bluuebunny/crossref_metadata_2025 huggingface dataset:
Drosophila growth cones: A genetically tractable platform for the analysis of axonal growth dynamics
Authors: Sánchez‐Soriano Natalia , Gonçalves‐Pimentel Catarina , Beaven Robin , Haessler Ulrike , Ofner‐Ziegenfuss Lisa , Ballestrem Christoph , Prokop Andreas
Published: November 2009

The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F‐actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross‐talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F‐actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin‐MT linking factor Short stop, thus identifying an essential molecular player in this context. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2010
Limitations of European Union policy and law for regulating use of lead shot and sinkers: comparisons with North American regulation
Authors: Thomas Vernon G. , Guitart Raimon
Published: November 2009

Extensive shooting and angling causes, indirectly, fatal lead poisoning of birds. European Union policy on this major source of pollution is inconsistent with its laws regulating other forms of lead in the environment. Only three countries have banned the use of lead shot completely in the European Union, despite availability of substitutes and evidence that their use is a major contributor to bird conservation. The European Commission uses the criterion of amount of lead deposited and corroded, and the concentration of lead in water and soil, as the basis of their decisions. The USA and Canada used the prevalence of lead poisoning among birds as the basis of policy and law allowing them to reduce lead use at the continental level. The EU and North American policies and law on lead reduction are compared in this study, on the basis of which recommendations are developed indicating how the EU could revise its approach and resolve this environmental problem. Reluctance to act on lead reduction by the European Parliament and its member states reflects the current vested interests of the sporting communities. Companies in eight European countries already produce non‐toxic materials for hunting and shooting, and are not the limiting factor in this issue. Copyright © 2009 John Wiley & Sons, Ltd and ERP Environment.
Measurement of IPTV traffic from an operative network
Authors: Mellia Marco , Meo Michela
Published: November 2009

In this paper, we present measurement results of IP TeleVision (IPTV) multicast traffic collected from real of the FastWeb network. FastWeb is an ISP provider, which is the main broadband telecommunication company in Italy. The network relies on an IP architecture and, besides television channels, delivers to the user services such as data transfer and VoIP over a single broadband connection. The traffic we focus on consists of 83 high‐quality digital TV channels encoded using different MPEG‐2 encoders. The results show that, depending on the encoder and based on the bitrate, flows can be classified as being: 1‐VBR, i.e. with one typical value of the mean bit rate, 2‐VBR, i.e. two typical bitrate values are observed, and VBR. Measurement of the packet loss, jitter and inter‐packet gap show that, independently from the class, packet generation process of the flows can have various degrees of burstiness. Despite the packet level burstiness, average jitter is limited to few milliseconds and no packet loss was ever observed in the backbone. Moreover, the evolution of inter‐packet gap over time remains basically unchanged when observing a flow in different time periods. Our results prove that the FastWeb network is properly carrying IPTV traffic, and that today technology is mature to make TV over IP feasible and of good quality. Copyright © 2009 John Wiley & Sons, Ltd.
Neuronal network pharmacodynamics of GABAergic modulation in the human cortex determined using pharmaco‐magnetoencephalography
Authors: Hall Stephen D. , Barnes Gareth R. , Furlong Paul L. , Seri Stefano , Hillebrand Arjan
Published: November 2009

Neuronal network oscillations are a unifying phenomenon in neuroscience research, with comparable measurements across scales and species. Cortical oscillations are of central importance in the characterization of neuronal network function in health and disease and are influential in effective drug development. Whilst animal in vitro and in vivo electrophysiology is able to characterize pharmacologically induced modulations in neuronal activity, present human counterparts have spatial and temporal limitations. Consequently, the potential applications for a human equivalent are extensive. Here, we demonstrate a novel implementation of contemporary neuroimaging methods called pharmaco‐magnetoencephalography. This approach determines the spatial profile of neuronal network oscillatory power change across the cortex following drug administration and reconstructs the time course of these modulations at focal regions of interest. As a proof of concept, we characterize the nonspecific GABAergic modulator diazepam, which has a broad range of therapeutic applications. We demonstrate that diazepam variously modulates θ (4–7 Hz), α (7–14 Hz), β (15–25 Hz), and γ (30–80 Hz) frequency oscillations in specific regions of the cortex, with a pharmacodynamic profile consistent with that of drug uptake. We examine the relevance of these results with regard to the spatial and temporal observations from other modalities and the various therapeutic consequences of diazepam and discuss the potential applications of such an approach in terms of drug development and translational neuroscience. Hum Brain Mapp, 2010. © 2009 Wiley‐Liss, Inc.
Association of trait emotional intelligence and individual fMRI‐activation patterns during the perception of social signals from voice and face
Authors: Kreifelts Benjamin , Ethofer Thomas , Huberle Elisabeth , Grodd Wolfgang , Wildgruber Dirk
Published: November 2009

Multimodal integration of nonverbal social signals is essential for successful social interaction. Previous studies have implicated the posterior superior temporal sulcus (pSTS) in the perception of social signals such as nonverbal emotional signals as well as in social cognitive functions like mentalizing/theory of mind. In the present study, we evaluated the relationships between trait emotional intelligence (EI) and fMRI activation patterns in individual subjects during the multimodal perception of nonverbal emotional signals from voice and face. Trait EI was linked to hemodynamic responses in the right pSTS, an area which also exhibits a distinct sensitivity to human voices and faces. Within all other regions known to subserve the perceptual audiovisual integration of human social signals (i.e., amygdala, fusiform gyrus, thalamus), no such linked responses were observed. This functional difference in the network for the audiovisual perception of human social signals indicates a specific contribution of the pSTS as a possible interface between the perception of social information and social cognition. Hum Brain Mapp, 2010. © 2009 Wiley‐Liss, Inc.
Cellular transformation by cigarette smoke extract involves alteration of glycolysis and mitochondrial function in esophageal epithelial cells
Authors: Kim Myoung Sook , Huang Yiping , Lee Juna , Zhong Xiaoli , Jiang Wei‐Wen , Ratovitski Edward A. , Sidransky David
Published: November 2009

Cigarette‐smoking increases the risk of developing various types of human cancers including esophageal cancers. To test the effects of chronic cigarette smoke exposure directly on esophageal epithelium, cellular resistance to mainstream extract (MSE), or sidestream smoke extract (SSE) was developed in chronically exposed nonmalignant Het‐1A cells. Anchorage‐independent growth, in vitro invasion capacity and proliferation of the resistant cells increased compared with the unexposed, sensitive cells. An epithelial marker E‐cadherin was down‐regulated and mesenchymal markers N‐cadherin and vimentin were up‐regulated in the resistant cells. Het‐1A cells resistant to MSE or SSE consumed more glucose, and produced more lactate than the sensitive cells. The increased anchorage‐independent cell growth of the resistant cells was suppressed by a glycolysis inhibitor, 2‐deoxy‐D‐glucose, indicating that these cells are highly dependent on the glycolytic pathway for survival. Decreased mitochondrial membrane potential and ATP production in the resistant cells indicate the presence of mitochondrial dysfunction induced by chronic exposure of cigarette smoke extract. Increased expression of nuclear genes in the glycolytic pathway and decreased levels of mitochondrial genes in the resistant cells support the notion that cigarette smoking significantly contributes to the transformation of nonmalignant esophageal epithelial cells into a tumorigenic phenotype.
NY‐CO‐58/KIF2C is overexpressed in a variety of solid tumors and induces frequent T cell responses in patients with colorectal cancer
Authors: Gnjatic Sacha , Cao Yanran , Reichelt Uta , Yekebas Emre F. , Nölker Christina , Marx Andreas H. , Erbersdobler Andreas , Nishikawa Hiroyoshi , Hildebrandt York , Bartels Katrin , Horn Christiane , Stahl Tanja , Gout Ivan , Filonenko Valeriy , Ling Khoon‐Lin , Cerundolo Vincenzo , Luetkens Tim , Ritter Gerd , Friedrichs Kay , Leuwer Rudolf , Hegewisch‐Becker Susanna , Izbicki Jakob R. , Bokemeyer Carsten , Old Lloyd J. , Atanackovic Djordje
Published: November 2009

NY‐CO‐58/KIF2C has been identified as a tumor antigen by screening antibody responses in patients with colorectal cancer. However, expression had not consequently been examined, and nothing was known about its ability to induce spontaneous T cell responses, which have been suggested to play a role in the development of colorectal cancer. We analyzed 5 colorectal cancer cell lines, and tumor samples and adjacent healthy tissues from 176 patients with epithelial cancers for the expression of NY‐CO‐58/KIF2C by RT‐PCR and Western Blot. T cell responses of 43 colorectal cancer patients and 35 healthy donors were evaluated by ELISpot following stimulation with 30mer peptides or full‐length protein. All cell lines and tumor samples from colorectal cancer patients expressed NY‐CO‐58/KIF2C on the protein and RNA level, and expression levels correlated strongly with Ki‐67 expression (r = 0.69; p = 0.0003). Investigating NY‐CO‐58/KIF2C‐specific T cell responses, CD8+ T cells directed against 1 or more peptides were found in less than 10% of patients, whereas specific CD4+ T cells were detected in close to 50% of patients. These T cells were of high avidity, recognized the naturally processed antigen and secreted IFN‐γ and TNF‐α. Depletion of CD4+CD25+ T cells before stimulation significantly increased the intensity of the preexisting response. NY‐CO‐58/KIF2C is significantly overexpressed in colorectal and other epithelial cancers and expression levels correlate with the proliferative activity of the tumor. Importantly, NY‐CO‐58/KIF2C was able to induce spontaneous CD4+ T cell responses of the Th1‐type, which were tightly controlled by peripheral T regulatory cells.
Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP‐1 in human multiple myeloma cells
Authors: Pandey Manoj K. , Sung Bokyung , Aggarwal Bharat B.
Published: November 2009

STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulate the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid‐induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP‐1 and silencing of the SHP‐1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescued betulinic acid‐induced cell death. Betulinic acid also downregulated the expression of STAT3‐regulated gene products such as bcl‐xL, bcl‐2, cyclin D1 and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub‐G1 cell population and an increase in caspase‐3‐induced PARP cleavage. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the betulinic acid‐induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10 to 55%) and bortezomib (from 5 to 70%) in MM cells. Overall, our results suggest that betulinic acid downregulates STAT3 activation through upregulation of SHP‐1, and this may have potential in sensitization of STAT3 overexpressing tumors to chemotherapeutic agents.
Tumor‐stromal crosstalk in invasion of oral squamous cell carcinoma: a pivotal role of CCL7
Authors: Jung Da‐Woon , Che Zhong Min , Kim Jinmi , Kim Kyungshin , Kim Ki‐Yeol , Williams Darren , Kim Jin
Published: November 2009

Recent studies have shown that stromal fibroblasts have a more profound influence on the initiation and progression of carcinoma than was previously appreciated. This study aimed at investigating the reciprocal relationship between cancer cells and their associated fibroblasts at both the molecular and cellular level in oral squamous cell carcinoma (OSCC). To identify key molecular regulators expressed by carcinoma‐associated fibroblasts (CAF) that promote cancer cell invasion, microarrays were performed by comparing cocultured OSCC cells and CAF with monoculture controls. Microarray and real‐time PCR analysis identified marked upregulation of the chemokine (C‐C motif) ligand 7 (CCL7) in cocultured CAF. ELISA showed an elevated level of CCL7 secretion from CAF stimulated by coculture with OSCC cells. CCL7 promoted the invasion and migration of OSCC cells, and the invasiveness was inhibited by treatment with CCL7 neutralizing antibody. OSCC cells were shown to express CCR1, CCR2 and CCR3, receptors for CCL7, by RT‐PCR. In addition, treatment with anti‐CCR1 or anti‐CCR3 antibody inhibited CCL7‐induced OSCC cell migration, implicating that CCL7 promotes cancer cell migration through CCR1 and CCR3 on OSCC cells. Cytokine antibody array analysis of the supernatant from OSCC cell culture revealed that interleukin‐1α was an inducer of CCL7 secretion by CAF. This study confirms the reciprocal relationship of the molecular crosstalk regulating the invasion of OSCC and describes new potential targets for future therapy.
Recombinant single‐chain variable fragment antibodies against extracellular epitopes of human multidrug resistance protein MRP3 for targeting malignant gliomas
Authors: Kuan Chien‐Tsun , Srivastava Nidhi , McLendon Roger E. , Marasco Wayne A. , Zalutsky Michael R. , Bigner Darell D.
Published: November 2009

Multidrug resistance protein 3 (MRP3), a multidrug resistance protein identified by serial analysis of gene expression as a glioblastoma multiforme (GBM)‐associated molecule, is highly expressed in GBM, but not in normal brain cells. Thus, MRP3 is a candidate for GBM immunotargeting, but to date, no monoclonal antibody has been isolated that can target an extracellular MRP3 epitope. By phage display, we have isolated 3 recombinant, fully human, single‐chain Fv (scFv) antibodies, M25, M58 and M89, which specifically react with the extracellular N‐terminus of human MRP3. In ELISA, these scFvs reacted only with the peptide used for screening and not with other MRP3‐derived peptides. Flow cytometric analysis revealed that these scFv fragments bind specifically to viable human GBM cells displaying different MRP3 expression levels, but not to MRP3‐null cells. Furthermore, these scFv antibodies failed to react with tumor cells overexpressing other MRP proteins, including MRP1, MRP2, MRP4 and MRP5. M25 and M58 also bound to viable neurospheres. Iodogen‐labeled scFvs demonstrated a yield of 56–76%. The immunoreactive fractions of the radiolabeled M25, M58 and M89 scFvs were 32, 52 and 69%, respectively. M25 exhibited 20% internalization into D2159MG neurospheres, M58, 33% into D54MG cells and M89, 26% into D247MG. Immunohistochemical evaluation of human gliomas to determine the localization of MRP3 antigen using scFvs M25 and M58 showed a dense cytoplasmic and membranous staining pattern. These Fv‐based recombinant antibodies, which possess superior tumor penetration capabilities and selectively target tumor cells that express MRP3, may potentially be used in immunotherapy and diagnosis for brain tumors and other cancers.
Parametric and non‐parametric approaches to exits from fixed exchange rate regimes
Authors: Aşıcı Ahmet Atıl
Published: November 2009

Following the demise of the Bretton‐Woods, increasing number of countries has been opting for flexible exchange rate regimes. Exiting from fixed regimes however is not without costs. Regime transitions have often been occurred in the midst of a crisis, which has considerable economic costs in terms of output contraction and exchange rate depreciation. Given the big number of countries having still fixed regimes and financial markets that are fairly close and expected to be liberalized sooner or later, issue of exiting a peg without incurring crisis is a real challenge confronting these countries. The aim of this paper is to determine the conditions under which orderly exit is possible. The paper employs Binary Recursive Tree and standard regression frameworks. Analysis shows that countries with higher output gap and overvalued real exchange rate, among others, are doomed to exit in a disorderly way. Following their exit, output collapses and exchange rate depreciates considerably. The ill‐managed financial liberalization and macroeconomic stabilization programs seem to lay the seeds of instability. An interesting finding is that the conventional strengths of parametric regression analysis can be dramatically improved by utilizing findings of non‐parametric BRT technology. Sample contains all countries depending on the data availability, and covers 1975–2004 period. Copyright © 2009 John Wiley & Sons, Ltd.
A comparative analysis of the evolution of a taxonomy for best practices: a case for ‘knowledge efficiency’
Authors: O'Leary Daniel E.
Published: November 2009

Taxonomies play an increasingly important role in knowledge management of business best practices, providing a basis by which to index, find and communicate knowledge. However, knowledge continues to evolve over time. As a result, taxonomies must also continue to evolve as organizations innovate and change.

Reportedly, firms customize best‐practice taxonomies to meet their unique organization needs. Accordingly, we might expect organizations to generate dissimilar best‐practice taxonomies. However, taxonomies must also reflect the state of knowledge in the area being categorized, and thus are likely to be similar in many ways in different organizations.

The purpose of this paper is to study how taxonomies change in different organizations and how they stay the same. In order to explain the parallels in organizational taxonomies, the notion of ‘knowledge artefact efficiency’ (or knowledge efficiency) is suggested to capture the concept that new knowledge is rapidly adopted by many organizations in their knowledge management systems. Copyright © 2009 John Wiley & Sons, Ltd.
The effects of extracellular nucleotides on [Ca2+]isignalling in a human‐derived renal proximal tubular cell line (HKC‐8)
Authors: Turvey Matthew R. , Wang Yanyun , Gu Yuchun
Published: November 2009

HKC‐8 cells are a human‐derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca2+]isignalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca2+]iin HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca2+]ithat was dependent on an intracellular source of Ca2+, while prolonged activation of P2Y receptors induced a rise in [Ca2+]ithat was dependent on intra‐ and extracellular sources of Ca2+. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca2+entry in coupling to activation of P2Y in HKC‐8 cells. U73221, an inhibitor of PI‐PLC, did not inhibit the initial ATP‐induced response; whereas D609, an inhibitor of PC‐PLC, caused a significant decrease in the initial ATP‐induced response, suggesting that P2Y receptors are coupled to PC‐PLC. Although P2X were present in HKC‐8, The P2X agonist, α,β me‐ATP, failed to cause a rise in [Ca2+]i. However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca2+]i. Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca2+]ielevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.
Determinants of pluripotency: From avian, rodents, to primates
Authors: Martins‐Taylor Kristen , Xu Ren‐He
Published: November 2009

Since mouse embryonic stem (ES) cells was first derived in 1981, the ability of this unprecedented cell type to self‐renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore, they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest, due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However, this is no longer a barrier, with the recent discovery of methods that can convert differentiated somatic cells into ES‐like cells or induced pluripotent stem (iPS) cells, by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo‐derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells, as well as the different determinants identified thus far to sustain their pluripotency. J. Cell. Biochem. 109: 16–25, 2010. © 2009 Wiley‐Liss, Inc.
The intermediate filament vimentin regulates chondrogenesis of adult human bone marrow‐derived multipotent progenitor cells
Authors: Bobick Brent E. , Tuan Rocky S. , Chen Faye H.
Published: November 2009

Cytoskeletal proteins play important regulatory roles in a variety of cellular processes, including proliferation, migration, and differentiation. However, whereas actin and tubulin have established roles regulating developmental chondrogenesis, there is no evidence supporting a function for the intermediate filament vimentin in embryonic cartilage formation. We hypothesized that vimentin may regulate the chondrogenic differentiation of adult multipotent progenitor cells (MPCs), such as those involved in cartilage formation during bone fracture repair. As our model of adult progenitor cell chondrogenesis, we employed high‐density pellet cultures of human bone marrow‐derived MPCs. siRNA‐mediated knockdown of vimentin mRNA and protein triggered a reduction in the extent of MPC cartilage formation, as evidenced by depressed accumulation of mRNAs for the cartilage‐specific marker genes aggrecan and collagen type II, as well as reduced levels of Alcian blue‐stainable proteoglycan and collagen II protein in the extracellular matrix. Moreover, mRNA and protein levels for the chondro‐regulatory transcription factors SOX5, SOX6, and SOX9 were diminished by vimentin knockdown. Depleted cellular vimentin also induced a drastic reduction in PKA phosphorylation levels but did not affect the phosphorylation of multiple other chondro‐regulatory kinases and transcription factors, including ERK1/2, p38, Smad2, and Smad1/5/8. Importantly, siRNA‐mediated knockdown of PKA C‐α mRNA and protein mimicked the reduction in chondrogenesis caused by diminished cellular vimentin. Finally, overexpression of vimentin in MPCs significantly enhanced the activity of a transfected collagen II promoter‐luciferase reporter gene. In conclusion, we describe a novel role for the intermediate filament vimentin as a positive regulator of adult human bone marrow‐derived MPC chondrogenesis. J. Cell. Biochem. 109: 265–276, 2010. Published 2009 Wiley‐Liss, Inc.
Ceramide plays a prominent role in MDA‐7/IL‐24‐induced cancer‐specific apoptosis
Authors: Sauane Moira , Su Zao‐zhong , Dash Rupesh , Liu Xiang , Norris James S. , Sarkar Devanand , Lee Seok‐Geun , Allegood Jeremy C. , Dent Paul , Spiegel Sarah , Fisher Paul B.
Published: November 2009

Melanoma differentiation associated gene‐7/interleukin‐24 (mda‐7/IL‐24) uniquely displays broad cancer‐specific apoptosis‐inducing activity through induction of endoplasmic reticulum (ER) stress. We hypothesize that ceramide, a promoter of apoptosis, might contribute to mda‐7/IL‐24 induction of apoptosis. Ad.mda‐7‐infected tumor cells, but not normal cells, showed increased ceramide accumulation. Infection with Ad.mda‐7 induced a marked increase in various ceramides (C16, C24, C24:1) selectively in prostate cancer cells. Inhibiting the enzyme serine palmitoyltransferase (SPT) using the potent SPT inhibitor myriocin (ISP1), impaired mda‐7/IL‐24‐induced apoptosis and ceramide production, suggesting that ceramide formation caused by Ad.mda‐7 occurs through de novo synthesis of ceramide and that ceramide is required for mda‐7/IL‐24‐induced cell death. Fumonisin B1 (FB1) elevated ceramide formation as well as apoptosis induced by Ad.mda‐7, suggesting that ceramide formation may also occur through the salvage pathway. Additionally, Ad.mda‐7 infection enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin in cancer cells. ASMase silencing by RNA interference inhibited the decreased cell viability and ceramide formation after Ad.mda‐7 infection. Ad.mda‐7 activated protein phosphatase 2A (PP2A) and promoted dephosphorylation of the anti‐apoptotic molecule BCL‐2, a downstream ceramide‐mediated pathway of mda‐7/IL‐24 action. Pretreatment of cells with FB1 or ISP‐1 abolished the induction of ER stress markers (BiP/GRP78, GADD153 and pospho‐eIF2α) triggered by Ad.mda‐7 infection indicating that ceramide mediates ER stress induction by Ad.mda‐7. Additionally, recombinant MDA‐7/IL‐24 protein induced cancer‐specific production of ceramide. These studies define ceramide as a key mediator of an ER stress pathway that may underlie mda‐7/IL‐24 induction of cancer‐specific killing. J. Cell. Physiol. 222: 546–555, 2010. © 2009 Wiley‐Liss, Inc.
2‐methoxyestradiol inhibits atorvastatin‐induced rounding of human vascular smooth muscle cells
Authors: Yin Hao , Gui Yu , Zheng Xi‐Long
Published: November 2009

The cardiovascular benefits of statins, including atorvastatin (ATV), have been reported to be gender‐dependent, but the underlying mechanism is unclear. In this study we examine whether estrogen and its metabolite, 2‐methoxyestradiol (2ME), affect the rounding response of human vascular smooth muscle cells (SMCs) induced by ATV. Twenty‐four hour treatment with ATV (10–100 µM) induced rounding of cultured human SMCs. Addition of 2ME (1–20 µM), but not 17β‐estradiol, for 2 h induced re‐spreading of rounded cells. Our further studies showed that the effects of 2ME were mimicked by microtubule‐disrupting drugs and inhibited by taxol. Inhibition of RhoA and ROCK (Rho‐kinase) by C3‐toxin and H‐1152, respectively, blocked 2ME effects. 2ME effects were also blocked by treatment with either actin‐interfering drugs, such as cytochalasin D and jasplakinolide, or myosin inhibitor blebbistatin. ML‐7 and ‐9, the inhibitors for myosin light chain kinase, inhibited 2ME effect as well. ATV treatment induced a decrease of F‐actin content and Thr18/Ser19 dual phosphorylation of myosin regulatory light chain (MRLC), which was rescued by 2ME or mevalonate. The rescue effects of 2ME on F‐actin content and MRLC dual phosphorylation were abolished by taxol or H‐1152. In addition, kinesin Eg5 inhibitor monastrol and dynein inhibitor erythro‐9‐3‐(2‐hydroxynonyl) adenine (EHNA) significantly blocked 2ME effects. Finally, our results revealed that 2ME inhibited the migration of SMCs induced by ATV (0.1 µM) in wound healing assay and Boyden chamber assay. In summary, our data show that 2ME, but not estrogen, inhibits ATV‐induced rounding of human SMCs through induction of microtubule disassembly and activation of the Rho‐ROCK‐actinomyosin pathway. J. Cell. Physiol. 222: 556–564, 2010. © 2009 Wiley‐Liss, Inc.
7‐ketocholesterol and 5,6‐secosterol modulate differently the stress‐activated mitogen‐activated protein kinases (MAPKs) in liver cells
Authors: Anticoli Simona , Arciello Mario , Mancinetti Adriano , De Martinis Massimo , Ginaldi Lia , Iuliano Luigi , Balsano Clara
Published: November 2009

Enhanced oxidative stress is a common feature of liver diseases and contributes to chronic liver disease (CLD) progression by inducing fibrogenesis during liver regeneration. Peroxidation products of cholesterol metabolism, named oxysterols, are new and reliable markers of oxidative stress in vivo. Patients affected by CLDs present high plasma levels of oxysterols, raising the question of the origin and biological relevance of these compounds in the pathophysiology of chronic liver damage. The aim of this study was to examine the molecular basis of the biological effects of oxysterols on liver‐derived cells, HepG2 and Huh7. Cells were treated with different concentrations (10−9 to 10−5 M) of 7‐ketocholesterol used as a reference, and 5,6‐secosterol, a recently discovered oxysterol. FACS investigations, caspase‐3 activation, and Sytox Green immunofluorescent assay showed that pathological concentrations of oxysterols induced necrosis (30–50%) after 48 h of treatment. The two analyzed compounds displayed a similar, but not identical, behavior. In fact, 5,6‐secosterol, but not 7‐ketocholesterol, induced cell senescence. Notably, low concentrations of 5,6‐secosterol caused a sustained activation of ERK1/2, inducing cell proliferation, this unexpected behavior should be better characterized by further studies. Since enhanced oxidative stress is known to worsen liver chronic hepatitis and frequently results in overall decreased cellular survival, our data suggest the important and different role oxysterols may have in interfering with physiological liver tissue regeneration in injured human liver. Antioxidant treatment may provide a highly specific and effective mean to counteract the common consequences of oxidative stress on chronic hepatitis, such as fibrosis/cirrhosis and liver failure. J. Cell. Physiol. 222: 586–595, 2010. © 2009 Wiley‐Liss, Inc.
MAPK pathway activation and the origins of pediatric low‐grade astrocytomas
Authors: Tatevossian Ruth G. , Lawson Andrew R.J. , Forshew Tim , Hindley Guy F.L. , Ellison David W. , Sheer Denise
Published: November 2009

Low‐grade astrocytomas (LGAs) are the most common type of brain tumor in children. Until recently, very little was known about the underlying biology and molecular genetics of these tumors. However, within the past year a number of studies have shown that the MAPK pathway is constitutively activated in a high proportion of LGAs. Several genetic aberrations which generate this deregulation of the MAPK pathway have been identified, most notably gene fusions between KIAA1549 and BRAF. In this review we summarize these findings, discuss how these gene fusions may arise and consider possible implications for diagnosis and treatment. J. Cell. Physiol. 222: 509–514, 2010. © 2009 Wiley‐Liss, Inc.
Evaluation of critical size defects of mouse calvarial bone: An organ culture study
Authors: Wu Xiaohong , Downes Sandra , Watts David C.
Published: November 2009

Mouse calvarial organ culture has been used widely for the study of bone biology. The purpose of this study was to evaluate the healing potential of neonatal mouse parietal defects in different culture media. The critical size defect (CSD) was also investigated. The parietal bones of neonatal mice were used. Full‐thickness, 0.8‐mm circular defects were created through the bones from one litter of mice. The bones were divided into three groups: Dulbecco's Modified Eagle Medium (DMEM) group, DMEM/osteogenic medium (OM) group, and OM group. Cultures were analyzed with microcomputed‐tomography, dissecting‐microscope, phase‐contrast‐microscope, Von Kossa stain, scanning‐electron‐microscopy, and energy‐dispersive‐X‐ray. Continuous bone formation of parietal bones was observed in all groups. Defects in the DMEM/OM group showed the highest healing potential and exhibited woven bone formation. Defects in the OM group showed limited bone healing at the defect edge. Defects in the DMEM group showed fibrous healing. The most effective culture medium (DMEM/OM) was used to determine the CSD of mouse calvaria in a separate experiment. Circular defects (diameters: 0.8, 1.0, and 1.5 mm) were made in the parietal bones from another litter of mice. The bones were analyzed with microcomputed‐tomography, and phase‐contrast‐microscopy. The bone filling percentages of different size defects were statistically significant: 1.5‐mm defects (4.49%), 1.0‐mm defects (47.65%), and 0.8‐mm defects (73.45%). In three culture conditions, DMEM/OM was the most effective approach to repair bone defects. A 1.5 mm in diameter, full‐thickness parietal defect was found to be the CSD under the DMEM/OM culture conditions. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.
Sensilla on the antennae, legs and ovipositor of Spathius agrili Yang (Hymenoptera: Braconidae), a parasitoid of the emerald ash borer Agrilus planipennis Fairmaire (Coleoptera: Buprestidae)
Authors: Wang Xiao‐Yi , Yang Zhong‐Qi , Gould Juli R.
Published: November 2009

To investigate the host‐finding mechanisms of Spathius agrili Yang, a parasitoid of the emerald ash borer, Agrilus planipennis Fairmaire, a highly concealed insect pest of Fraxinus spp. The morphology and ultrastructure of sensilla on the antennae and legs of both sexes and on the ovipositors and ovipositor sheaths of females were observed using SEM. Results showed that abundant sensilla trichodea (ST) are distributed on the antennae of both females and males, with the sharp trichoid with a socket at the base being the most numerous sensillum. There are 6–7 sensilla placodea on each segment of the flagellum, arranged lengthways in parallel rows. There are very few sensilla styloconica on the female antennae. The tibiae have mostly sensilla chaetica, whereas three types of ST and the relatively plentiful sensilla chaetica are present on the tarsi. Besides ST, a mass of sensilla squamiformia and small short spines are distributed on the pulvilli. Basically there are no sensilla on the female ovipositor, only steering mechanisms for drilling bark and a variety of v‐shaped grooves. However, a large numbers of short sensilla chaetica and microtrichia are found on the internal surface of ovipositor sheath. The outer surface of the ovipositor sheath also has ST. In addition, a clear spring‐like extension structure can be observed on the fore parts of ovipositor sheath. These findings suggest that the main function of sensilla on the antennae is olfaction, while the sensilla on legs and ovipositor sheaths are mechanoreceptors and tactile sensors. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.
Aberrant distribution of junctional complex components in retinoic acid receptor alpha‐deficient mice
Authors: Chung Sanny S.W. , Choi Cindy , Wang Xiangyuan , Hallock Loretta , Wolgemuth Debra J.
Published: November 2009

Retinoic acid receptor alpha (RARα)‐deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In this study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell–cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli‐cell barrier in the tubules of RARα‐deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)‐responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO‐1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin‐40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RARα‐deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RARα‐deficient testes may correlate with a disrupted cyclic expression of RA‐responsive structural components, including vimentin, a downregulation of connexin‐40 in spermatogenic cells, and delayed assembly of ZO‐1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.
Synaptic membrane proteins form stable microdomains in early endosomes
Authors: Geumann Ulf , Schäfer Christina , Riedel Dietmar , Jahn Reinhard , Rizzoli Silvio O.
Published: November 2009

In the plasma membrane, membrane proteins are frequently organized in microdomains that are stabilized both by protein‐protein and protein‐lipid interactions, with the membrane lipid cholesterol being instrumental for microdomain stability. However, it is unclear whether such microdomains persist during endocytotic membrane trafficking. We used stimulated emission‐depletion microscopy to investigate the domain structure of the endosomes. We developed a semiautomatic method for counting the individual domains, an approach that we have validated by immunoelectron microscopy. We found that in endosomes derived from neuroendocrine PC12 cells synaptophysin and several SNARE proteins are organized in microdomains. Cholesterol depletion by methyl‐β‐cyclodextrin disintegrates most of the domains. Interestingly, no change in the frequency of microdomains was observed when endosomes were fused with protein‐free liposomes of similar size (in what constitutes a novel approach in modifying acutely the lipid composition of organelles), regardless of whether the membrane lipid composition of the liposomes was similar or very different from that of the endosomes. Similarly, Rab depletion from the endosome membranes left the domain structure unaffected. Furthermore, labeled exogenous protein, introduced into endosomes by liposome fusion, equilibrated with the corresponding microdomains. We conclude that synaptic membrane proteins are organized in stable but dynamic clusters within endosomes, which are likely to persist during membrane recycling. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.
Optimized time‐gated generalized polarization imaging of Laurdan and di‐4‐ANEPPDHQ for membrane order image contrast enhancement
Authors: Owen Dylan M. , Gaus Katharina
Published: November 2009

The membrane dyes Laurdan and di‐4‐ANEPPDHQ can be used to image membrane order due to a spectral blue‐shift in the fluorescence emission between the liquid‐ordered and liquid‐disordered phases. These images typically take the form of a normalized intensity ratio image known as a generalized polarization (GP) plot. Here, we exploit the known excited state photophysics and time‐resolved data acquisition via time‐correlated single‐photon counting (TCSPC) to demonstrate GP contrast enhancement for these two probes of 7 and 31%, respectively. This improvement in image contrast enhancement will be invaluable when studying the role of lipid rafts in fixed and live cell systems. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.
A metallothionein mimetic peptide protects neurons against kainic acid‐induced excitotoxicity
Authors: Sonn Katrin , Pankratova Stanislava , Korshunova Irina , Zharkovsky Alexander , Bock Elisabeth , Berezin Vladimir , Kiryushko Darya
Published: November 2009

Metallothioneins I and II (MTI/II) are metal‐binding proteins overexpressed in response to brain injury. Recently, we have designed a peptide, termed EmtinB, which is modeled after the β‐domain of MT‐II and mimics the biological effects of MTI/II in vitro. Here, we demonstrate the neuroprotective effect of EmtinB in the in vitro and in vivo models of kainic acid (KA)‐induced neurotoxicity. We show that EmtinB passes the blood–brain barrier and is detectable in plasma for up to 24 hr. Treatment with EmtinB significantly attenuates seizures in C57BL/6J mice exposed to moderate (20 mg/kg) and high (30 mg/kg) KA doses and tends to decrease mortality induced by the high KA dose. Histopathological evaluation of hippocampal (CA3 and CA1) and cortical areas of mice treated with 20 mg/kg KA shows that EmtinB treatment reduces KA‐induced neurodegeneration in the CA1 region. These findings establish EmtinB as a promising target for therapeutic development. © 2009 Wiley‐Liss, Inc.
Selection of reference genes for real‐time quantitative reverse transcription‐polymerase chain reaction in hippocampal structure in a murine model of temporal lobe epilepsy with focal seizures
Authors: Pernot Fabien , Dorandeu Frédéric , Beaup Claire , Peinnequin André
Published: November 2009

Reference genes are often used to normalize expression of data from real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR), and only a validation of their stability during a given experimental paradigm leads to reliable interpretations. The present study was thus designed to validate potential reference genes in a mouse model of mesiotemporal lobe epilepsy (MTLE) with focal seizures after unilateral intrahippocampal injection of kainate (KA). Ipsilateral and contralateral hippocampi were removed during nonconvulsive status epilepticus (5 hr), epileptogenesis (7 days), and the chronic period of recurrent focal seizures (21 days). Naive animals were equally studied. The stability of eight potential reference genes (hypoxanthine phosphoribosyltransferase, Hprt1; peptidylprolyl isomerase A, Ppia; TATA box binding protein, Tbp; beta‐actin, Actb; acidic ribosomal phosphoprotein P0, Arbp; glyceraldehyde‐3‐phosphate dehydrogenase, Gapdh; ribosomal RNA 18S, 18S rRNA; and glucuronidase beta, Gusb) were determined using geNorm and NormFinder software. The first five (Hprt1, Ppia, Tbp, Actb, and Arbp) were found to be stable across the different phases of the disease and appeared adequate for normalizing RT‐qPCR data in this model. This was in contrast to the other three (18S rRNA, Gapdh, and Gusb), which showed unstable expressions and should be avoided. The analysis of KA‐induced changes in the expression of glial fibrillary acidic protein (Gfap) gene resulted in various relative expressions or even a completely different pattern when unstable reference genes were used. These results highlight the absolute need to validate the reference genes for a correct interpretation of mRNA quantification. © 2009 Wiley‐Liss, Inc.
Calpain activation and Na+/Ca2+exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate
Authors: Brustovetsky Tatiana , Bolshakov Alexey , Brustovetsky Nickolay
Published: November 2009

Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na+/Ca2+exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of Western blotting. Degradation of NCX3 was detected only after 1 hr of incubation with glutamate, whereas DCD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate‐induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA subtype of glutamate receptors, added shortly after the initial glutamate‐induced jump in cytosolic Ca2+, completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate‐treated hippocampal neurons, the initial increase in cytosolic Ca2+that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD. © 2009 Wiley‐Liss, Inc.
Glutamate transporters in the central nervous system of a pond snail
Authors: Hatakeyama Dai , Mita Koichi , Kobayashi Suguru , Sadamoto Hisayo , Fujito Yutaka , Hiripi László , Elekes Károly , Ito Etsuro
Published: November 2009

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of 3H‐labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm. © 2009 Wiley‐Liss, Inc.