bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Authors: Ashokkumar Balasubramaniem , Nabokina Svetlana M. , Ma Thomas Y. , Said Hamid M.
Published: July 2009

The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant ( P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant ( P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC.
Experimental Study of the Effects of Marrow Mesenchymal Stem Cells Transfected with Hypoxia‐Inducible Factor‐1α Gene
Authors: Yang Jinfu , Tang Tao , Li Feng , Zhou Wenwu , Liu Jian , Tan Zhiping , Zheng Wei , Yang Yifeng , Zhou Xinmin , Hu Jianguo
Published: July 2009

Objective. To construct the eukaryotic expression vector hypoxia‐inducible factor 1α‐pcDNA3.1 and to investigate its transfective efficiency into mesenchymal stem cells (MSCs) in vitro and the expression of HIF‐1α gene in MSCs. Methods. mRNA of Wistar Rats′ myocardial cells was extracted, and cDNA was synthesized with Reverse Transcription Kit, HIF‐1α was amplified by polymerase chain reaction (PCR), and constructed into pcDNA3.1. Transfected HIF‐1α‐pcDNA3.1 into MSCs by liposome mediated method. The expression of HIF‐1α in the cells was detected by Western Blot Analysis and ELISA. Results. Eukaryotic expression vector HIF‐1α‐pcDNA3.1 was constructed successfully. Analyzed by flow cytometer, The MSCs′ surfaces mark were CD44+, SH3(CD73)+, CD34−, CD45− and the CD44+ cells and SH3(CD73)+ cells were 94.7% and 97.3%, respectively, showing the high purity of the cultured MSCs. After inducing, the cultured MSCs can differentiate into osteoblasts and adipocytes successfully. In HIF‐1α gene transfected MSCs, the expression of HIF‐1α mRNA and HIF‐1α protein were both increased obviously. Conclusion. HIF‐1α was cloned successfully. HIF‐1α‐pcDNA3.1 can be transfected into MSCs by liposome‐mediated method effectively and which resulting stable expression of
HIF‐1α in transfected MSCs.
Numerical Simulation of 980 nm-LD-Pumped Yb3+-Er3+-Tm3+-Codoped Fiber Amplifier for 1500 nm and 1600 nm Bands
Authors: Jiang Chun
Published: June 2009

The theoretical model of Yb3+-Er3+-Tm3+-codoped fiber amplifier pumped by 980 nm laser
is proposed, and the rate and power propagation equations are
numerically solved to analyze the dependences of the gains at
1500 nm and 1600 nm bands on the activator
concentrations, fiber length, pump power, and signal wavelength.
The numerical results show that our model is in good agreement
with experimental result, and with pump power of 200 mW and
fiber length varying from 0.15 to 1.5 m, the gains at the
two bands may reach 10.0–20.0 dB when the codoping
concentrations of Yb3+, Er3+, and Tm3+ are in the ranges 1.0–3.0×1025, 1.0–3.0×1024, and 1.0–3.0×1024 ions/m3, respectively. The fiber parameters may be optimized to flatten the gain spectra.
Crystal Structural and Functional Analysis of the Putative Dipeptidase fromPyrococcus horikoshiiOT3
Authors: Jeyakanthan Jeyaraman , Takada Katsumi , Sawano Masahide , Ogasahara Kyoko , Mizutani Hisashi , Kunishima Naoki , Yokoyama Shigeyuki , Yutani Katsuhide
Published: June 2009

The crystal structure of a putative dipeptidase (Phdpd) fromPyrococcus horikoshiiOT3 was solved using X-ray data at 2.4 Å resolution. The protein is folded into two distinct entities. The N-terminal domain consists of the general topology of theα/βfold, and the C-terminal domain consists of five long mixed strands, four helices, and two310helices. The structure ofPhdpd is quite similar to reported structures of prolidases fromP. furiosus(Zn-Pfprol) andP. horikoshii(Zn-Phdpd), where Zn ions are observed in the active site resulting in an inactive form. However,Phdpd did not contain metals in the crystal structure and showed prolidase activity in the absence of additional Co ions, whereas the specific activities increased by 5 times in the presence of a sufficient concentration (1.2 mM) of Co ions. The substrate specificities (X-Pro) ofPhdpd were broad compared with those of Zn-Phdpd in the presence of Co ions, whose relative activities are 10% or less for substrates other than Met-Pro, which is the most favorable substrate. The binding constants of Zn-Phdpd with three metals (Zn, Co, and Mn) were higher than those ofPhdpd and that with Zn was higher by greater than 2 orders, which were determined by DSC experiments. From the structural comparison of both forms and the above experimental results, it could be elucidated why the protein withZn2+ions is inactive.
Recovering Decay Rates from Noisy Measurements with Maximum Entropy in the Mean
Authors: Gzyl Henryk , Ter Horst Enrique
Published: June 2009

We present a new method, based on the method of maximum entropy in the
mean, which builds upon the standard method of maximum entropy, to improve the parametric estimation of a decay rate when the measurements are corrupted by large level of
noise and, more importantly, when the number of measurements is small. The method is
developed in the context on a concrete example: that of estimation of the parameter in an
exponential distribution. We show how to obtain an estimator with the noise filtered out,
and using simulated data, we compare the performance of our method with the Bayesian
and maximum likelihood approaches.
The Estimation of the Time Constant of the Human Inner Ear Pressure Change by Noninvasive Technique
Authors: Traboulsi Raghida , Poumarat Georges , Chazal Jean , Avan Paul , Mom Thierry , Ronchan-Cole Isabelle , Traboulsi Salam
Published: June 2009

We propose a noninvasive method to estimate the time constant. The calculation of this factor permits us to understand the pressure variations of the inner ear and also predict the behavior of the flow resistance of the cochlear aqueduct. A set of mathematical relationships incorporating the intralabyrinthine pressure, the intracranial pressure, and the time constant was applied. The modeling process describes the hydrodynamic effects of the cerebrospinal fluid in the intralabyrinthine fluid space, where the input and output of the created model are, respectively, the sinusoidal variation of the respiration signal and the distortion product of otoacoustic emissions. The obtained results were compared with those obtained by different invasive techniques. A long time constant was detected each time when the intracranial pressure increased; this phenomenon is related to the role of the cochlear aqueduct described elsewhere. The interpretation of this model has revealed the ability of these predictions to provide a greater precision for hydrodynamic variation of the inner ear, consequently the variation of the dynamic process of the cerebrospinal fluid.
On Rational Approximations to Euler′s Constant γ and to γ + log (a/b)
Authors: Elsner Carsten
Published: June 2009

The author continues to study series transformations for
the Euler‐Mascheroni constant γ. Here, we discuss in detail recently published
results of A. I. Aptekarev and T. Rivoal who found rational approximations to γ and γ + log q (q ∈ ℚ>0) defined by linear recurrence formulae. The main purpose of
this paper is to adapt the concept of linear series transformations with integral
coefficients such that rationals are given by explicit formulae which approximate γ and γ + log q. It is shown that for every q ∈ ℚ>0 and every integer d ≥ 42 there are infinitely many rationals am/bm for m = 1, 2, … such that |γ + log q − am/bm|≪ and bm | Zm with log Zm ~ 12d2m2 for m tending to infinity.
Flow through a Two‐Scale Porosity Material
Authors: Andersson A. G. , Westerberg L. G. , Papathanasiou T. D. , Lundström T. Staffan
Published: July 2009

Flow through a two‐scale porous medium is here investigated by a unique comparison between simulations performed with computational fluid dynamics and the boundary element method with microparticle image velocimetry in model geometries.
Convergence Comparison of Several Iteration Algorithms for the Common Fixed Point Problems
Authors: Song Yisheng , Liu Xiao
Published: June 2009

We discuss the following viscosity approximations with the weak contraction "Equation missing" for a non-expansive mapping sequence "Equation missing", "Equation missing", "Equation missing". We prove that Browder's and Halpern's type convergence theorems imply Moudafi's viscosity approximations with the weak contraction, and give the estimate of convergence rate between Halpern's type iteration and Mouda's viscosity approximations with the weak contraction.
Convergence Theorems of Three-Step Iterative Scheme for a Finite Family of Uniformly Quasi-Lipschitzian Mappings in Convex Metric Spaces
Authors: You-xian Tian , Chun-de Yang
Published: June 2009

We consider a new Noor-type iterative procedure with errors for approximating the common fixed point of a finite family of uniformly quasi-Lipschitzian mappings in convex metric spaces. Under appropriate conditions, some convergence theorems are proved for such iterative sequences involving a finite family of uniformly quasi-Lipschitzian mappings. The results presented in this paper extend, improve and unify some main results in previous work.
Increased Manganese Superoxide Dismutase Expression or Treatment with Manganese Porphyrin Potentiates Dexamethasone-Induced Apoptosis in Lymphoma Cells
Authors: Jaramillo Melba C. , Frye Jennifer B. , Crapo James D. , Briehl Margaret M. , Tome Margaret E.
Published: July 2009

Glucocorticoid-induced apoptosis is exploited for the treatment of hematologic malignancies. Innate and acquired resistance limits treatment efficacy; however, resistance mechanisms are not well understood. Previously, using WEHI7.2 murine thymic lymphoma cells, we found that increasing the resistance to hydrogen peroxide (H2O2) by catalase transfection or selection for H2O2 resistance caused glucocorticoid resistance. This suggests the possibility that increasing H2O2 sensitivity could sensitize the cells to glucocorticoids. In other cell types, increasing manganese superoxide dismutase (MnSOD) can increase intracellular H2O2. The current study showed that increased expression of MnSOD sensitized WEHI7.2 cells to glucocorticoid-induced apoptosis and H2O2. Treatment of WEHI7.2 cells with the catalytic antioxidant Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+), a manganoporphyrin, mimicked the effects of increased MnSOD expression. MnTE-2-PyP5+ also sensitized WEHI7.2 cells to cyclophosphamide and inhibited cell growth; it had no effect on the WEHI7.2 cell response to doxorubicin or vincristine. In primary follicular lymphoma cells, MnTE-2-PyP5+ increased cell death due to dexamethasone. Treatment of H9c2 cardiomyocytes with MnTE-2-PyP5+ inhibited doxorubicin cytotoxicity. The profile of MnTE-2-PyP5+ effects suggests MnTE-2-PyP5+ has potential for use in hematologic malignancies that are treated with glucocorticoids, cyclophosphamide, and doxorubicin. [Cancer Res 2009;69(13):5450–7]
A Ruthenium-Containing Organometallic Compound Reduces Tumor Growth through Induction of the Endoplasmic Reticulum Stress GeneCHOP
Authors: Meng Xiangjun , Leyva Mili L. , Jenny Marjorie , Gross Isabelle , Benosman Samir , Fricker Bastien , Harlepp Sébastien , Hébraud Pascal , Boos Anne , Wlosik Pauline , Bischoff Pierre , Sirlin Claude , Pfeffer Michel , Loeffler Jean-Philippe , Gaiddon Christian
Published: July 2009

Cisplatin-derived anticancer therapy has been used for three decades despite its side effects. Other types of organometallic complexes, namely, some ruthenium-derived compounds (RDC), which would display cytotoxicity through different modes of action, might represent alternative therapeutic agents. We have studied both in vitro and in vivo the biological properties of RDC11, one of the most active compounds of a new class of RDCs that contain a covalent bond between the ruthenium atom and a carbon. We showed that RDC11 inhibited the growth of various tumors implanted in mice more efficiently than cisplatin. Importantly, in striking contrast with cisplatin, RDC11 did not cause severe side effects on the liver, kidneys, or the neuronal sensory system. We analyzed the mode of action of RDC11 and showed that RDC11 interacted poorly with DNA and induced only limited DNA damages compared with cisplatin, suggesting alternative transduction pathways. Indeed, we found that target genes of the endoplasmic reticulum stress pathway, such as Bip, XBP1, PDI, and CHOP, were activated in RDC11-treated cells. Induction of the transcription factor CHOP, a crucial mediator of endoplasmic reticulum stress apoptosis, was also confirmed in tumors treated with RDC11. Activation of CHOP led to the expression of several of its target genes, including proapoptotic genes. In addition, the silencing of CHOP by RNA interference significantly reduced the cytotoxicity of RDC11. Altogether, our results led us to conclude that RDC11 acts by an atypical pathway involving CHOP and endoplasmic reticulum stress, and thus might provide an interesting alternative for anticancer therapy. [Cancer Res 2009;69(13):5458–66]
Lessons from an Aggressive Cancer: Evolutionary Dynamics in Esophageal Carcinoma
Authors: Klein Christoph A. , Stoecklein Nikolas H.
Published: July 2009

Rapid progression to metastatic disease and an intrinsic resistance to any type of systemic therapy are hallmarks of aggressive solid cancers. The molecular basis for this phenotype is not clear. A detailed study of the somatic progression from local to early systemic esophageal cancer revealed rapid diversification of cancer cells isolated from various sites, but also evidence for early clonal expansion. These findings have implications for diagnostic pathology and therapeutic decision making. [Cancer Res 2009;69(13):5285–8]
Breast Cancer Lung Metastasis Requires Expression of Chemokine Receptor CCR4 and Regulatory T Cells
Authors: Olkhanud Purevdorj B. , Baatar Dolgor , Bodogai Monica , Hakim Fran , Gress Ronald , Anderson Robin L. , Deng Jie , Xu Mai , Briest Susanne , Biragyn Arya
Published: July 2009

Cancer metastasis is a leading cause of cancer morbidity and mortality. More needs to be learned about mechanisms that control this process. In particular, the role of chemokine receptors in metastasis remains controversial. Here, using a highly metastatic breast cancer (4T1) model, we show that lung metastasis is a feature of only a proportion of the tumor cells that express CCR4. Moreover, the primary tumor growing in mammary pads activates remotely the expression of TARC/CCL17 and MDC/CCL22 in the lungs. These chemokines acting through CCR4 attract both tumor and immune cells. However, CCR4-mediated chemotaxis was not sufficient to produce metastasis, as tumor cells in the lung were efficiently eliminated by natural killer (NK) cells. Lung metastasis required CCR4+ regulatory T cells (Treg), which directly killed NK cells using β-galactoside–binding protein. Thus, strategies that abrogate any part of this process should improve the outcome through activation of effector cells and prevention of tumor cell migration. We confirm this prediction by killing CCR4+ cells through delivery of TARC-fused toxins or depleting Tregs and preventing lung metastasis. [Cancer Res 2009;69(14):5996–6004]
The Liver Is a Site for Tumor-Induced Myeloid-Derived Suppressor Cell Accumulation and Immunosuppression
Authors: Ilkovitch Dan , Lopez Diana M.
Published: July 2009

Tumor-induced immunosuppression plays a key role in tumor evasion of the immune system. A key cell population recognized as myeloid-derived suppressor cells (MDSC) contributes and helps orchestrate this immunosuppression. MDSC can interact with T cells, macrophages, and natural killer cells to create an environment favorable for tumor progression. In various tumor models, their presence at high levels has been reported in the bone marrow, blood, spleen, and tumor. We report for the first time that MDSC accumulate and home to the liver in addition to the other organs. Liver MDSC suppress T cells and accumulate to levels comparable with splenic MDSC. Additionally, hematopoiesis in the liver contributes to the dramatic expansion of MDSC in this organ. Furthermore, MDSC in the liver interact with macrophages, also known as Kupffer cells, and cause their up-regulation of PD-L1, a negative T-cell costimulatory molecule. The liver is thus an organ in which MDSC accumulate and can contribute to immunosuppression directly and indirectly. MDSC play a role in various pathologic states in addition to cancer, and these results contribute to our understanding of their biology and interactions with immune-related cells. [Cancer Res 2009;69(13):5514–21]
Identification of Inhibitors of ABCG2 by a Bioluminescence Imaging–Based High-Throughput Assay
Authors: Zhang Yimao , Byun Youngjoo , Ren Yunzhao R. , Liu Jun O. , Laterra John , Pomper Martin G.
Published: July 2009

ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)–based assay was developed to allow high-throughput compound screening. This assay exploits our finding that d-luciferin, the substrate of firefly luciferase (fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of d-luciferin and enhance bioluminescence signal by increasing intracellular d-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty-seven compounds showed BLI enhancement, a measure of anti-ABCG2 activity, of ≥5-fold, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g., mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from a FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing. [Cancer Res 2009;69(14):5867–75]
Depletion of Embryonic Stem Cell Signature by Histone Deacetylase Inhibitor in NCCIT Cells: Involvement of Nanog Suppression
Authors: You Jueng Soo , Kang Jae Ku , Seo Dong-Wan , Park Jae Hyun , Park Jong Woo , Lee Jae Cheol , Jeon Yae Jee , Cho Eun Jung , Han Jeung-Whan
Published: July 2009

The embryonic stem cell-like gene expression signature has been shown to be associated with poorly differentiated aggressive human tumors and has attracted great attention as a potential target for future cancer therapies. Here, we investigate the potential of the embryonic stem cell signature as molecular target for the therapy and the strategy to suppress the embryonic stem cell signature. The core stemness gene Nanog is abnormally overexpressed in human embryonic carcinoma NCCIT cells showing gene expression profiles similar to embryonic stem cells. Down-regulation of the gene by either small interfering RNAs targeting Nanog or histone deacetylase inhibitor apicidin causes reversion of expression pattern of embryonic stem cell signature including Oct4, Sox2, and their target genes, leading to cell cycle arrest, inhibition of colony formation in soft agar, and induction of differentiation into all three germ layers. These effects are antagonized by reintroduction of Nanog. Interestingly, embryonic carcinoma cells (NCCIT, NTERA2, and P19) exhibit a higher sensitivity to apicidin in down-regulation of Nanog compared with embryonic stem cells. Furthermore, the down-regulation of Nanog expression by apicidin is mediated by a coordinated change in recruitment of epigenetic modulators and transcription factors to the promoter region. These findings indicate that overexpression of stemness gene Nanog in NCCIT cells is associated with maintaining stem cell-like phenotype and suggest that targeting Nanog might be an approach for improved therapy of poorly differentiated tumors. [Cancer Res 2009;69(14):5716–25]
Cell Survival under Stress Is Enhanced by a Mitochondrial ATP-Binding Cassette Transporter That Regulates Hemoproteins
Authors: Lynch John , Fukuda Yu , Krishnamurthy Partha , Du Guoqing , Schuetz John D.
Published: July 2009

The ATP-binding cassette (ABC) transporter ABCB6 localizes to the mitochondria, where it imports porphyrins and up-regulates de novo porphyrin synthesis. If ABCB6 also increases the intracellular heme concentration, it may broadly affect the regulation and physiology of cellular hemoproteins. We tested whether the ability of ABCB6 to accelerate de novo porphyrin biosynthesis alters mitochondrial and extramitochondrial heme levels. ABCB6 overexpression increased the quantity of cytosolic heme but did not affect mitochondrial heme levels. We then tested whether the increased extramitochondrial heme would increase the concentration and/or activity of cellular hemoproteins (hemoglobin, catalase, and cytochrome c oxidase). ABCB6 overexpression increased the activity and quantity of hemoproteins found in several subcellular compartments, and reduction of ABCB6 function (by small interfering RNA or knockout) reversed these findings. In complementary studies, suppression of ABCB6 expression sensitized cells to stress induced by peroxide and cyanide, whereas overexpression of ABCB6 protected against both stressors. Our findings show that the ability of ABCB6 to increase cytosolic heme levels produces phenotypic changes in hemoproteins that protect cells from certain stresses. Collectively, these findings have implications for the health and survival of both normal and abnormal cells, which rely on heme for multiple cellular processes. [Cancer Res 2009;69(13):5560–7]
Negative Regulation of β4 Integrin Transcription by Homeodomain-Interacting Protein Kinase 2 and p53 Impairs Tumor Progression
Authors: Bon Giulia , Di Carlo Selene E. , Folgiero Valentina , Avetrani Paolo , Lazzari Chiara , D'Orazi Gabriella , Brizzi Maria Felice , Sacchi Ada , Soddu Silvia , Blandino Giovanni , Mottolese Marcella , Falcioni Rita
Published: July 2009

Increased expression of α6β4 integrin in several epithelial cancers promotes tumor progression; however, the mechanism underlying its transcriptional regulation remains unclear. Here, we show that depletion of homeodomain-interacting protein kinase 2 (HIPK2) activates β4 transcription that results in a strong increase of β4-dependent mitogen-activated protein kinase and Akt phosphorylation, anchorage-independent growth, and invasion. In contrast, stabilization of HIPK2 represses β4 expression in wild-type p53 (wtp53)-expressing cells but not in p53-null cells or cells expressing mutant p53, indicating that HIPK2 requires a wtp53 to inhibit β4 transcription. Consistent with our in vitro findings, a strong correlation between β4 overexpression and HIPK2 inactivation by cytoplasmic relocalization was observed in wtp53-expressing human breast carcinomas. Under loss of function of HIPK2 or p53, the p53 family members TAp63 and TAp73 strongly activate β4 transcription. These data, by revealing that β4 expression is transcriptionally repressed in tumors by HIPK2 and p53 to impair β4-dependent tumor progression, suggest that loss of p53 function favors the formation of coactivator complex with the TA members of the p53 family to allow β4 transcription. [Cancer Res 2009;69(14):5978–86]
The Inhibitor of Apoptosis Protein Livin (ML-IAP) Plays a Dual Role in Tumorigenicity
Authors: Abd-Elrahman Ihab , Hershko Klilah , Neuman Tzahi , Nachmias Boaz , Perlman Riki , Ben-Yehuda Dina
Published: July 2009

The inhibitor of apoptosis protein (IAP) family can inhibit apoptosis induced by a variety of stimuli. We and others previously described the IAP Livin (ML-IAP). We found that Livin is unique among the IAP members as, on a strong apoptotic stimulus, it is specifically cleaved by caspases to produce a truncated protein with paradoxical proapoptotic activity (tLivin). We also showed that Livin encodes two splicing variants, termed Livin α and β, with diverse antiapoptotic effects in vitro. In this study, we compared the Livin isoforms in vivo. An animal model was established and the effects of Livin α and β on the initiation and development of tumors were compared. In the animal model, Livin α promotes tumor initiation in comparison with control. Interestingly, the growth of tumors originating from cells expressing Livin β was inhibited. In these tumors, Livin β was cleaved and produced a high level of the proapoptotic tLivin β that repressed tumor development. When we eliminated the proapoptotic effect of Livin β by point mutations, the resulting antiapoptotic Livin β mutants contributed to tumor progression. In terms of mechanism, we show that Livin β tumors develop only in mice lacking natural killer (NK) cell activity. Thus, from the animal model, we can conclude that Livin plays a major role in tumorigenicity and that NK cells induce cleavage of Livin to its proapoptotic truncated protein that in turn inhibits tumor growth. Therefore, Livin and tLivin may serve as potential targets for cancer therapy. [Cancer Res 2009;69(13):5475–80]
Protein Kinase D Regulates Cell Migration by Direct Phosphorylation of the Cofilin Phosphatase Slingshot 1 Like
Authors: Peterburs Philipp , Heering Johanna , Link Gisela , Pfizenmaier Klaus , Olayioye Monilola A. , Hausser Angelika
Published: July 2009

Protein kinase D (PKD) has been identified as a negative regulator of epithelial cell migration; however, its molecular substrates and downstream signaling pathways that mediate this activity have remained elusive. In this study, we provide evidence that the cofilin phosphatase slingshot 1 like (SSH1L), an important regulator of the complex actin remodeling machinery, is a novel in vivo PKD substrate. PKD-mediated phosphorylation of serines 937 and 978 regulates SSH1L subcellular localization by binding of 14-3-3 proteins and thus impacts the control of local cofilin activation and actin remodeling during cell migration. In line with this, we show that the loss of PKD decreases cofilin phosphorylation, induces a more spread cell morphology, and stimulates chemotactic migration of breast cancer cells in an SSHL1-dependent fashion. Our data thus identify PKD as a central regulator of the cofilin signaling network via direct phosphorylation and regulation of SSH1L. [Cancer Res 2009;69(14):5634–8]
Development of Tumor-Reactive T Cells After Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplant for Chronic Lymphocytic Leukemia
Authors: Nishida Tetsuya , Hudecek Michael , Kostic Ana , Bleakley Marie , Warren Edus H. , Maloney David , Storb Rainer , Riddell Stanley R.
Published: July 2009

Purpose: Allogeneic nonmyeloablative hematopoietic stem cell transplant (NM-HSCT) can result in durable remission of chronic lymphocytic leukemia (CLL). It is thought that the efficacy of NM-HSCT is mediated by recognition of tumor cells by T cells in the donor stem cell graft. We evaluated the development of CTLs specific for CLL after NM-HSCT to determine if their presence correlated with antitumor efficacy.

Experimental Design: Peripheral blood mononuclear cells obtained from 12 transplant recipients at intervals after NM-HSCT were stimulated in vitro with CLL cells. Polyclonal T-cell lines and CD8+ T-cell clones were derived from these cultures and evaluated for lysis of donor and recipient target cells including CLL. The presence and specificity of responses was correlated with clinical outcomes.

Results: Eight of the 12 patients achieved remission or a major antitumor response and all 8 developed CD8+ and CD4+ T cells specific for antigens expressed by CLL. A clonal analysis of the CD8+ T-cell response identified T cells specific for multiple minor histocompatibility (H) antigens expressed on CLL in six of the responding patients. A significant fraction of the CD8+ T-cell response in some patients was also directed against nonshared tumor-specific antigens. By contrast, CLL-reactive T cells were not detected in the four patients who had persistent CLL after NM-HSCT, despite the development of graft-versus-host disease.

Conclusions: The development of a diverse T-cell response specific for minor H and tumor-associated antigens expressed by CLL predicts an effective graft-versus-leukemia response after NM-HSCT.
Id1 Is Down-Regulated by Hepatocyte Growth Factor via ERK-Dependent and ERK-Independent Signaling Pathways, Leading to Increased Expression of p16INK4a in Hepatoma Cells
Authors: Ushio Kazutaka , Hashimoto Tomio , Kitamura Naomi , Tanaka Toshiaki
Published: July 2009

Hepatocyte growth factor (HGF) inhibits the proliferation of several tumor cell lines and tumor growth in vivo. We showed previously that HGF induces cell cycle arrest at G1 in a human hepatoma cell line, HepG2, by up-regulating the expression of p16INK4a through strong activation of extracellular signal-regulated kinase (ERK). However, although essential, the activation was not sufficient for the up-regulation of p16. In this study, we examined regulatory mechanisms of p16 expression through a transcription factor, Ets, which has been shown previously to bind to the promoter. The treatment of HepG2 cells with HGF induced ERK-dependent phosphorylation of Ets, which leads to its activation, before the up-regulation of p16, suggesting that another factor suppresses Ets activity. We found that HGF reduces the amount of Id1, which is a dominant-negative inhibitor of Ets, leading to a decrease in Ets associated with Id1. Id1 was down-regulated via transcriptional regulation not via the ubiquitin-proteasome-mediated pathway. Inhibition of the HGF-induced high-intensity ERK activity had a modest effect on the Id1 down-regulation, and inhibition of the phosphatidylinositol 3-kinase pathway had no effect, showing that Id1 is regulated by ERK-dependent and -independent pathways other than the phosphatidylinositol 3-kinase pathway. Exogenously expressed Id1 suppressed the up-regulation of p16 by HGF and the antiproliferative effect of HGF. Knockdown of Id1 significantly enhanced the activity of the p16 promoter coordinately with the activation of ERK. Our results indicated that down-regulation of Id1 plays a key role in the inhibitory effect of HGF on cell proliferation and provides a molecular basis for cancer therapy with HGF. (Mol Cancer Res 2009;7(7):1179–88)
Adolescent Learning and the Internet
Authors: Strom Paris , Strom Robert , Wing Charlotte , Beckert Troy
Published: July 2009

Students were electronically polled about the influence of the Internet in their education and ways teachers could use this resource to motivate them and increase their learning. Responses from the 956 adolescents showed that the school quest to increase student engagement could more often be met through online assignments that facilitate self-directed learning. The practice of teachers spending much of their time preparing and presenting lessons should become more balanced to reflect the powerful presence of the Internet as the main source of student information. This shift requires a new focus in teacher preparation and in-service development for local schools. Specific changes in the roles of principals and teachers to offer greater school support for Internet learning are identified.
BCR-ABL promotes the frequency of mutagenic single-strand annealing DNA repair
Authors: Fernandes Margret S. , Reddy Mamatha M. , Gonneville Jeffrey R. , DeRoo Scott C. , Podar Klaus , Griffin James D. , Weinstock David M. , Sattler Martin
Published: July 2009

Intracellular oxidative stress in cells transformed by the BCR-ABL oncogene is associated with increased DNA double-strand breaks. Imprecise repair of these breaks can result in the accumulation of mutations, leading to therapy-related drug resistance and disease progression. Using several BCR-ABL model systems, we found that BCR-ABL specifically promotes the repair of double-strand breaks through single-strand annealing (SSA), a mutagenic pathway that involves sequence repeats. Moreover, our results suggest that mutagenic SSA repair can be regulated through the interplay between BCR-ABL and extrinsic growth factors. Increased SSA activity required Y177 in BCR-ABL, as well as a functional PI3K and Ras pathway downstream of this site. Furthermore, our data hint at a common pathway for DSB repair whereby BCR-ABL, Tel-ABL, Tel-PDGFR, FLT3-ITD, and Jak2V617F all increase mutagenic repair. This increase in SSA may not be sufficiently suppressed by tyrosine kinase inhibitors in the stromal microenvironment. Therefore, drugs that target growth factor receptor signaling represent potential therapeutic agents to combat tyrosine kinase-induced genomic instability.
Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion
Authors: Anong William A. , Franco Taina , Chu Haiyan , Weis Tahlia L. , Devlin Emily E. , Bodine David M. , An Xiuli , Mohandas Narla , Low Philip S.
Published: July 2009

The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3–ankyrin–β-spectrin and glycophorin C–protein 4.1–β-spectrin.1–7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C–protein 4.1 interaction has little effect on membrane stability.8 We demonstrate the existence of a novel band 3–adducin–spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3–adducin–spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane.
Mitogen-activated protein kinases and NFκB are involved in SP-A-enhanced responses of macrophages to mycobacteria
Authors: Lopez Joseph P , Vigerust David J , Shepherd Virginia L
Published: July 2009

Background

Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFκB in mediating the enhanced signaling in response to BCG in the presence of SP-A.

Methods

Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 μg of SP-A to 5 × 105BCG for 30 min at 37°C. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined.

Results

Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFκB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IκB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively.

Conclusion

These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFκB.
Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH)
Authors: Chaudhuri Roy R , Allen Andrew G , Owen Paul J , Shalom Gil , Stone Karl , Harrison Marcus , Burgis Timothy A , Lockyer Michael , Garcia-Lara Jorge , Foster Simon J , Pleasance Stephen J , Peters Sarah E , Maskell Duncan J , Charles Ian G
Published: July 2009

Background

In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth.

Results

We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes.

Conclusion

We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.
Effects of clinical pathways in the joint replacement: a meta-analysis
Authors: Barbieri A , Vanhaecht K , Van Herck P , Sermeus W , Faggiano F , Marchisio S , Panella M
Published: July 2009

Background

A meta-analysis was performed to evaluate the use of clinical pathways for hip and knee joint replacements when compared with standard medical care. The impact of clinical pathways was evaluated assessing the major outcomes of in-hospital hip and knee joint replacement processes: postoperative complications, number of patients discharged at home, length of in-hospital stay and direct costs.

Methods

Medline, Cinahl, Embase and the Cochrane Central Register of Controlled Trials were searched. The search was performed from 1975 to 2007. Each study was assessed independently by two reviewers. The assessment of methodological quality of the included studies was based on the Jadad methodological approach and on the New Castle Ottawa Scale. Data analysis abided by the guidelines set out by The Cochrane Collaboration regarding statistical methods. Meta-analyses were performed using RevMan software, version 4.2.

Results

Twenty-two studies met the study inclusion criteria and were included in the meta-analysis for a total sample of 6,316 patients. The aggregate overall results showed significantly fewer patients suffering postoperative complications in the clinical pathways group when compared with the standard care group. A shorter length of stay in the clinical pathway group was also observed and lower costs during hospital stay were associated with the use of the clinical pathways. No significant differences were found in the rates of discharge to home.

Conclusion

The results of this meta-analysis show that clinical pathways can significantly improve the quality of care even if it is not possible to conclude that the implementation of clinical pathways is a cost-effective process, because none of the included studies analysed the cost of the development and implementation of the pathways. Based on the results we assume that pathways have impact on the organisation of care if the care process is structured in a standardised way, teams critically analyse the actual organisation of the process and the multidisciplinary team is highly involved in the re-organisation. Further studies should focus on the evaluation of pathways as complex interventions to help to understand which mechanisms within the clinical pathways can really improve the quality of care. With the need for knee and hip joint replacement on the rise, the use of clinical pathways might contribute to better quality of care and cost-effectiveness.
Clinical Implications of Microsatellite Instability and MLH1 Gene Inactivation in Sporadic Insulinomas
Authors: Mei Mei , Deng Dajun , Liu Tong-Hua , Sang Xin-Ting , Lu Xin , Xiang Hong-Ding , Zhou Jing , Wu HaiYan , Yang YingMai , Chen Jie , Lu Chong-Mei , Chen Yuan-Jia
Published: July 2009

Context: The molecular pathogenesis of sporadic insulinomas is unknown. There is a lack of biomarker to distinguish benign and malignant form of insulinoma.

Objective: Our objective was to confirm the occurrence of microsatellite instability (MSI) in insulinomas, to identify alterations of mismatch repair (MMR) genes in the tumors, and to evaluate the possibility to distinguish benign and malignant insulinoma or to predict the clinical outcome of patients with these alterations.

Design and Patients: We detected MSI and inactivation of MLH1 gene in 55 sporadic insulinomas by PCR, immunohistochemical staining, allelic typing, analysis of promoter methylation, and exon mutations. Their correlations with clinicopathological characteristics were analyzed with univariate and multivariate statistic analysis.

Results: A high rate of MSI (MSI-H) was found in 33% of sporadic insulinomas. Reduced expression of mutL homolog 1 (MLH1) protein was observed in 36% of insulinomas and correlated with MSI-H (P = 0.008). Promoter methylation and loss of heterozygosity of MLH1 gene was found in 31 and 49% of insulinomas, respectively. Reduced expression of MLH1 and MSI-H were significantly associated with both tumor malignancy (P = 0.033 and P = 4.8 × 10−6, respectively) and incurable disease (P = 0.006 and P = 0.001, respectively).

Conclusion: High frequency of MSI occurred in sporadic insulinomas. The silencing of MLH1 gene may partially contribute to the MSI-H in the tumors. Assessing MSI-H and expressions of MLH1 could be used to distinguish benign and malignant insulinomas and to predict the outcome of patients.

Detecting of a high rate of microsatellite instability can be used to distinguish malignancy from benign, and predict clinical outcome of the sporadic insulinomas.
Adrenal Function during Childhood and Puberty in Daughters of Women with Polycystic Ovary Syndrome
Authors: Maliqueo Manuel , Sir-Petermann Teresa , Pérez Virginia , Echiburú Bárbara , Ladrón de Guevara Amanda , Gálvez Carla , Crisosto Nicolás , Azziz Ricardo
Published: July 2009

Context: In some patients, PCOS may develop as a consequence of an exaggerated adrenarche during pubertal development.

Objective: The aim of the study was to assess adrenal function during childhood and pubertal development in daughters of women with PCOS (PCOSd).

Design: We included 98 PCOSd [64 during childhood (ages 4–8 yr) and 34 during the peripubertal period (ages 9–13 yr)] and 51 daughters of control women (Cd) [30 during childhood and 21 during the peripubertal period]. In both groups, an acute ACTH-(1–24) stimulation test (0.25 mg) and an oral glucose tolerance test were performed. Bone age and serum concentrations of cortisol, androstenedione, 17-hydroxyprogesterone, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), glucose, and insulin were determined.

Results: PCOSd and Cd were similar in age and body mass index. During the peripubertal period, basal and poststimulated DHEAS concentrations were higher in PCOSd compared to Cd. Among PCOSd, 12.5% of girls in childhood and 32.4% in peripuberty presented biochemical evidence of exaggerated adrenarche. Stimulated insulin was higher in PCOSd compared to Cd during childhood (P = 0.03) and peripuberty (P = 0.03). An advancement of 8 months between bone and chronological age was observed in peripubertal PCOSd compared to Cd.

Conclusions: In PCOSd, basal and stimulated DHEAS concentrations were higher during the onset of puberty. Around 30% of the PCOSd demonstrated an exacerbated adrenarche, which may reflect increased P450c17 activity. In addition, a modest advance in bone age was observed, probably secondary to the hyperinsulinemia and/or adrenal hyperandrogenism.

Daughters of women with PCOS exhibit increased dehydroepiandrosterone sulfate serum concentrations and biochemical evidence of an exacerbated adrenarche.