bluuebunny/crossref_metadata_2025 huggingface dataset:
Preclinical Assessment of Simultaneous Targeting of Epidermal Growth Factor Receptor (ERBB1) and ERBB2 as a Strategy for Cholangiocarcinoma Therapy†,‡
Authors: Zhang Zichen , Oyesanya Regina A. , Campbell Deanna J. W. , Almenara Jorge A. , DeWitt Jennifer L. , Sirica Alphonse E.
Published: May 2010

Overexpression of epidermal growth factor receptor (ErbB1) and/or ErbB2 has been implicated in the pathogenesis of cholangiocarcinoma, suggesting that combined ErbB1/ErbB2 targeting might serve as a target-based therapeutic strategy for this highly lethal cancer. To test this strategy, we investigated targeting with the ErbB1 inhibitor tryphostin AG1517 and the ErbB2 inhibitor tryphostin AG879, in combination and alone, as well as with the dual ErbB1/ErbB2 inhibitor lapatinib, to assess the effectiveness of simultaneous targeting of ErbB1 and ErbB2 signaling over single inhibitor treatments in suppressing cholangiocarcinoma cell growth

in vitro

and the therapeutic efficacy of lapatinib

in vivo

. Our

in vitro

studies were carried out using rat (BDEneu and C611B) and human (HuCCT1 and TFK1) cholangiocarcinoma cell lines. The efficacy of lapatinib to significantly suppress liver tumor growth was tested in an orthotopic, syngeneic rat model of intrahepatic cholangiocarcinoma progression. Our results demonstrated that simultaneous targeting of ErbB1 and ErbB2 signaling was significantly more effective in suppressing the

in vitro

growth of both rat and human cholangiocarcinoma cells than individual receptor targeting. Lapatinib was an even more potent inhibitor of cholangiocarcinoma cell growth and inducer of apoptosis than either tryphostin when tested

in vitro

against these respective cholangiocarcinoma cell lines, regardless of differences in their levels of ErbB1 or ErbB2 protein expression and/or mechanism of activation. Lapatinib treatment also produced a significant suppression of intrahepatic cholangiocarcinoma growth when administered early to rats, but was without effect in inhibiting liver tumor growth in rats with more advanced tumors.

Conclusion:

Our findings suggest that simultaneous targeting of ErbB1 and ErbB2 could be a potentially selective strategy for cholangiocarcinoma therapy, but is likely to be ineffective by itself against advanced cancer.
Role of tau protein on neocortical and hippocampal oscillatory patterns
Authors: Cantero Jose L. , Moreno‐Lopez Bernardo , Portillo Federico , Rubio Alicia , Hita‐Yañez Eva , Avila Jesus
Published: May 2010

Tau is a neuronal microtubule‐associated protein implicated in microtubules stabilization, axonal establishment and elongation during neuronal morphogenesis. Because of its elevated expression in neocortical regions and hippocampus, tau might play a role in sculpting collective neural responses underlying slow and fast brain oscillations and/or long‐range synchronization patterns between hippocampus and neocortex. To test this hypothesis, local field potentials were recorded in tau‐deficient (tau−/−) and wild‐type mice from different neocortical regions and from the hippocampus during spontaneous motor exploratory behavior. We found that tau−/− mice showed hippocampal theta slowing and reduced levels of gamma long‐range synchronization involving the frontal cortex. We hypothesize that the lack of normal phosphorylated tau during early stages of development might influence the maturation of parvalbumin interneurons affecting the spatiotemporal structure of long‐range gamma synchronization. Also, the proper functioning of gap‐junction channels might be compromised by the absence of tau in hippocampal networks. Altogether, these results provide novel insights into the functional role of tau protein in the formation of collective neural responses and emergence of neocortical‐hippocampal interactions in the mammalian brain. © 2010 Wiley‐Liss, Inc.
Preserved hippocampal novelty responses following anterior temporal‐lobe resection that impairs familiarity but spares recollection
Authors: Bowles Ben , O'Neil Edward B. , Mirsattari Seyed M. , Poppenk Jordan , Köhler Stefan
Published: May 2010

Although it is well established that the integrity of the medial temporal lobe (MTL) is critical for declarative memory, the functional organization of the MTL remains a matter of intense debate. One issue that has received little consideration so far is whether the hippocampus can function normally in the presence of a lesion to perirhinal cortex that produces noticeable memory impairments. This question is intriguing as the MTL forms a hierarchical system, in which perirhinal cortex represents one of the critical nodes in the reciprocal projections between neocortical association areas and the hippocampus. Here, we used functional magnetic resonance imaging to examine whether NB, an individual who underwent surgical resection of the left anterior temporal lobe that included large aspects of perirhinal and entorhinal cortex but spared the hippocampus, exhibits intact hippocampal novelty responses to auditory sentences. Our results revealed such evidence in NB's left and right hippocampus. They complement previous behavioral work in NB, indicating that recollective processes considered to rely on hippocampal integrity are also preserved. Further analyses revealed intact novelty responses in structures that provide neuroanatomical input to the hippocampus, including remaining perirhinal cortex and surgically spared parahippocampal cortex. These findings point to viable neuroanatomical mechanisms as to how functional integrity in the hippocampus may be maintained in the face of widespread, but incomplete removal of its input structures. © 2010 Wiley‐Liss, Inc.
Pattern separation deficits associated with increased hippocampal CA3 and dentate gyrus activity in nondemented older adults
Authors: Yassa Michael A. , Lacy Joyce W. , Stark Shauna M. , Albert Marilyn S. , Gallagher Michela , Stark Craig E.L.
Published: May 2010

There is widespread evidence that memory deteriorates with aging, however the exact mechanisms that underlie these changes are not well understood. Given the growing size of the aging population, there is an imperative to study age‐related neurocognitive changes in order to better parse healthy from pathological aging. Using a behavioral paradigm that taxes pattern separation (the ability to differentiate novel yet similar information from previously learned information and thus avoid interference), we investigated age‐related neural changes in the human hippocampus using high‐resolution (1.5 mm isotropic) blood‐oxygenation level‐dependent fMRI. Recent evidence from animal studies suggests that hyperactivity in the CA3 region of the hippocampus may underlie behavioral deficits in pattern separation in aged rats. Here, we report evidence that is consistent with findings from the animal studies. We found a behavioral impairment in pattern separation in a sample of healthy older adults compared with young controls. We also found a related increase in CA3/dentate gyrus activity levels during an fMRI contrast that stresses pattern separation abilities. In a detailed analysis of behavior, we also found that the pattern of impairment was consistent with the predictions of the animal model, where larger changes in the input (greater dissimilarity) were required in order for elderly adults to successfully encode new information as distinct from previously learned information. These findings are also consistent with recent fMRI and behavioral reports in healthy aging, and further suggest that a specific functional deficit in the CA3/dentate network contributes to memory difficulties with aging. © 2010 Wiley‐Liss, Inc.
Brain‐derived neurotrophic factor val66met polymorphism and hippocampal activation during episodic encoding and retrieval tasks
Authors: Dennis Nancy A. , Cabeza Roberto , Need Anna C. , Waters‐Metenier Sheena , Goldstein David B. , LaBar Kevin S.
Published: May 2010

Brain‐derived neurotrophic factor (BDNF) is a neurotrophin which has been shown to regulate cell survival and proliferation, as well as synaptic growth and hippocampal long‐term potentiation. A naturally occurring single nucleotide polymorphism in the human BDNF gene (val66met) has been associated with altered intercellular trafficking and regulated secretion of BDNF in met compared to val carriers. Additionally, previous studies have found a relationship between the BDNF val66met genotype and functional activity in the hippocampus during episodic and working memory tasks in healthy young adults. Specifically, studies have found that met carriers exhibit both poorer performance and reduced neural activity within the medial temporal lobe (MTL) when performing episodic memory tasks. However, these studies have not been well replicated and have not considered the role of behavioral differences in the interpretation of neural differences. The current study sought to control for cognitive performance in investigating the role of the BDNF val66met genotype on neural activity associated with episodic memory. Across item and relational memory tests, met carriers exhibited increased MTL activation during both encoding and retrieval stages, compared to noncarriers. The results suggest that met carriers are able to recruit MTL activity to support successful memory processes, and reductions in cognitive performance observed in prior studies are not a ubiquitous effect associated with variants of the BDNF val66met genotype. © 2010 Wiley‐Liss, Inc.
Ultrastructural modifications of spine and synapse morphology by SAP97
Authors: Poglia Lorenzo , Muller Dominique , Nikonenko Irina
Published: May 2010

Synaptic scaffolding proteins from membrane‐associated guanylate kinases (MAGUK) family are implicated in synapse formation and functioning. To better understand the role of one of the proteins of this family, SAP97, we studied with electron microscopy the effects of its overexpression on spine and synapse morphology in CA1 pyramidal neurons of rat organotypic hippocampal slice cultures. Dramatic spine enlargement induced by SAP97 overexpression was accompanied by marked morphological changes, with spines enwrapping and engulfing presynaptic terminals. The size and complexity of the PSD was also significantly increased. Similar to PSD‐95, SAP97 promoted formation of multi‐innervated spines (MIS). In addition, both MAGUK proteins induced multiple excitatory contacts on dendritic shafts suggesting a mechanism for shaft synapse formation. Formation of MIS and shaft synapses was blocked by the nitric oxide synthase (NOS) inhibitor L‐NAME. Immunochemistry revealed that overexpression of SAP97 was associated with overexpression of PSD‐95 and recruitment of nNOS to the synapse. These data provide evidence for both common and distinct structural alterations produced by overexpression of SAP97 and PSD‐95 and demonstrate strong interactions between these two proteins to regulate contact formation through nitric oxide signaling. © 2010 Wiley‐Liss, Inc.
Major ion geochemistry and nutrient behaviour in the mixing zone of the Changjiang (Yangtze) River and its tributaries in the Three Gorges Reservoir
Authors: Ran Xiangbin , Yu Zhigang , Yao Qingzheng , Chen Hongtao , Mi Tiezhu
Published: May 2010

Inorganic ions and nutrients were measured at different depths of the Xiangxi and Daninghe Rivers to explore the mixing processes of representative bays in the Three Gorges Reservoir (TGR). HCO3−and Ca2+are the dominant ions. Carbonate weathering is the most important mechanism controlling the ion water chemistry; however, important differences exist between the main channel and its tributaries. Major ion levels in the TGR bays depend on hydrological mixing. Results show that the major ions of Ca2+, Mg2+, Na+, K+, Sr2+, SO42−and Cl−show chemically conservative behaviour during transit through the bays of the TGR. This means the ions can be used as tracers in the same way that salinity is used in estuaries to explore behaviour of other non‐conservative elements and to indicate specific source waters. In contrast, nutrients are not conserved in the mixing zone. The mixing of the main channel and tributaries and biological utilization in backwater reaches were the key factor controlling nutrient distributions in Xiangxi and Daninghe Bays. Copyright © 2010 John Wiley & Sons, Ltd.
Associations of aspirin, nonsteroidal anti‐inflammatory drug and paracetamol use with PSA‐detected prostate cancer: Findings from a large, population‐based, case–control study (the ProtecT study)
Authors: Murad Ali S. , Down Liz , Davey Smith George , Donovan Jenny L. , Athene Lane Janet , Hamdy Freddie C. , Neal David E. , Martin Richard M.
Published: May 2010

Evidence from laboratory studies suggests that chronic inflammation plays an important role in prostate cancer aetiology. This has resulted in speculation that nonsteroidal anti‐inflammatory drugs may protect against prostate cancer development. We analysed data from a cross‐sectional case–control study (ncases= 1,016; ncontrols= 5,043), nested within a UK‐wide population‐based study that used prostate specific antigen (PSA) testing for identification of asymptomatic prostate cancers, to investigate the relationship of aspirin, nonsteroidal anti‐inflammatory drug (NSAID) and paracetamol use with prostate cancer. In conditional logistic regression models accounting for stratum matching on age (5‐year age bands) and recruitment centre, use of non‐aspirin NSAIDs [odds ratio (OR) = 1.32; 95% confidence interval (CI): 1.04–1.67] or all NSAIDs (OR = 1.25; 95% CI = 1.07–1.47) were positively associated with prostate cancer. There were weaker, not conventionally statistically significant, positive associations of aspirin (OR = 1.13; 95% CI = 0.94–1.36) and paracetamol (OR = 1.20; 95% CI = 0.90–1.60) with prostate cancer. Mutual adjustment for aspirin, non‐aspirin NSAIDs or paracetamol made little difference to these results. There was no evidence of confounding by age, family history of prostate cancer, body mass index or self‐reported diabetes. Aspirin, NSAID and paracetamol use were associated with reduced serum PSA concentrations amongst controls. Our findings do not support the hypothesis that NSAIDs reduce the risk of PSA‐detected prostate cancer. Our conclusions are unlikely to be influenced by PSA detection bias because the inverse associations of aspirin, NSAID and paracetamol use with serum PSA would have attenuated (not generated) the observed positive associations.
CHI3L1 (YKL‐40) is expressed in human gliomas and regulates the invasion, growth and survival of glioma cells
Authors: Ku Bo Mi , Lee Yeon Kyung , Ryu Jinhyun , Jeong Joo Yeon , Choi Jungil , Eun Keyoung Mi , Shin Hye Young , Kim Dong Gyu , Hwang Eun Mi , Yoo Jae cheal , Park Jae‐Yong , Roh Gu Seob , Kim Hyun Joon , Cho Gyeong Jae , Choi Wan Sung , Paek Sun Ha , Kang Sang Soo
Published: May 2010

Chitinase 3‐like 1 (CHI3L1) is a secreted glycoprotein that has pleiotropic activity in aggressive cancers. In our study, we examined the expression and function of CHI3L1 in glioma cells. CHI3L1 was highly expressed in human glioma tissue, whereas its expression in normal brain tissue was very low. CHI3L1 suppression by shRNA reduced glioma cell invasion, anchorage‐independent growth and increased cell death triggered by several anticancer drugs, including cisplatin, etoposide and doxorubicin, whereas CHI3L1 overexpression had the opposite effect in glioma cells. Because the invasive nature of glioma cells plays a critical role in the high morbidity of glioma, we have further defined the role of CHI3L1 in the process of glioma invasion. Downregulation of CHI3L1 results in decreased cell–matrix adhesion and causes a marked increase in stress fiber formation and cell size with fewer cellular processes. Furthermore, the expression and activity of matrix metalloproteinase‐2 was also decreased in glioma cells in which CHI3L1 was knocked down. Taken together, these results suggest that CHI3L1 plays an important role in the regulation of malignant transformation and local invasiveness in gliomas. Thus, targeting the CHI3L1 molecule may be a potential therapeutic molecular target for gliomas.
Novel immunogenic antigens increase classification accuracy in meningioma to 93.84%
Authors: Ludwig Nicole , Keller Andreas , Heisel Sabrina , Leidinger Petra , Rheinheimer Stefanie , Andres Claudia , Stephan Bernhard , Steudel Wolf‐Ingo , Donauer Erich , Graf Norbert , Burgeth Bernhard , Weickert Joachim , Lenhof Hans‐Peter , Meese Eckart
Published: May 2010

There is growing evidence that simultaneous analysis of multiple autoantibody reactions can be utilized for diagnosis of neoplasms. Using a set of 57 meningioma‐associated antigens, we recently separated meningioma patients from individuals without known disease with an accuracy of 90.3%. Here, we ask whether a largely increased set of immunogenic antigens can further improve this discrimination. We used an array with 1,827 human recombinant clones and measured reactivity of serum autoantibodies against the clones by a novel automated image analysis procedure. We were able to separate meningioma sera from sera of healthy controls with a specificity of 95.62%, a sensitivity of 91.83% and an accuracy of 93.84%. Of the analyzed clones, 23 in‐frame clones were highly informative for the classification of meningiomavs. normal sera as shown by their AUC values. These results demonstrate that the accuracy of a serum‐based diagnostic can be readily and considerably improved by screening extended sets of proteins.
The association between change in body mass index and upper aerodigestive tract cancers in the ARCAGE project: Multicenter case–control study
Authors: Park Sungshim Lani , Lee Yuan‐Chin Amy , Marron Manuela , Agudo Antonio , Ahrens Wolfgang , Barzan Luigi , Bencko Vladimir , Benhamou Simone , Bouchardy Christine , Canova Cristina , Castellsague Xavier , Conway David I. , Healy Claire M. , Holcátová Ivana , Kjaerheim Kristina , Lagiou Pagona , Lowry Raymond J. , Macfarlane Tatiana V. , Macfarlane Gary J. , McCartan Bernard E. , McKinney Patricia A. , Merletti Franco , Pohlabeln Hermann , Richiardi Lorenzo , Simonato Lorenzo , Sneddon Linda , Talamini Renato , Trichopoulos Dimitrios , Znaor Ariana , Brennan Paul , Hashibe Mia
Published: May 2010

Previous studies reported an inverse relationship between body mass index (BMI) and upper aerodigestive tract (UADT) cancers. Examining change in BMI over time may clarify these previous observations. We used data from 2,048 cases and 2,173 hospital‐ and population‐based controls from ten European countries (alcohol‐related cancers and genetic susceptibility in Europe study) to investigate the relationship with BMI and adult change in BMI on UADT cancer risk. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for associations between BMI at three time intervals and BMI change on UADT cancer development, adjusting for center, age, sex, education, fruit and vegetable intake, smoking and alcohol consumption. We found an inverse relationship between UADT cancers and BMI at time of interview and 2 years before interview. No association was found with BMI at 30 years of age. Regarding BMI change between age 30 and 2 years before interview, BMI decrease (BMI change <−5%) vs. BMI stability (−5% ≤ BMI change <5%) showed no overall association with UADT cancers (OR = 1.15; 95% CI = 0.89, 1.49). An increase in BMI (BMI change ≥+5%) was inversely associated with UADT cancers (OR = 0.74; 95% CI = 0.62, 0.89). BMI gain remained inversely associated across all subsites except for esophageal cancer. When stratified by smoking or by drinking, association with BMI gain was detected only in drinkers and smokers. In conclusion, BMI gain is inversely associated with UADT cancers. These findings may be influenced by smoking and/or drinking behaviors and/or the development of preclinical UADT cancers and should be corroborated in studies of a prospective nature.
Liver specific overexpression of platelet‐derived growth factor‐B accelerates liver cancer development in chemically induced liver carcinogenesis
Authors: Maass Thorsten , Thieringer Florian R. , Mann Amrit , Longerich Thomas , Schirmacher Peter , Strand Dennis , Hansen Torsten , Galle Peter R. , Teufel Andreas , Kanzler Stephan
Published: May 2010

A genetic basis of hepatocellular carcinoma (HCC) has been well‐established and major signaling pathways, such as p53, Wnt‐signaling, transforming growth factor‐β (TGF‐β) and Ras pathways, have been identified to be essential to HCC development. Lately, the family of platelet‐derived growth factors (PDGFs) has shifted to the center of interest. We have reported on spontaneously developing liver fibrosis in PDGF‐B transgenic mice. Since HCC rarely occurs in healthy liver, but dramatically increases at the cirrhosis stage of which liver fibrosis is a preliminary stage, we investigated liver cancer development in chemically induced liver carcinogenesis in these mice. HCC induction was performed by treatment of the mice with diethylnitrosamine and phenobarbital. At an age of 6 months, the tumor development of these animals was analyzed. Not only the development of dysplastic lesions in PDGF‐B transgenic mice was significantly increased but also their malignant transformation to HCC. Furthermore, we were able to establish a key role of PDGF‐B signaling at diverse stages of liver cancer development. Here, we show that development of liver fibrosis is likely through upregulation of TGF‐β receptors by PDGF‐B. Additionally, overexpression of PDGF‐B also leads to an increased expression of β‐catenin as well as vascular endothelial growth factor and platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31), all factors with established roles in carcinogenesis. We were able to extend the understanding of key genetic regulators in HCC development by PDGF‐B and decode essential downstream signals.
The accuracy of colposcopic biopsy: Analyses from the placebo arm of the Gardasil clinical trials
Authors: Stoler Mark H. , Vichnin Michelle D. , Ferenczy Alex , Ferris Daron G. , Perez Gonzalo , Paavonen Jorma , Joura Elmar A. , Djursing Henning , Sigurdsson Kristján , Jefferson Lucy , Alvarez Frances , Sings Heather L. , Lu Shuang , James Margaret K. , Saah Alfred , Haupt Richard M. ,   for the FUTURE I, II and III Investigators
Published: May 2010

We evaluated the overall agreement between colposcopically directed biopsies and the definitive excisional specimens within the context of three clinical trials. A total of 737 women aged 16–45 who had a cervical biopsy taken within 6 months before their definitive therapy were included. Per‐protocol, colposcopists were to also obtain a representative cervical biopsy immediately before definitive therapy. Using adjudicated histological diagnoses, the initial biopsies and the same day biopsies were correlated with the surgically excised specimens. The overall agreement between the biopsies taken within 6 months of definitive therapy, and the definitive therapy diagnoses was 42% (weighted kappa = 0.34) (95% CI: 0.29–0.39). The overall underestimation of cervical intraepithelial neoplasia grade 2/3 or adenocarcinoma in situ (CIN2‐3/AIS) and CIN3/AIS was 26 and 42%, respectively. When allowing for one degree of variance in the correlation, the overall agreement was 92% for CIN2‐3/AIS. The overall agreement between the same day biopsy and definitive therapy specimen was 56% (weighted kappa = 0.41) (95% CI: 0.36–0.47), and the underestimation of CIN2‐3/AIS was 57%. There were significant associations in the agreement between biopsies and excisional specimen diagnoses when patients were stratified by age, number of biopsies, lesion size, presence of human papillomavirus (HPV)16/18 and region. Of 178 diagnostic endocervical curettages performed, 14 (7.9%) found any HPV disease. Colposcopic accuracy improved when CIN2 and CIN3/AIS were grouped as a single predictive measure of high‐grade disease. Colposcopy functioned well when allowed a one‐degree difference between the biopsy and the surgical histologic interpretations, as done in clinical practice. Taking more than one biopsy improved colposcopic accuracy and could improve patient management.
The Kennedy pathway—De novo synthesis of phosphatidylethanolamine and phosphatidylcholine
Authors: Gibellini Federica , Smith Terry K.
Published: May 2010

The Kennedy Pathway. See The Kennedy Pathway–De Novo Synthesis of Phosphatidylethanolamine and Phosphatidylcholine by Federica Gibellini and Terry K. Smith, pp. 414–428.
Electrospun nanofibrous matrix improves the regeneration of dense cortical bone
Authors: Cai You Zhi , Wang Lin Lin , Cai Hong Xin , Qi Yi Ying , Zou Xiao Hui , Ouyang Hong Wei
Published: May 2010

Numerous in vitro studies have indicated the potential of using electrospun nanofibrous scaffolds for tissue regeneration. However, few reports have demonstrated their utility in real tissue repair models. The present investigation tested the hypothesis that electrospun poly‐L‐lactic acid (PLLA) nanofibrous membrane leads to dense cortical bone regeneration and improves the efficacy of currently‐used collagenous guided bone regeneration (GBR) membrane. In vitro, the function of bone marrow‐derived mesenchymal stem cells (BMSCs) on nanofibrous scaffolds was evaluated. In an in vivo experiment, large bony defects were created in rabbit tibia and treated with a nanofiber‐reinforced bilayer membrane, nanofibrous membrane, or collagenous membrane alone. Three and six weeks after operation, bone defect healing was assessed radiologically and histologically. In vitro differentiation studies showed that BMSCs had much higher expression of Runx2 and collagen type I, alpha 1 mRNAs, when cultured on nanofibrous scaffolds. The radiographic and histological data both showed that the group treated with bilayer membrane had more bony tissue formation at 3 weeks. Moreover, at 6 weeks, only the bilayer membrane‐treated bone defects displayed better regeneration of cortical bone tissue, whereas in the other groups the defects were filled with spongy bone‐like tissue. The results demonstrated that electrospun nanofibrous membrane improves the regeneration of cortical bone, suggesting that this type of membrane can be combined with current collagenous GBR membrane to improve guided bone regeneration technology. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.
Differences in the mechanism of collagen lattice contraction by myofibroblasts and smooth muscle cells
Authors: Dallon J.C. , Ehrlich H. Paul
Published: May 2010

Both rat derived vascular smooth muscle cells (SMC) and human myofibroblasts contain α smooth muscle actin (SMA), but they utilize different mechanisms to contract populated collagen lattices (PCLs). The difference is in how the cells generate the force that contracts the lattices. Human dermal fibroblasts transform into myofibroblasts, expressing α‐SMA within stress fibers, when cultured in lattices that remain attached to the surface of a tissue culture dish. When attached lattices are populated with rat derived vascular SMC, the cells retain their vascular SMC phenotype. Comparing the contraction of attached PCLs when they are released from the culture dish on day 4 shows that lattices populated with rat vascular SMC contract less than those populated with human myofibroblast. PCL contraction was evaluated in the presence of vanadate and genistein, which modify protein tyrosine phosphorylation, and ML‐7 and Y‐27632, which modify myosin ATPase activity. Genistein and ML‐7 had no affect upon either myofibroblast or vascular SMC‐PCL contraction, demonstrating that neither protein tyrosine kinase nor myosin light chain kinase was involved. Vanadate inhibited myofibroblast‐PCL contraction, consistent with a role for protein tyrosine phosphatase activity with myofibroblast‐generated forces. Y‐27632 inhibited both SMC and myofibroblast PCL contraction, consistent with a central role of myosin light chain phosphatase. J. Cell. Biochem. 111: 362–369, 2010. © 2010 Wiley‐Liss, Inc.
Tgif1 represses apolipoprotein gene expression in liver
Authors: Melhuish Tiffany A. , Chung David D. , Bjerke Glen A. , Wotton David
Published: May 2010

TG‐interacting factor (Tgif1) represses gene expression by interaction with general corepressors, and can be recruited to target genes by transforming growth factor beta (TGFβ) activated Smads, or by the retinoid X receptor (RXR). Here we show that Tgif1 interacts with the LXRα nuclear receptor and can repress transcription from a synthetic reporter activated by LXRα. In cultured cells reducing endogenous Tgif1 levels resulted in increased expression of LXRα target genes. To test the in vivo role of Tgif1, we analyzed LXRα‐dependent gene expression in mice lacking Tgif1. In the livers of Tgif1 null mice, we observed significant derepression of the apolipoprotein genes, Apoa4 and Apoc2, suggesting that Tgif1 is an important in vivo regulator of apolipoprotein gene expression. In contrast, we observed relatively minimal effects on expression of other LXR target genes. This work suggests that Tgif1 can regulate nuclear receptor complexes, in addition to those containing retinoic acid receptors, but also indicates that there is some specificity to which NR target genes are repressed by Tgif1. J. Cell. Biochem. 111: 380–390, 2010. © 2010 Wiley‐Liss, Inc.
FGF‐2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3‐K/GSK3β signaling
Authors: Ding Vanessa M.Y. , Ling Ling , Natarajan Subaashini , Yap Miranda G.S. , Cool Simon M. , Choo Andre B.H.
Published: May 2010

Fibroblast growth factor‐2 (FGF‐2) is widely used to culture human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells. Despite its importance in maintaining undifferentiated hESC phenotype, a lack of understanding in the role of FGF‐2 still exists. Here, we investigate the signaling events in hESC following the addition of exogenous FGF‐2. In this study, we show that hESC express all forms of fibroblast growth factor receptors (FGFRs) which co‐localize on Oct3/4 positive cells. Furthermore, downregulation of Oct3/4 in hESC occurs following treatment with an FGFR inhibitor, suggesting that FGF signaling may regulate Oct3/4 expression. This is also observed in iPS cells. Also, downstream of FGF signaling, both mitogen activated protein kinase (MAPK) and phosphoinositide 3‐kinase pathways (PI3‐K) are activated following FGF‐2 stimulation. Notably, inhibition of MAPK and PI3‐K signaling using specific kinase inhibitors revealed that activated PI3‐K, rather than MAPK, can mediate pluripotent marker expression. To understand the importance of PI3‐K activation, activation of Wnt/β‐catenin by FGF‐2 was investigated. Wnt signaling had been implicated to have a role in maintaining of pluripotent hESC. We found that upon FGF‐2 stimulation, GSK3β is phosphorylated following which nuclear translocation of β‐catenin and TCF/LEF activation occurs. Interestingly, inhibition of the Wnt pathway with Dikkopf‐1 (DKK‐1) resulted in only partial suppression of the FGF‐2 induced TCF/LEF activity. Prolonged culture of hESC with DKK‐1 did not affect pluripotent marker expression. These results suggest that FGF‐2 mediated PI3‐K signaling may have a direct role in modulating the downstream of Wnt pathway to maintain undifferentiated hESC. J. Cell. Physiol. 225: 417–428, 2010. © 2010 Wiley‐Liss, Inc.
Survivin is regulated by interleukin‐4 in colon cancer stem cells
Authors: Di Stefano A.B. , Iovino F. , Lombardo Y. , Eterno V. , Höger T. , Dieli F. , Stassi G. , Todaro M.
Published: May 2010

Colorectal cancer has provided an important model to test the stem cell hypothesis of cancer origin, which implies that cancer arises as a result of genetic aberrations in stem cells leading to deregulation of the proliferation/differentiation balance. We and others have demonstrated that, similarly to other solid tumors, colon carcinogenesis and progression are dictated by highly apoptosis‐resistant stem‐like cells. Our data have suggested that protection from apoptosis is achieved by autocrine production of interleukin‐4 (IL‐4) through up‐regulation of anti‐apoptotic mediators. In this study, we extend our analysis to another apoptosis inhibitor widely expressed in tumors, namely survivin (also known as BIRC‐5, baculoviral IAP repeat‐containing protein 5). We show that this protein, with important roles in cell death counteraction and mitotic progression control, is regulated by the IL‐4 pathway in colon rectal cancer stem cells (CR‐CSC). Hence, the presence of IL‐4 increases survivin levels in our model while cytokine neutralization has opposing effects. Treatment with cytokine neutralizing agent or with leflunomide, Stat6 inhibitor, have similar consequences on survivin localization, increasing its nuclear pool, an observation known to be correlated with a good prognosis in colon cancer patients. These results demonstrate that IL‐4, through activation of the STAT‐6 signaling pathway, is involved in survivin expression levels as well as its localization. These findings shed more light on the molecular mechanisms involved in IL‐4‐mediated chemoresistance. J. Cell. Physiol. 225: 555–561, 2010. © 2010 Wiley‐Liss, Inc.
FRNK overexpression limits the depth and frequency of vascular smooth muscle cell invasion in a three‐dimensional fibrin matrix
Authors: Brewster L.P. , Ucuzian A.A. , Brey E.M. , Liwanag M. , Samarel A.M. , Greisler H.P.
Published: May 2010

Pathological vascular smooth muscle cell (VSMC) behavior after vascular interventions such as angioplasty or bypass is initiated within the 3D environment of the vessel media. Here VSMCs proliferate, invade the surrounding matrix, migrate adluminally, and deposit substantial amounts of matrix, leading to myointimal hyperplasia and decreased blood flow to critical organs and tissue. Since focal adhesion kinase (FAK) mediates many of the VSMC responses to these pathologic events, it provides a reasonable pharmacologic target to limit this invasive VSMC behavior and to better understand the cellular pathophysiology of this disease. Here we quantified the effectiveness of disabling FAK in VSMCs with its dominant‐negative inhibitor, FAK‐related nonkinase (FRNK), in a clinically relevant 3D assay. We found that FRNK overexpression decreased VSMC invasion (both the length and frequency) in this matrix. These effects were demonstrated in the presence and absence of chemical mitotic inhibition, suggesting that FAK's effect on cellular matrix invasion, migration, and proliferation utilize separate and/or redundant signaling cascades. Mechanistically, FAK inhibition decreased its localization to focal adhesions which led to a significant decrease in FAK autophosphorylation and the phosphorylation of the serine/threonine kinase, AKT. Together these findings suggest that disruption of FAK signaling may provide a pharmaceutical tool that limits pathological VSMC cell behavior. J. Cell. Physiol. 225: 562–568, 2010. © 2010 Wiley‐Liss, Inc.
The human SWI/SNF complex associates with RUNX1 to control transcription of hematopoietic target genes
Authors: Bakshi Rachit , Hassan Mohammad Q. , Pratap Jitesh , Lian Jane B. , Montecino Martin A. , van Wijnen Andre J. , Stein Janet L. , Imbalzano Anthony N. , Stein Gary S.
Published: May 2010

The acute myeloid leukemia 1 (AML1, RUNX1) transcription factor is a key regulator of hematopoietic differentiation that forms multi‐protein complexes with co‐regulatory proteins. These complexes are assembled at target gene promoters in nuclear microenvironments to mediate phenotypic gene expression and chromatin‐related epigenetic modifications. Here, immunofluorescence microscopy and biochemical assays are used to show that RUNX1 associates with the human ATP‐dependent SWI/SNF chromatin remodeling complex. The SWI/SNF subunits BRG1 and INI1 bind in vivo to RUNX1 target gene promoters (e.g., GMCSF, IL3, MCSF‐R, MIP, and p21). These interactions correlate with histone modifications characteristic of active chromatin, including acetylated H4 and dimethylated H3 lysine 4. Downregulation of RUNX1 by RNA interference diminishes the binding of BRG1 and INI1 at selected target genes. Taken together, our findings indicate that RUNX1 interacts with the human SWI/SNF complex to control hematopoietic‐specific gene expression. J. Cell. Physiol. 225: 569–576, 2010. © 2010 Wiley‐Liss, Inc.
Cannabinoid receptor 1 mediates palmitic acid‐induced apoptosis via endoplasmic reticulum stress in human renal proximal tubular cells
Authors: Lim J.C. , Lim S.K. , Han H.J. , Park S.H.
Published: May 2010

The endocannabinoid system (ECS) is activated at the onset of obesity and diverse metabolic diseases. Endocannabinoids mediate their physiological and behavioral effects by activating specific cannabinoid receptors, mainly cannabinoid receptor 1 (CB1R). Diabetic nephropathy (DN) is induced by hyperlipidemia, and renal proximal tubule cells are an important site for the onset of DN. However, the pathophysiology of CB1R, especially in the hyperlipidemia of DN, has not been elucidated. Therefore, we examined the effect of palmitic acid (PA) on CB1R expression and its related signal pathways in human renal proximal tubular cells (HK‐2 cells). PA significantly increased CB1R mRNA and protein levels and induced CB1R internalization. PA‐induced activation of CB1R is prevented by the treatment of AACOCF3 (a cPLA2 inhibitor), indomethacin and NS398 (a COX 2 inhibitors). Indeed, PA increased cPLA2, and COX‐2 but not COX‐1. We also investigated whether the PA‐induced activation of CB1R is linked to apoptosis. As a result, AM251 (a CB1R antagonist) attenuated PA‐mediated apoptosis in a concentration‐dependent manner. Furthermore, PA decreased GRP78 expression and induced increases in the endoplasmic reticulum (ER) stress signaling pathways p‐PERK, p‐eIF2α, p‐ATF4, and CHOP, which were blocked by AM251 treatment. Moreover, PA increased the Bax/Bcl‐2 ratio, cleaved PARP, and caspase‐3 levels. The PA‐induced apoptotic effects were decreased with CB1R‐specific antagonist (AM251) treatment and CB1 si‐RNA transfection. In conclusion, PA induced apoptosis through ER stress via CB1R expression in human proximal tubule cells. Our results provide evidence that CB1R blockade may be a potential anti‐diabetic therapy for the treatment of DN. J. Cell. Physiol. 225: 654–663, 2010. © 2010 Wiley‐Liss, Inc.
A novel vascular targeting strategy for brain‐derived endothelial cells using a TCR mimic antibody
Authors: Bhattacharya Raktima , Xu Yan , Rahman Md. Ashequr , Couraud Pierre‐Olivier , Romero Ignacio A. , Weksler Babette B. , Weidanz Jon A. , Bickel Ulrich
Published: May 2010

Organ‐specific vascular targeting, for example, to the blood–brain barrier, requires the identification of unique molecular addresses on a subset of endothelial cells. The present study describes a crucial step towards tapping the exquisite specificity of the peptide/HLA class I system for this goal. We utilized a novel T‐cell receptor (TCR) mimic antibody of high affinity and specificity, which is restricted by HLA‐A2 and has been generated to recognize a peptide epitope derived from p68 RNA helicase (YLLPAIVHI). The parent protein is highly expressed by brain endothelial cells. Flow cytometry and confocal imaging showed that the antibody binds to HLA‐A2‐positive human brain‐derived endothelial cells, both immortalized hCMEC/D3 cells and primary cells. The TCR mimic antibody undergoes internalization into vesicles, where significant colocalization occurs with the early endosomal marker EEA‐1, but barely with caveolin‐1. To our knowledge internalization of neither MHC class I protein nor TCR mimics by brain endothelial cells has been previously observed. Knock down of p68 protein expression by siRNA reduced the presentation of YLLPAIVHI‐peptide/HLA‐A2 complexes on the cell membrane by half as measured by flow cytometry 48 h later. We also found that brain endothelial cells isolated from HLA‐A2 transgenic mouse strains express the A2 transgene, and brain endothelial cells of one of these strains also present YLLPAIVHI‐peptide/HLA‐A2, making these mouse strains suitable models for studying TCR mimic antibodies in vivo. In conclusion, these data strongly support the notion that TCR mimic antibodies could be a new class of therapeutic targeting agents in a wide variety of diseases. J. Cell. Physiol. 225: 664–672, 2010. © 2010 Wiley‐Liss, Inc.
Shed HER2 extracellular domain in HER2‐mediated tumor growth and in trastuzumab susceptibility
Authors: Ghedini Gaia C. , Ciravolo Valentina , Tortoreto Monica , Giuffrè Sarah , Bianchi Francesca , Campiglio Manuela , Mortarino Mimosa , Figini Mariangela , Coliva Angela , Carcangiu Maria L. , Zambetti Milvia , Piazza Tiziana , Ferrini Silvano , Ménard Sylvie , Tagliabue Elda , Pupa Serenella M.
Published: May 2010

The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti‐HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2‐driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110‐kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, ‐2, and ‐3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2‐driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2‐driven SKOV3 tumor growth but not that of HER2‐negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD‐facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2‐disease and in the susceptibility to Trastuzumab‐based therapy. J. Cell. Physiol. 225: 256–265, 2010. © 2010 Wiley‐Liss, Inc.
Ultrasound‐assisted dispersive liquid–liquid microextraction coupled with capillary gas chromatography for simultaneous analysis of nine pyrethroids in domestic wastewaters
Authors: Yan Hongyuan , Du Jingjing , Zhang Xiguo , Yang Gengliang , Row Kyung Ho , Lv Yunkai
Published: May 2010

A simple and rapid ultrasound‐assisted dispersive liquid–liquid microextraction method coupled with GC‐flame ionization detection was developed for simultaneous determination of nine pyrethroids in domestic wastewater samples. An ultrasound‐assisted process was applied to accelerate the formation of the fine cloudy solution using small volume of disperser solvent, which markedly increased the extraction efficiency and reduced the equilibrium time. Various parameters affecting the extraction efficiency were investigated, including the type and volume of extraction solvent and disperser solvent, extraction and ultrasonic time. Good linearity was obtained for all analytes in the range of 0.8–100 μg/L with the correlation coefficient (r2)≥0.998. The recoveries at three spiking levels ranged from 75.3 to 101.2% with the RSD less than 8.7% (n=5). Under the optimum condition, the enrichment factors for the nine pyrethroids ranged from 728‐ to 1725‐fold. This method offered a good alternative for routine analysis due to its simplicity and reliability.
Sample complexity reduction aids efficient detection of low‐abundant proteins from human amniotic fluid
Authors: Alam Mashkoor , Selladurai Masilamani , Nagpal Saurabh , Tomar Anil Kumar , Saraswat Mayank , Raziuddin M. , Mittal Sunita , Singh Tej P. , Yadav Savita
Published: May 2010

Working with biological fluids poses a challenge of visualizing proteins present in lower concentrations. This study describes a batch‐mode chromatographic method for the fractionation of human amniotic fluid (AF). This method is easy to use with minimal sample quantity, resin volume and sample processing time. For albumin depletion, two methods were evaluated. The results demonstrated that specific depletion of albumin, using affinity‐ligand‐based resin, is more efficient than the conventional dye‐based method. The albumin‐depleted human AF was fractionated by strong anion‐exchange resin in spin devices, for sample, complexity reduction and enrichment of low‐abundant proteins. Analysis of four eluate fractions generated after this step shows enrichment of few low‐abundant proteins. Two novel low‐abundant proteins, Rab GDP dissociation inhibitor β and peptide methionine sulfoxide reductase, were identified from human AF. α‐1‐B Glycoprotein was successfully identified by this strategy, whereas the published literature reports that it was not identified by strong anion‐exchange FPLC followed by SDS‐PAGE. Therefore, the current method has distinct advantages over the conventional column‐based chromatography. This study also reports altered expression of some proteins in Rh‐isoimmunized AF samples in comparison with normal AF.
Analysis of cocaine and benzoylecgonine in urine by using multisyringe flow injection analysis‐gas chromatography‐mass spectrometry system
Authors: Brunetto María del Rosario , Delgado Yelitza , Clavijo Sabrina , Contreras Yaritza , Torres Dina , Ayala Carlos , Gallignani Máximo , Forteza Rafael , Martin Víctor Cerdà
Published: May 2010

In this paper, a method was described to determine cocaine (COC) and benzoylecgonine (BZE) in human urine samples by GC‐MS detection. The extraction of analytes from urine samples was achieved in an Oasis hydrophilic–lipophilic balance column (20 mm×3.9 mm id, dp=25 μm; Waters, USA), incorporated in a multisyringe flow injection system, used for the sample treatment. Finally, to improve the volatility of the BZE, an in‐line derivatization reaction withN,O‐bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane was made microwave‐assisted in order to reduce the reaction time. The results showed that the proposed method is a good alternative for the analysis of COC and BZE in urine samples because it offers advantages compared with those described in the literature, which include simplicity in the sample treatment, the sensitivity and selectivity necessary to determine the analytes of interest at low levels in the urine and high sample throughput.
Application of accelerated solvent extraction to the investigation of saikosaponins from the roots of Bupleurum falcatum
Authors: Li Wei , Liu Zhengbo , Wang Zi , Chen Li , Sun Yinshi , Hou Jingang , Zheng Yinan
Published: May 2010

Accelerated solvent extraction (ASE) was applied to the extraction of saikosaponin a, saikosaponin c and saikosaponin d from the roots of Bupleurum falcatum. Main extraction parameters such as the extraction solvents, extraction temperature and static extraction time were investigated and optimized. The optimized procedure employed 70% methanol as extraction solvent, 120°C of extraction temperature, 10 min of static extraction time, 60% of flush volume and the extraction recoveries of the three compounds were near to 100% with one extraction cycle. The extracted samples were analyzed by HPLC with UV detector. The HPLC conditions were as follows: Hypersil ODS2 (4.6 mm×250 mm, 5 μm) column, acetonitrile and water as mobile phase, flow rate of 1.0 mL/min, UV detection wavelength of 204 nm and injection volume of 20 μL. Compared with the traditional methods including heat‐reflux extraction and ultrasonic‐assisted extraction, the proposed ASE method was more efficient and faster to be operated. The results indicated that ASE was an alternative method for extracting saikosaponins from the roots of B. falcatum.