,25-Dihydroxyvitamin D3 [1α,25-(OH)2D3; calcitriol] is best known as a hormone involved in calcium homeostasis but is also a potent antiproliferative agent in many cell types, particularly epithelial cells. 1α,25(OH)2D3 mediates its actions through a classic steroid hormone-like transcriptional mechanism by influencing the expression of hundreds of genes. Effects of 1α,25(OH)2D3 have been observed on expression of cell cycle regulators, growth factors and their receptors, apoptotic machinery, metastatic potential, and angiogenesis; all of which have some effect on hyperproliferative conditions. This minireview focuses on the anticancer potential of 1α,25(OH)2D3 and its analogues by summarizing the promising data from animal and human trials of 1α,25(OH)2D3 and some of the more interesting synthetic vitamin D analogues in the treatment of a variety of different animal cancer models and in human patients with advanced cancer. Optimal administration of vitamin D analogues is only just being achieved with high-dose intermittent administration overcoming bioavailability and hypercalcemia problems and combination therapy with cytotoxic agents (taxols and cisplatins), antiresorptive agents (bisphosphonates), or cytochrome P450 inhibitors being attempted. Although the potential of vitamin D as an antiproliferative drug has been realized in the treatment of psoriasis and in parathyroid cell hyperplasia associated with secondary hyperparathyroidism, the search for an anticancer treatment incorporating a vitamin D analogue remains elusive. [Mol Cancer Ther 2006;5(4):797–808]
Intracellular uptake and intracavitary targeting of folate-conjugated liposomes in a mouse lymphoma model with up-regulated folate receptors
Authors: Shmeeda Hilary , Mak Lidia , Tzemach Dina , Astrahan Peleg , Tarshish Mark , Gabizon Alberto
Published: April 2006

The folate receptor is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anticancer agents to folate receptor–expressing tumors. This study examines folate-lipid conjugates as a means of enhancing the tumor selectivity of liposome-encapsulated drugs in a mouse lymphoma model. Folate-derivatized polyethylene glycol (PEG3350)-distearoyl-phosphatidylethanolamine was post-loaded at various concentrations into the following preparations: radiolabeled PEGylated liposomes, PEGylated liposomes labeled in the aqueous compartment with dextran fluorescein, and PEGylated liposomal doxorubicin (PLD, Doxil). We incubated folate-targeted radiolabeled or fluorescent liposomes with mouse J6456 lymphoma cells up-regulated for their folate receptors (J6456-FR) to determine the optimal ligand concentration required in the lipid bilayer for liposomal cell association, and to examine whether folate-targeted liposomes are internalized by J6456-FR cells in suspension. Liposomal association with cells was quantified based on radioactivity and fluorescence-activated cell sorting analysis, and internalization was assessed by confocal fluorescence microscopy. We found an optimal ligand molar concentration of ∼0.5% using our ligand. A substantial lipid dose-dependent increase in cell-associated fluorescence was found in folate-targeted liposomes compared with nontargeted liposomes. Confocal depth scanning showed that a substantial amount of the folate-targeted liposomes are internalized by J6456-FR cells. Binding and uptake of folate-targeted PLD by J6456-FR cells were also observed in vivo after i.p. injection of folate-targeted PLD in mice bearing ascitic J6456-FR tumors. The drug levels in ascitic tumor cells were increased by 17-fold, whereas those in plasma were decreased by 14-fold when folate-targeted PLD were compared with nontargeted PLD in the i.p. model. Folate-targeted liposomes represent an attractive approach for the intracellular delivery of drugs to folate receptor–expressing lymphoma cells and seem to be a promising tool for in vivo intracavitary drug targeting. [Mol Cancer Ther 2006;5(4):818–24]
Interleukin-12-deficient mice are at greater risk of UV radiation–induced skin tumors and malignant transformation of papillomas to carcinomas
Authors: Meeran Syed M. , Mantena Sudheer K. , Meleth Sreelatha , Elmets Craig A. , Katiyar Santosh K.
Published: April 2006

Solar UV radiation–induced immunosuppression is a risk factor for nonmelanoma skin cancer. Interleukin (IL)-12 has been shown to possess antitumor activity and inhibit the immunosuppressive effects of UV radiation in mice. In this study, we generated IL-12 knockout (KO) mice on a C3H/HeN background to characterize the role of IL-12 in photocarcinogenesis. After exposure of the mice to UVB (180 mJ/cm2) radiation thrice a week for 35 weeks, the development of UV-induced tumors was more rapid and the tumor multiplicity and tumor size were significantly higher in IL-12 KO mice than their wild-type (WT) counterparts (P < 0.05-0.001). Moreover, the malignant transformation of UVB-induced papillomas to carcinomas was higher in IL-12 KO mice in terms of carcinoma incidence (55%, P < 0.001), carcinoma multiplicity (77%, P < 0.001), and carcinoma size (81%, P < 0.001). As IL-12 has the ability to repair UV-induced DNA damage, we determined this effect in our in vivo IL-12 KO mouse model. We found that UVB-induced DNA damage in the form of cyclobutane pyrimidine dimers was removed or repaired more rapidly in WT mice than IL-12 KO mice. Similarly, the UVB-induced sunburn cell formation is primarily a consequence of DNA damage. It was observed that UVB-induced sunburn cells were repaired rapidly in WT mice compared with IL-12 KO mice. The rapid removal or repair of UV-induced cyclobutane pyrimidine dimers or sunburn cells will result in reduced risk of photocarcinogenesis. Taken together, our data show that IL-12 deficiency is associated with the greater risk of photocarcinogenesis in mice, and this may be due to reduction in damaged DNA repair ability. [Mol Cancer Ther 2006;5(4):825–32]
Methylation of p16 and Ras Association Domain Family Protein 1a during Colorectal Malignant Transformation
Authors: Umetani Naoyuki , de Maat Michiel F.G. , Sunami Eiji , Hiramatsu Suzanne , Martinez Steve , Hoon Dave S.B.
Published: April 2006

Accurate assessment of gene methylation in formalin-fixed, paraffin-embedded archived tissue (FF-PEAT) by microdissection remains challenging because the tissue volume is small and DNA is damaged. In addition, methods for methylation assessment, such as methylation-specific PCR (MSP), require sodium bisulfite modification (SBM) on purified DNA, which causes major loss of DNA. On-slide SBM, in which DNA is modified in situ before isolation of tumor cells, eliminates DNA purification steps and allows histology-oriented assessment of gene methylation. This study describes a protocol and use of on-slide SBM using 20 FF-PEAT of colorectal cancers with intratumoral adenoma components to detect accumulation of gene methylation during colorectal malignant transformation. Deparaffinized tissue sections were incubated in sodium bisulfite solution for 8 hours at 60°C, stained with hematoxylin, and then microdissected. Proteinase K lysate was directly used as a template in subsequent PCR. Using on-slide SBM, 282-bp-long bisulfite direct sequencing was possible. Yield of modified DNA was 2.6-fold greater than standard SBM on average. The mean conversion rate was 97%, and false-positive or false-negative results were not observed in subsequent MSP. Intratumoral heterogeneity by accumulation of p16 and Ras association domain family protein 1a methylation during malignant transformation were shown by MSP comparing cancer with adenoma parts within a single section. On-slide SBM is applicable in most methylation studies using FF-PEAT. It allows detailed, intratumoral analysis of methylation heterogeneity within solid tumors. On-slide SBM will significantly improve our approach and understanding of epigenetic events in minimal disease and the carcinogenic process. (Mol Cancer Res 2006;4(5):303–9)
Promoter Demethylation and Histone Acetylation Mediate Gene Expression of MAGE-A1, -A2, -A3, and -A12 in Human Cancer Cells
Authors: Wischnewski Frank , Pantel Klaus , Schwarzenbach Heidi
Published: April 2006

The broad range of expression of cancer-testis antigens in various tumor types makes the proteins encoded by human MAGE gene family promising targets for anticancer immunotherapy. However, a major drawback is their heterogeneous expression. In the current study, we have examined the influence of the DNA methylase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) together with the histone deacetylase inhibitor trichostatin A on the expression of MAGE-A1, -A2, -A3, and -A12 genes in different cell lines. Reverse transcription-PCR, Western blot analyses, and immunocytochemical staining show that trichostatin A was able to significantly up-regulate 5-aza-CdR-induced MAGE gene expression. Transient transfection assays with methylated reporter plasmids containing promoter fragments of the different MAGE genes show that trichostatin A was able to overcome gene silencing. In addition, the methylation status of the MAGE promoters was assessed by sodium bisulfite mapping in the various cell lines before and after stimulation with 5-aza-CdR and/or trichostatin A. In contrast to the methylation patterns, which clearly correlated with the basal MAGE RNA transcripts, up-regulation of the MAGE-A mediated by both agents only resulted in a reduction in promoter methylation ranging between 1% and 19%. In conclusion, our data show for the first time that not only hypermethylation but also histone deacetylation is responsible for the mechanism underlying MAGE gene silencing. (Mol Cancer Res 2006;4(5):339–49)
Properties of surface layer generated by new combined process of burnishing and nitriding
Authors: Wróblewski G. , Skalski K.
Published: April 2006

The results of a metal surface layer (MSL) investigation are presented in the present article. The surface layer was created using a new technological process. This process is the result of a combination of mechanical treatment (i.e. burnishing) and thermochemical treatment (i.e. nitriding). As a result of increasing the dislocation density during a burnishing treatment, which is followed by a nitriding treatment, the diffusion process in MSL is intensified. In the present article, specimens made of Armco iron (as a model material) were investigated. Four different stages of the surface layer development were investigated, i.e. initial material (after annealing), material after burnishing, material after nitriding and material treated by the combined technology. The main goal of the investigations carried out was a comparison of the changes in properties of the surface layers as a result of the different technologies. Investigations were performed on the metallography of different MSL structures, nitrogen concentration and hardness together with stress measurements.
Plasma surface engineering of precipitation hardening stainless steels
Authors: Esfandiari M. , Dong H.
Published: April 2006

Low temperature plasma nitriding of 17-4 PH martensitic and A286 austenitic precipitation hardening stainless steels has been investigated over a wide range of treatment temperatures and times. Experimental results have shown that the microstructural characteristics and properties are highly process condition dependent and there exits the possibility to provide considerable improvement in wear resistance without compromising corrosion resistance properties.
Influence of ion bombardment on structure and tribological performance of nanoscale multilayer C/Cr PVD coatings
Authors: Kok Y. N. , Wen J. G. , Petrov I. , Hovsepian P.Eh.
Published: April 2006

Low friction C/Cr coatings have been successfully deposited by the combined steered cathodic arc/unbalanced magnetron sputtering technique. The present paper focuses on the properties, tribological performance and microstructure evolution of C/Cr coatings as a function of the bias voltage Ub ranging from −65 to −350 V. As the bias voltage increases from −65 to −95 V, the structure changes from columnar to dense structure which comprises of randomly distributed onionlike carbon clusters. Further increase in the bias voltage to −350 V led to segregation and self-organisation of the carbon atoms induced by the high energy ion bombardment and finally to the formation of a new type self-organised multilayer structure. On the other hand, the phase composition transforms from graphitic (sp2 C–C bonded) to Me carbon (Cr–C bonded), where the content of the carbide phase increases with increasing the bias voltage. The C/Cr coatings showed an excellent adhesion ( Lc>70 N), with hardness ranging from 8·23 to 25 GPa depending on the bias voltage. Pin on disc tests showed that the friction coefficient was reduced from 0·22 to 0·16 when the Ub was increased from −65 to −95 V. A strong correlation was found between the microstructure, the residual stress, the sp2/sp3 content and the coating friction behaviour. Dry high speed milling trials on automotive aluminium alloy (Al–Si8Cu3Fe) showed that C/Cr coated cemented carbide end mills enhance the tool performance and the tool life compared with the uncoated tools by a factor of 2, suggesting the potential for dry high speed machining of ‘sticky’ alloys.
Preliminary study on plasma surface modification of medical grade 316LVM and high nitrogen austenitic stainless steels
Authors: Rayner B. , Li X. Y. , Dong H.
Published: April 2006

Specimens of 316LVM (F138) and high N (F1586) medical grade austenitic stainless steels (ASS) have been low temperature plasma surface alloyed with nitrogen and carbon, at temperatures between 370–550°C, to enhance their tribological properties and corrosion resistance. Systematic materials characterisation was conducted to investigate the microstructure, phase constituents, composition and surface mechanical properties of the plasma alloyed layers. The tribological and corrosion properties of the surface engineered materials have been evaluated by pin on disc tribometer and electrochemical corrosion tests. Experimental results showed that the surface hardness of both materials was significantly improved by all plasma treatments. Wear resistance was increased by up to 109 times, and wear volumes differed only slightly between treatments. Wear modes were altered from severe abrasive wear to mild oxidative wear. Anodic polarisation test results revealed that when nitrided at ≤400°C and carburised at ≤450°C corrosion properties are maintained or improved. Therefore, it is feasible to achieve combined improvements in wear and corrosion resistance for medical grade austenitic stainless, which has a great potential for the production of cost effective, long life ASS joint prostheses.
High temperature oxidation and erosion–oxidation behaviour of HVOF sprayed Ni–20Cr, WC–20Cr–7Ni and Cr3C2–Ni–20Cr coatings
Authors: Kunioshi C. T. , Correa O. V. , Ramanathan L. V.
Published: April 2006

The oxidation and erosion–oxidation (E–O) behaviour of high velocity oxy fuel sprayed Ni–20Cr alloy as well as WC and Cr3C2 cermet coatings on a steel substrate were studied. The E–O tests were carried out in a rig with specimen assemblies that were rotated through a fluidized bed of erodent particles in the temperature range 500–850°C and with erodent impact velocities of 2·5– 19·5 m s−1. Alumina powder (∼200 μm) was used as the erodent. The oxidation behaviour of the coatings was determined gravimetrically. The Ni–20Cr and Cr3C2–25(Ni–20Cr) coatings showed significantly higher oxidation resistance than the WC–20Cr–7Ni coating, especially at temperatures above 800°C. The E–O resistance of the coatings was determined by wastage, as a function of temperature. The three coatings did not show any significant change in E–O at temperatures up to 500–600°C. At higher temperatures, wastage increased with temperature, reached a maximum at 700°C and then decreased with further increase in temperature. Different E–O regimes were identified. The specimen surfaces were examined in a scanning electron microscope and their roughness determined. E–O maps have been drawn to define conditions under which the coatings undergo low, moderate and severe wastage.
Surface morphology and properties of zirconia thin films prepared at different deposition rates
Authors: Zhang D. P. , Qi H. J. , Shao S. Y. , He H. B. , Shao J. D. , Fan Z. X.
Published: April 2006

ZrO2 thin films were prepared on BK7 glass substrates by electron beam evaporation deposition method. The influence of deposition rate varying from 1·2 to 6·3 nm s−1 on surface morphology and other properties of ZrO2 films were examined. With increasing deposition rate, the surface defect density increased. The decrease in half width at full maximum in X-ray diffraction pattern with deposition rate indicates an increase in crystal dimension with increasing deposition rate. Electron beam deposited ZrO2 films are known to be inhomogeneous. From the change of the peak transmittance value, it can be deduced that the inhomogeneity of ZrO2 films strengthened gradually with increasing deposition rate. The type of surface defects changed from nodules to craters when the deposition rate was high enough.
Re-designing emergency services: the 'EGS' system
Authors: Donlon M , Cooper JC
Published: April 2006

The emergency general surgical workload is a major component of hospital service involving over a million admissions and 300,000 emergency operations per year in the UK. Providing appropriate management and continuity of care to these patients is placing an increasing strain on hospitals.
Patients' rights and responsibilities
Authors: Scowen Patricia
Published: April 2006

Patients undergoing treatment are vulnerable and have a right to expect a high level of care. There are also certain things patients can do to help ensure they receive high-quality care. The Patient Liaison Group (PLG) believes it is important for patients to understand both their rights and their responsibilities in this process and has designed Patients' Rights and Responsibilities to provide details of both. We hope this publication will be useful to surgeons and all hospital staff dealing with surgical patients, as well as to patients themselves.
Training pathway for primary care dentists in orthodontics
Authors: Drake Sharon
Published: April 2006

In some parts of the UK there is a lack of availability of orthodontic care to meet the treatment needs. With the advent of Dentists with Special Interests (DwSIs), Primary Care Trusts (PCTs) will have further opportunity to develop their clinical network in orthodontics to meet the needs of their local population through the appointment of DwSIs.
Professional indemnity – are we protected during our professional activities?
Authors: Harley Kathryn
Published: April 2006

Practising dentists must maintain 'adequate' and 'appropriate' indemnity. Following the Dentists Act 1984 (Amendment) Order 2005, this will be a legal requirement for all registered dentists, hygienists, therapists, dental nurses, technicians and clinical dental technicians. It has been introduced to protect patients from the risk of finding themselves uncompensated if they suffer harm during treatment.
Predicting stochastic gene expression dynamics in single cells
Authors: Mettetal Jerome T. , Muzzey Dale , Pedraza Juan M. , Ozbudak Ertugrul M. , van Oudenaarden Alexander
Published: April 2006

Fluctuations in protein numbers (noise) due to inherent stochastic effects in single cells can have large effects on the dynamic behavior of gene regulatory networks. Although deterministic models can predict the average network behavior, they fail to incorporate the stochasticity characteristic of gene expression, thereby limiting their relevance when single cell behaviors deviate from the population average. Recently, stochastic models have been used to predict distributions of steady-state protein levels within a population but not to predict the dynamic, presteady-state distributions. In the present work, we experimentally examine a system whose dynamics are heavily influenced by stochastic effects. We measure population distributions of protein numbers as a function of time in the
Escherichia coli
lactose uptake network (
lac
operon). We then introduce a dynamic stochastic model and show that prediction of dynamic distributions requires only a few noise parameters in addition to the rates that characterize a deterministic model. Whereas the deterministic model cannot fully capture the observed behavior, our stochastic model correctly predicts the experimental dynamics without any fit parameters. Our results provide a proof of principle for the possibility of faithfully predicting dynamic population distributions from deterministic models supplemented by a stochastic component that captures the major noise sources.
Essential role of retinoblastoma protein in mammalian hair cell development and hearing
Authors: Sage Cyrille , Huang Mingqian , Vollrath Melissa A. , Brown M. Christian , Hinds Philip W. , Corey David P. , Vetter Douglas E. , Chen Zheng-Yi
Published: April 2006

The retinoblastoma protein pRb is required for cell-cycle exit of embryonic mammalian hair cells but not for their early differentiation. However, its role in postnatal hair cells is unknown. To study the function of pRb in mature animals, we created a new conditional mouse model, with the
Rb
gene deleted primarily in the inner ear. Progeny survive up to 6 months. During early postnatal development,
pRb
−/−
hair cells continue to divide and can transduce mechanical stimuli. However, adult
pRb
−/−
mice exhibit profound hearing loss due to progressive degeneration of the organ of Corti. We show that pRb is required for the full maturation of cochlear outer hair cells, likely in a gene-specific manner, and is also essential for their survival. In addition, lack of pRb results in cell division in postnatal auditory supporting cells. In contrast, many
pRb
−/−
vestibular hair cells survive and continue to divide in adult mice. Significantly, adult
pRb
−/−
vestibular hair cells are functional, and
pRb
−/−
mice maintain partial vestibular function. Therefore, the functional adult vestibular
pRb
−/−
hair cells, derived from proliferation of postnatal hair cells, are largely integrated into vestibular pathways. This study reveals essential yet distinct roles of pRb in cochlear and vestibular hair cell maturation, function, and survival and suggests that transient block of pRb function in mature hair cells may lead to propagation of functional hair cells.
A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC
Authors: Lai Johnathan C. , Tan Wenzhi , Benimetskaya Luba , Miller Paul , Colombini Marco , Stein C. A.
Published: April 2006

G3139, an 18-mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA, can induce caspase-dependent apoptosis via the intrinsic mitochondrial pathway in 518A2 and other melanoma cells. G3139-mediated apoptosis appears to be independent of its ability to down-regulate the expression of Bcl-2 protein, because the release of mitochondrial cytochromecprecedes in time the down-regulation of Bcl-2 protein expression. In this study, we demonstrate the ability of G3139 and other phosphorothioate oligonucleotides to bind directly to mitochondria isolated from 518A2 cells. Furthermore, we show that this interaction leads to the release of cytochromecin the absence of a mitochondrial membrane permeability transition. Our data further demonstrate that there is an interaction between G3139 and VDAC, a protein that can facilitate the physiologic exchange of ATP and ADP across the outer mitochondrial membrane. Evidence from the electrophysiologic evaluation of VDAC channels reconstituted into phospholipid membranes demonstrates that G3139 is capable of producing greatly diminished channel conductance, indicating a closed state of the VDAC. This effect is oligomer length-dependent, and the ability of phosphorothioate homopolymers of thymidine of variable lengths to cause the release of cytochromecfrom isolated mitochondria of 518A2 melanoma cells can be correlated with their ability to interact with VDAC. Because it has been suggested that the closure of VDAC leads to the opening of another outer mitochondrial membrane channel through which cytochromeccan transit, thus initiating apoptosis, it appears that VDAC may be an important pharmacologic target of G3139.
Voltage sensor conformations in the open and closed states in
rosetta
structural models of K
+
channels
Authors: Yarov-Yarovoy Vladimir , Baker David , Catterall William A.
Published: April 2006

Voltage-gated ion channels control generation and propagation of action potentials in excitable cells. Significant progress has been made in understanding structure and function of the voltage-gated ion channels, highlighted by the high-resolution open-state structure of the voltage-gated potassium channel, K
v
1.2. However, because the structure of the closed state is unknown, the gating mechanism remains controversial. We adapted the
rosetta
membrane method to model the structures of the K
v
1.2 and KvAP channels using homology,
de novo
, and domain assembly methods and selected the most plausible models using a limited number of experimental constraints. Our model of K
v
1.2 in the open state is very similar in overall topology to the x-ray structure of this channel. Modeling of KvAP in the open state suggests that orientation of the voltage-sensing domain relative to the pore-forming domain is considerably different from the orientation in the K
v
1.2 open state and that the magnitude of the vertical movement of S4 is significantly greater. Structural modeling of closed state of K
v
1.2 suggests gating movement that can be viewed as a sum of two previously suggested mechanisms: translation (2–4 Å) plus rotation (≈180°) of the S4 segment as proposed in the original “sliding helix” or “helical screw” models coupled with a rolling motion of the S1–S3 segments around S4, similar to recent “transporter” models of gating. We propose a unified mechanism of voltage-dependent gating for K
v
1.2 and KvAP in which this major conformational change moves the gating charge across the electric field in an analogous way for both channels.
Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance
Authors: Earley Keith , Lawrence Richard J. , Pontes Olga , Reuther Rachel , Enciso Angel J. , Silva Manuela , Neves Nuno , Gross Michael , Viegas Wanda , Pikaard Craig S.
Published: April 2006

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.
Lentinula edodes tlg1 Encodes a Thaumatin-Like Protein That Is Involved in Lentinan Degradation and Fruiting Body Senescence
Authors: Sakamoto Yuichi , Watanabe Hisayuki , Nagai Masaru , Nakade Keiko , Takahashi Machiko , Sato Toshitsugu
Published: April 2006

Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising β-1,3-glucan with β-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichiacoli and Aspergillus oryzae exhibited β-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.
Polygalacturonase-Inhibiting Protein Interacts with Pectin through a Binding Site Formed by Four Clustered Residues of Arginine and Lysine
Authors: Spadoni Sara , Zabotina Olga , Di Matteo Adele , Mikkelsen Jørn Dalgaard , Cervone Felice , De Lorenzo Giulia , Mattei Benedetta , Bellincampi Daniela
Published: April 2006

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein that inhibits fungal polygalacturonases (PGs) and retards the invasion of plant tissues by phytopathogenic fungi. Here, we report the interaction of two PGIP isoforms from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) with both polygalacturonic acid and cell wall fractions containing uronic acids. We identify in the three-dimensional structure of PvPGIP2 a motif of four clustered arginine and lysine residues (R183, R206, K230, and R252) responsible for this binding. The four residues were mutated and the protein variants were expressed in Pichia pastoris. The ability of both wild-type and mutated proteins to bind pectins was investigated by affinity chromatography. Single mutations impaired the binding and double mutations abolished the interaction, thus indicating that the four clustered residues form the pectin-binding site. Remarkably, the binding of PGIP to pectin is displaced in vitro by PGs, suggesting that PGIP interacts with pectin and PGs through overlapping although not identical regions. The specific interaction of PGIP with polygalacturonic acid may be strategic to protect pectins from the degrading activity of fungal PGs.