bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain
Authors: Ureña Jesús Mariano , La Torre Anna , Martínez Albert , Lowenstein Eve , Franco Neus , Winsky‐Sommerer Raphaelle , Fontana Xavier , Casaroli‐Marano Ricardo , Ibáñez‐Sabio Miguel Angel , Pascual Marta , Del Rio José Antonio , de Lecea Luis , Soriano Eduardo
Published: July 2005

Cytosolic tyrosine kinases play a critical role both in neural development and in adult brain function and plasticity. Here we isolated a cDNA with high homology to human Ack1 and mouse Tnk2. This cDNA directs the expression of a 125‐kD protein that can be autophosphorylated in tyrosines. Initially, this clone was named Pyk1 for proline‐rich tyrosine kinase (Lev et al., 1995); however, since it corresponds to the mouse homolog of Ack1, here we called it Ack1/Pyk1. In this study we show that Ack1/Pyk1 mRNA and protein is highly expressed in the developing and adult brain. The highest levels of Ack1/Pyk1 expression were detected in the hippocampus, neocortex, and cerebellum. Electron microscopy studies showed that Ack1/Pyk1 protein is expressed in these regions both at dendritic spines and presynaptic axon terminals, indicating a role in synaptic function. Furthermore, we demonstrate that Ack1/Pyk1 mRNA levels are strongly upregulated by increased neural activity, produced by intraperitoneal kainate injections. During development, Ack1/Pyk1 was also expressed in the proliferative ventricular zones and in postmitotic maturing neurons. In neuronal cultures, Ack1/Pyk1 was detected in developing dendrites and axons, including dendritic tips and growth cones. Moreover, Ack1/Pyk1 colocalized with Cdc42 GTPase in neuronal cultures and coimmunoprecipitated with Cdc42 in HEK 293T cells. Altogether, our findings indicate that Ack1/Pyk1 tyrosine kinase may be involved both in adult synaptic function and plasticity and in brain development. J. Comp. Neurol. 490:119–132, 2005. © 2005 Wiley‐Liss, Inc.
Water deprivation increases the expression of neuronal nitric oxide synthase (nNOS) but not orexin‐A in the lateral hypothalamic area of the rat
Authors: Yao Song T. , Gouraud Sabine , Paton Julian F.R. , Murphy David
Published: July 2005

The discovery that the lateral hypothalamic area (LHA) might be important in modulating drinking behavior and fluid balance has led to numerous studies aimed at identifying the key neurotransmitters/neuromodulators and pathways involved. While past studies have demonstrated the presence of neuronal nitric oxide synthase (nNOS) within the LHA, its role in the regulation of fluid homeostasis is not known. In light of this, and the mounting evidence suggesting a role for nitric oxide in osmotic regulation within the hypothalamus, this study sought to determine the effects of 24‐ and 72‐hours of water deprivation on nNOS protein expression within the LHA of the rat with immunohistochemistry. In euhydrated control animals we observed nNOS‐like immunoreactivity throughout all levels of the LHA. Following 24 hours of dehydration the number of nNOS‐like immunopositive neurons was significantly increased in the rostral but not the caudal regions of LHA. Seventy‐two hours of water deprivation lead to further increases in nNOS‐like immunoreactivity at different levels of the LHA. Interestingly, however, we observed increased nNOS‐like immunoreactivity in the caudal regions of the LHA that was not evident after 24 hours of water deprivation. Double‐labeling immunofluorescence histochemistry revealed that the nNOS‐like immunoreactive neurons were not colocalized with the orexin‐A‐containing neurons. These results suggest that an osmotic challenge leads to an upregulation of nNOS immunoreactivity within discrete areas of the LHA. This altered neurochemistry within the LHA further highlights the potential importance of nitric oxide and the LHA in central regulation of fluid homeostasis. J. Comp. Neurol. 490:180–193, 2005. © 2005 Wiley‐Liss, Inc.
Effective thermal actions and thermal properties of timber members in natural fires
Authors: König Jürgen
Published: July 2005

For the thermal analysis of structural or non‐structural timber members, using conventional simplified heat transfer models, thermal conductivity values of timber are normally calibrated to test results such that they implicitly take into account influences such as mass transport that are not included in the model. Various researchers and designers have used such effective thermal conductivity values, originally determined for standard fire exposure, to evaluate other fire scenarios such as natural fires. This paper discusses in qualitative terms some parameters that govern the burning of wood and their influence on effective conductivity values. Reviewing fire tests of timber slabs under natural fire conditions, the study explains why effective conductivity values, giving correct results for the ISO 834 standard fire scenario, should not be used in other fire scenarios. For this reason, the thermal properties of timber given in EN 1995‐1‐2 are limited to standard fire exposure. As shown by heat transfer calculations, the effective thermal conductivity of the char layer is strongly dependent on the charring rate and therefore varies during a natural fire scenario. It has also been shown that char oxidation during the decay phase in a natural fire has a significant influence on the temperature development in the timber member, since char surface temperatures exceed the gas temperature in the compartment or furnace. Using increased effective gas temperature as thermal action during the decay phase, and varying conductivity values for the char layer, fairly good agreement could be obtained regarding the temperature development in the timber member and the char depth. Copyright © 2005 John Wiley & Sons, Ltd.
Telomere dysfunction drives chromosomal instability in human mammary epithelial cells
Authors: Soler David , Genescà Anna , Arnedo Gema , Egozcue Josep , Tusell Laura
Published: July 2005

The development of genomic instability is an important step toward generating the multiple genetic changes required for cancer. Telomere dysfunction is one of the factors that contribute to tumorigenesis. Telomeres shorten with each cell division in the absence of telomerase. Human mammary epithelial cells (HMECs) obtained from normal human tissue demonstrate two growth phases. After an initial phase of active growth, HMECs exhibit a growth plateau termed selection. However, some cells can emerge from this growth plateau by spontaneously losing expression of the p16INK4a protein. These post‐selection HMECs are capable of undergoing an additional 20–50 population doublings in culture. Continued proliferation of these post‐selection HMECs leads to further telomere erosion, loss of the capping function, and the appearance of end‐to‐end chromosome fusions that can enter bridge‐fusion‐breakage (BFB) cycles, generating massive chromosomal instability before terminating in a population growth plateau termed agonescence. We have found that the chromosome arms carrying the shortest telomeres are those involved in telomere–telomere type rearrangements. In addition, we found that the risk of a particular chromosome being unstable differs between individuals. Most importantly, we identified sister chromatid fusion as a first event in generating genomic instability in HMECs. During post‐selection HMEC growth, double strand breaks are generated by both fused chromosomes as well as individual chromosomes with fused chromatids entering BFB cycles. These broken chromosome extremities are able to join other broken ends or eroded telomeres, producing massive chromosomal instability at the later passages of the cell culture. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat. © 2005 Wiley‐Liss, Inc.
Osteochondral repair in the rabbit model utilizing bilayered, degradable oligo(poly(ethylene glycol) fumarate) hydrogel scaffolds
Authors: Holland Theresa A. , Bodde Esther W. H. , Baggett L. Scott , Tabata Yasuhiko , Mikos Antonios G. , Jansen John A.
Published: July 2005

In this study, hydrogel scaffolds, based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF), were implanted into osteochondral defects in the rabbit model. Scaffolds consisted of two layers—a bottom, bone forming layer and a top, cartilage forming layer. Three scaffold formulations were implanted to assess how material composition and transforming growth factor‐β1 (TGF‐β1) loading affected osteochondral repair. Critical histological evaluation and scoring of the quantity and quality of tissue in the chondral and subchondral regions of defects was performed at 4 and 14 weeks. At both time points, no evidence of prolonged inflammation was observed, and healthy tissue was seen to infiltrate the defect area. The quality of this tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 14 weeks. A promising degree of Safranin O staining and chondrocyte organization was observed in the newly formed surface tissue, while the underlying subchondral bone was completely integrated with the surrounding bone at 14 weeks. Material composition within the bottom, bone‐forming layer did not appear to affect the rate of scaffold degradation or tissue filling. However, no bone upgrowth into the chondral region was observed with any scaffold formulation. TGF‐β1 loading in the top layer of scaffolds appeared to exert some therapeutic affect on tissue quality, but further studies are necessary for scaffold optimization. Yet, the excellent tissue filling and integration resulting from osteochondral implantation of these OPF‐based scaffolds demonstrates their potential in cartilage repair strategies. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005
A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus
Authors: Martínez‐Ramos Isy , Maya‐Mendoza Apolinar , Gariglio Patricio , Aranda‐Anzaldo Armando
Published: July 2005

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non‐coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR–NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton‐nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR‐based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c‐myc locus and the multigene locus PRM1 → PRM2 → TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM. © 2005 Wiley‐Liss, Inc.
Structure and function of the upstream promotor of the human Mafbx gene: The proximal upstream promotor modulates tissue‐specificity
Authors: Zhao Weidong , Wu Yong , Zhao Jingbo , Guo Shuang , Bauman William A. , Cardozo Christopher P.
Published: July 2005

Muscle loss has been linked to increased expression of an ubiquitin ligase termed muscle atrophy F‐box (MAFbx), a nuclear protein involved in degradation of MyoD. To gain insights into mechanisms by which the human MAFbx gene is controlled, the structure of its upstream promotor were studied, and its expression in cultured cells was characterized. Expression of MAFbx was found only in cells of muscle lineage. A reporter gene controlled by 948 bases of human MAFbx upstream promotor displayed similar cell‐type selectivity. MAFbx levels were greatly enhanced upon myogenic differentiation of C2C12 myoblasts, and differentiation markedly increased activity of a reporter gene constructed with 400 bp of upstream promotor from the MAFbx gene. The core promotor spanned approximately 160 bases beginning at −241 bp upstream of the first codon, included potential binding sites for MyoD and myogenin, and was highly conserved among mouse, rat, and humanMAFbx genes. The major transcription start site for the human MAFbx gene was 340 bases upstream of the ATG and was localized the highly conserved region of 140 bp. The findings indicate an important role for the immediate upstream promotor of the human MAFbx gene in mediating its developmental expression and tissue specificity. © 2005 Wiley‐Liss, Inc.
Angiogenesis and apoptosis in glioma: Two arenas for promising new therapies
Authors: Bögler Oliver , Mikkelsen Tom
Published: July 2005

New therapies for gliomas are urgently needed in view of the very marginal increase in patient survival that has been achieved over the past two decades, which is only somewhat mitigated by improvements in quality of life. Two relatively recent fields of research that hold out great promise in this area, are angiogenesis and apoptosis. Depriving growing tumors of the blood supply they need, or tipping the balance in the cancer cell towards cell death, both provide conceptually elegant approaches to therapy, with the hope of great efficacy and little toxicity. However, attempts at successfully translating exciting laboratory findings to the clinic have been slowed by the complexity of the underlying biology. In this article we examine some of the issues that have impeded progress, and examine the potential role that integrins may play as targets, with a role in both angiogenesis and apoptosis. © 2005 Wiley‐Liss, Inc.
Role of O‐linked β‐N‐acetylglucosamine modification in the subcellular distribution of alpha4 phosphoprotein and Sp1 in rat lymphoma cells
Authors: Dauphinee Shauna M. , Ma Marlene , Too Catherine K.L.
Published: July 2005

The mTOR alpha4 phosphoprotein is a prolactin (PRL)‐downregulated gene product that is found in the nucleus of PRL‐dependent rat Nb2 lymphoma cells. Alpha4 lacks a nuclear localization signal (NLS) and the mechanism of its nuclear targeting is unknown. Post‐translational modification by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) moieties has been implicated in the nuclear transport of some proteins, including transcription factor Sp1. The nucleocytoplasmic enzymes O‐β‐N‐acetylglucosaminyltransferase (OGT) and O‐β‐N‐acetylglucosaminidase (O‐GlcNAcase) adds or remove O‐GlcNAc moieties, respectively. If O‐GlcNac moieties contribute to the nuclear targeting of alpha4, a decrease in O‐GlcNAcylation (e.g., by inhibition of OGT) may redistribute alpha4 to the cytosol. The present study showed that alpha4 and Sp1 were both O‐GlcNAcylated in quiescent and PRL‐treated Nb2 cells. PRL alone or PRL + streptozotocin (STZ; an O‐GlcNAcase inhibitor) significantly (P ≤ 0.05) increased the O‐GlcNAc/alpha4 ratio above that in control quiescent cells. However, PRL + alloxan (ALX; an OGT inhibitor) or ALX alone did not decrease O‐GlcNAcylation of alpha4 below that of controls and alpha4 remained nuclear. In comparison, PRL (±ALX/STZ) greatly increased Sp1 protein levels, caused a significant decrease in the GlcNAc/Sp1 ratio (P ≤ 0.05, n = 3) as compared to controls and partially redistributed Sp1 to the cytosol. Finally, a 50% downregulation of OGT gene expression by small interfering RNA (i.e., siOGT) partially redistributed both alpha4 and Sp1 to the cytosol. The alpha4 protein partner PP2Ac had no detectable O‐GlcNAc moieties and its nuclear distribution was not affected by siOGT. In summary, alpha4 and Sp1 contained O‐GlcNAc moieties, which contributed to their nuclear targeting in Nb2 cells. © 2005 Wiley‐Liss, Inc.
(−)‐Epigallocatechin‐3‐gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms
Authors: Hung Chi‐Feng , Huang Tur‐Fu , Chiang Han‐Sun , Wu Wen‐Bin
Published: July 2005

It is increasingly evident that the stromal cells are involved in key metastatic processes of melanoma and some malignant solid tumors. (−)‐Epigallocatechin‐3‐gallate (EGCG), a polyphenolic compound from green tea, has been shown to have anti‐tumor activity, inhibiting adhesion, migration, and proliferation of tumor cells. However, little attention has been paid on its effects on stromal cells. In the present study, we determined the effects of EGCG on stromal fibroblasts. We showed that fibroblast adhesion to collagen, fibronectin, and fibrinogen were inhibited by EGCG. One of the possible mechanisms is binding of EGCG to fibronectin and fibrinogen but not to collagen. We then focused how EGCG affected fibroblast adhesion to collagen. EGCG treatment attenuated the antibody binding to fibroblast's integrin α2β1, indicating EGCG may affect the expression and affinity of integrin α2β1. Moreover, intracellular H2O2 level was decreased by EGCG treatment, suggesting that the tonic maintenance of intracellular H2O2 may be required for cell adhesion to collagen. In parallel, collagen‐induced FAK phosphorylation, actin cytoskeleton reorganization in fibroblasts, migration and matrix metalloproteinase(s) (MMPs) activity were also affected by EGCG. Tubular networks formed by melanoma cells grown on three‐dimensional Matrigel were also disrupted when fibroblasts were treated with EGCG in a non‐contact coculture system. Taken together, we provided here the first evidence that EGCG is an effective inhibitor on behaviors of the stromal fibroblasts, affecting their adhesion and migration. The inhibitory activity of EGCG may contribute to its anti‐tumor activity. The findings and concepts disclosed here may provide important basis for a further experiment towards understanding tumor–stroma interaction. © 2005 Wiley‐Liss, Inc.
Cholestane‐3β,5α,6β‐triol inhibits osteoblastic differentiation and promotes apoptosis of rat bone marrow stromal cells
Authors: Liu Hongmei , Yuan Lan , Xu Shanjin , Wang Kui , Zhang Tianlan
Published: July 2005

Converging lines of evidence suggest that oxidized lipids, long recognized as a risk factor in atherogenesis, also contribute to osteoporosis, but the underlying mechanism is not understood in detail. The effect of atherogenesis related factors including oxysterols on the differentiation and survival of marrow stromal cells (MSCs) would be very important in understanding the link between atherosclerosis and osteoporosis. In the present study, the effect of oxysterol cholestane‐3β,5α,6β‐triol (Triol) on osteoblastic differentiation and apoptosis of primary rat bone MSCs as well as the related mechanisms were studied. Triol inhibited MSCs osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, osteocalcin secretion, and matrix mineralization. In the other aspect, Triol promoted MSCs apoptosis, as characterized by condensed or fragmented nuclei as well as active externalization of phosphatidyl serine to the cell surface. In addition, Triol was found to induce increases of intracellular Ca2+ and Ca2+‐dependent reactive oxygen species generation in MSCs. These effects were involved in the action of Triol on apoptosis, but not on osteoblastic differentiation of MSCs. These results suggested that Triol might contribute to the decreased bone formation by inhibition of osteoblastic differentiation and promotion of apoptosis of MSCs, providing insights about common factors underlying the pathogenesis of atherosclerosis and osteoporosis. © 2005 Wiley‐Liss, Inc.
Tyrosine phosphatases as regulators of skeletal development and metabolism
Authors: Schiller Katherine R. , Mauro Laura J.
Published: July 2005

The protein tyrosine kinases (PTK) and the protein tyrosine phosphatases (PTPs) are enzymes which play an integral role in tyrosine phosphorylation‐dependent signaling cascades. By catalyzing the phosphorylation and dephosphorylation of cellular proteins, these enzymes direct the steady‐state levels of specific phosphoproteins and ultimately dictate the functional state of all cells. The importance of this type of signaling in the skeleton is accepted but poorly understood. The contribution of the PTKs to signaling events in bone has been well studied but, in contrast, the regulation by PTPs is poorly defined. The recent identification of 107 genes within the human genome which encode members of the PTP superfamily emphasizes the need to consider the importance of these proteins in skeletal tissue. In this prospective, we will summarize the present state of our knowledge regarding the function of this enzyme superfamily, illustrating its relevance to the development and maintenance of the skeleton and highlighting future directions that should improve our understanding of these critical signaling molecules. © 2005 Wiley‐Liss, Inc.
Repair of DNA interstrand crosslinks may take place at the nuclear matrix
Authors: Atanassov Boyko , Gospodinov Anastas , Stoimenov Ivaylo , Mladenov Emil , Russev George , Tsaneva Irina , Anachkova Boyka
Published: July 2005

Host cell reactivation assay using Trioxsalen‐crosslinked plasmid pEGFP‐N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP‐1, which did not contain the promoter of the egfp gene, and with pEGFP‐G−, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%–2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP‐N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose‐dependant way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment. © 2005 Wiley‐Liss, Inc.
Leptin expression in human primary skeletal muscle cells is reduced during differentiation
Authors: Solberg Rigmor , Aas Vigdis , Thoresen G. Hege , Kase Eili Tranheim , Drevon Christian A. , Rustan Arild C. , Reseland Janne E.
Published: July 2005

We found leptin to be strongly expressed in undifferentiated human myoblasts derived from biopsies of the thigh (Musculus vastus lateralis). Both mRNA expression and secretion of leptin were reduced during in vitro differentiation into primary myotubes. However, the expression of the leptin receptor (OB‐Rb) mRNA, was unchanged during differentiation of the muscle cells. Administration of recombinant leptin had no effect on leptin, myogenin, myoD, or GLUT4 mRNA expressions during the period of cellular differentiation. A functional leptin receptor was demonstrated by an acute leptin‐induced 1.5‐fold increase in ERK activity (P = 0.029). Although mRNA expression of regulation of suppressor of cytokine signaling‐3 (SOCS‐3) mRNA expression was unaltered, leptin significantly stimulated fatty acid oxidation after 6 h measured as acid soluble metabolites (ASM). Palmitic acid (PA), oleic acid (OA), and eicosapentaenoic acid (EPA), known to modulate leptin expression in other tissues, had no effect on mRNA expression or secretion of leptin from human myotubes. In conclusion, we demonstrate that leptin is highly expressed in undifferentiated human myoblasts and the expression is reduced during differentiation to mature myotubes. The role of leptin in these cells needs to be further characterized. © 2005 Wiley‐Liss, Inc.
Suppressive effect of regucalcin on cell differentiation and mineralization in osteoblastic MC3T3‐E1 cells
Authors: Yamaguchi Masayoshi , Kobayashi Mayumi , Uchiyama Satoshi
Published: July 2005

The role of regucalcin in the regulation of osteoblastic cell function was investigated. Osteoblastic MC3T3‐E1 cells with subconfluent monolayers were cultured in a medium containing regucalcin (10−10–10−8 M) without fetal bovine serum (FBS). The proliferation of osteoblastic cells was not significantly altered in the presence of regucalcin. The results of reverse transcription‐polymerase chain reaction (RT‐PCR) analysis with specific primers showed that the expression of Runx2 (Cbfa1) and insulin‐like growth factor‐I (IGF‐I) mRNAs in osteoblastic cells was significantly suppressed in the presence of regucalcin (10−10 or 10−9 M). Transforming growth factor‐β1 mRNA levels were significantly enhanced in the 24 h‐culture with regucalcin (10−10 or 10−9 M). α1(I) collagen and glyceroaldehyde‐3‐phosphate dehydrogenase (G3PDH) mRNA levels were not significantly changed by culture with regucalcin (10−10 or 10−9 M). Alkaline phosphatase activity was significantly decreased in the lysate of cells cultured for 24 or 48 h with regucalcin (10−10–10−8 M). Moreover, the expression of regucalcin in osteoblastic cells was demonstrated by RT‐PCR and Western blot analysis. When regucalcin (10−7 M) was added into the enzyme reaction mixture containing the lysate of osteoblastic cells cultured in the absence of regucalcin, alkaline phosphatase activity was significantly decreased. Also, Ca2+/calmodulin‐dependent nitric oxide (NO) synthase activity in the cell lysate was significantly decreased by addition of regucalcin (10−10–10−8 M) into the reaction mixture. The presence of anti‐regucalcin monoclonal antibody (5 or 10 ng/ml) in the enzyme reaction mixture caused a significant increase in NO synthase activity in the cell lysate in the presence or absence of Ca2+/calmodulin, suggesting a role of endogenous regucalcin. When osteoblastic cells with subconfluency were cultured in the presence of regucalcin (10−10 or 10−9 M) for 3, 9, or 18 days, the results with Alizarin red staining showed that the mineralization was markedly suppressed by culture with regucalcin for 3, 9, or 18 days. This study demonstrates that regucalcin regulates the function of osteoblastic cells, and that the protein suppresses cell differentiation and mineralization. © 2005 Wiley‐Liss, Inc.
Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells
Authors: Thullbery Matthew D. , Cox Holly D. , Schule Travis , Thompson Charles M. , George Kathleen M.
Published: July 2005

Acetylcholinesterase (AChE) expression is regulated in cell types at the transcriptional and translational levels. In this study, we characterized and compared AChE catalytic activity, mRNA, protein expression, and protein localization in a variety of neuronal (SH‐SY5Y neuroblastoma and primary cerebellar granule neurons (CGN)) and non‐neuronal (LLC‐MK2, HeLa, THP‐1, and primary astrocytes) cell types. All cell lines expressed AChE catalytic activity; however the levels of AChE‐specific activity were higher in neuronal cells than in the non‐neuronal cell types. CGN expressed significantly more AChE activity than SH‐SY5Y cells. All cell lines analyzed expressed AChE protein at equivalent levels, as well as mRNA splice variants. Localization of AChE was characterized by immunofluorescence and confocal microscopy. SH‐SY5Y, CGN, and nerve‐growth factor‐differentiated PC‐12 cells exhibited a pattern of AChE localization characterized as diffuse in the cytoplasm and punctate staining along neurites and on the plasma membrane. The localization in HeLa, LLC‐MK2, fibroblasts, and undifferentiated PC‐12 cells was significantly different than in neuronal cells—AChE was intensely localized in the perinuclear region, without staining near or on the plasma membrane. Based on the evidence presented here, we hypothesize that the presence of AChE protein doesn't correlate with catalytic activity, and the diffuse cytoplasmic and plasma membrane localization of AChE is a property of neuronal cell types. © 2005 Wiley‐Liss, Inc.
A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death
Authors: Węsierska‐Gądek Józefa , Gueorguieva Marieta , Ranftler Carmen , Zerza‐Schnitzhofer Gerlinde
Published: July 2005

The efficacy of distinct anti‐cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti‐proliferative and pro‐apoptotic action of a number of anti‐cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti‐proliferative and pro‐apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose‐ and time‐dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases‐3/7 in two separate microtiter plates using the CellTiter‐Glo™ Luminescent Cell Viability Assay and the Caspase‐Glo™ 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter‐Blue™ Cell Viability Assay and Caspase‐Glo™ 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases‐3/7. The new multiplexing assay offers a range of benefits over standard assays. © 2005 Wiley‐Liss, Inc.
Functional role of PKC in contraction of cultured human prostatic stromal cells
Authors: Takahashi Ryosuke , Nishimura Junji , Hirano Katsuya , Naito Seiji , Kanaide Hideo
Published: July 2005

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC‐potentiated protein phosphatase‐1 inhibitory protein (CPI‐17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI‐17 was over‐expressed, PDBu induced a large contraction, but the agonist‐induced contraction did not become larger than expected. In α‐toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI‐17 over‐expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI‐17 and also that the PKC‐CPI‐17 pathway does not appear to play a major role in the agonist‐induced contraction even when CPI‐17 was over‐expressed. © 2005 Wiley‐Liss, Inc.
Differential expression of antioxidant enzymes in various hepatocellular carcinoma cell lines
Authors: Yang Liang‐Yo , Chen Wei‐Li , Lin Jia‐Wei , Lee Shu‐Fen , Lee Chin‐Cheng , Hung Tsung I , Wei Yau‐Huei , Shih Chwen‐Ming
Published: July 2005

Recent evidence suggests that reactive oxygen species (ROS) play an important role in the pathogenesis of various illnesses, and the ROS and antioxidant enzymes are highly associated with cell differentiation and diseases. In this study, we tested the hypothesis that specific antioxidant enzymes are differentially expressed in hepatocellular carcinoma (HCC) cell lines with various degrees of differentiation. We compared the expression of several antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GRx), and glutathione peroxidase (GPx) in five HCC cell lines with well (Hep G2 and Hep 3B) or poor (HA22T/VGH, HA55T/VGH, and SK‐Hep‐1) differentiation. Our results showed that both well‐differentiated HCC cell lines expressed extremely higher CAT and GRx enzyme activities than all three poorly differentiated ones. Moreover, the protein and mRNA levels of CAT were much higher in two well‐differentiated HCC cell lines than in all three poorly differentiated ones. Both well‐differentiated HCC cell lines also showed a higher protein or mRNA expression of Cu/ZnSOD and MnSOD than three poorly differentiated ones. Our results demonstrate that specific antioxidant enzymes (especially, CAT and GRx) are differentially expressed in HCC cell lines with well or poor differentiation. These findings suggest that CAT and GRx are two potential differentiation markers for HCC. © 2005 Wiley‐Liss, Inc.
Cross‐talk among epidermal growth factor, Ap5A, and nucleotide receptors causing enhanced ATP Ca2+ signaling involves extracellular kinase activation in cerebellar astrocytes
Authors: Delicado Esmerilda G. , Jiménez Ana I. , Carrasquero Luz María G. , Castro Enrique , Miras‐Portugal Mª Teresa
Published: July 2005

In previous papers, we reported that ATP calcium responses in cerebellar astrocytes were strongly potentiated by preincubation with nanomolar concentrations of the diadenosine pentaphosphate Ap5A. However, the intracellular signaling pathway mediating this effect was not defined. We also showed that stimulation of astrocytes with the dinucleotide led to the activation of extracellular regulated kinases (ERKs). Here, we examined whether ERKs are involved in the potentiating mechanism and intracellular mechanism leading to their activation. Epidermal growth factor (EGF) exactly reproduced the potentiation displayed by the dinucleotide. Moreover, the potentiation of ATP responses by Ap5A and EGF was completely abolished by the MAP kinase (MEK) inhibitor U‐0126, indicating that ERK activation is a required step for the potentiation event. Our data also indicated that ERK activation and the potentiation of ATP calcium responses were sensitive to the src‐like kinase inhibitor herbimycin A, p21ras farnesyltransferase inhibitor peptide, and some PKC inhibitors. Taken together, our findings reveal that Ap5A triggers the potentiation of ATP calcium responses through an intracellular mechanism that is insensitive to pertussis toxin and that this potentiation requires src protein‐mediated ERK activation and the participation of an atypical protein kinase C isoform activated downstream from ERK. © 2005 Wiley‐Liss, Inc.
Nanocomposites of NLO chromophore‐modified layered silicates and polypropylene
Authors: Dudkina Marina M. , Tenkovtsev Andrey V. , Pospiech Doris , Jehnichen Dieter , Häußler Liane , Leuteritz Andreas
Published: July 2005

Intercalation of guest species into layered inorganic solids is a method of producing ordered inorganic–organic assemblies with unique microstructures controlled by host–guest and guest–guest interactions. Smectite clay minerals, such as montmorillonite, having appropriate functional molecules in between the silicate layers are supposed to exhibit a wide range of novel characteristics. Nanocomposite material based on maleic anhydride‐grafted polypropylene and dye‐modified layered silicate was developed. Characteristics of organo‐modified montmorillonite particles and polymer/clay hybrids have been investigated through FTIR, SAXS, DSC, UV measurements, and transmission electron microscopy. The results of the intercalation process, structural characterization, and thermal properties will be discussed in comparison with the intercalation and nanocomposite preparation results. The intercalation was successfully conducted by the ion‐exchange method. It was shown that intercalated dibenzilidene acetone type chromophores exist in the clay galleries in an aggregated form, probably as J‐aggregates. This feature strongly effects on optical and nonlinear optical properties of nanocomposites. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 2493–2502, 2005
Surface film formation on a graphite electrode in Li‐ion batteries: AFM and XPS study
Authors: Leroy S. , Blanchard F. , Dedryvère R. , Martinez H. , Carré B. , Lemordant D. , Gonbeau D.
Published: July 2005

The formation of a passivation film (solid electrolyte interphase, SEI) at the surface of the negative electrode of full LiCoO2/graphite lithium‐ion cells using LiPF6 (1M) in carbonate solvents as electrolyte was investigated by means of x‐ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The analyses were carried out at different potentials of the first and the fifth cycles, showing the potential‐dependent character of the surface‐film species formation. These species were mainly identified as Li2CO3 up to 3.8 V and LiF up to 4.2 V. This study shows the formation of the SEI during charging and its partial dissolution during discharge. Copyright © 2005 John Wiley & Sons, Ltd.
Grid interaction of offshore wind farms. Part 2. Case study simulations
Authors: Morren Johan , Pierik Jan T. G. , Haan Sjoerd W. H. de , Bozelie Jan
Published: July 2005

Owing to an increasing penetration of wind turbines and large wind farms in electrical power systems, the wind turbines start influencing the overall power system behaviour. Dynamic wind farm models for power system studies have been presented in Part 1 of the article. In this contribution, these models are applied in a number of case studies to determine the impact of wind farms on the power grid. Four turbine concepts are evaluated with respect to their grid interaction. Case studies involving normal behaviour, voltage dip behaviour, voltage control capability and behaviour during frequency deviations will be presented. Copyright © 2005 John Wiley & Sons, Ltd.
Large‐scale wind power integration in Norway and impact on damping in the Nordic grid
Authors: Hagstrøm Espen , Norheim Ian , Uhlen Kjetil
Published: July 2005

The Nordic energy market is in need of new power generation capacity. Norway is today in shortage of electrical energy, and the hydropower‐based system must rely on importing electrical power from its neighbours in a normal year of hydro inflow. One solution to this problem is to invest in new generation capacity, e.g. by integrating more wind power into the Norwegian grid. The Norwegian government has announced that 1000 MW of wind power will be installed in Norway by 2010, corresponding to an eventual energy production of approximately 3 TWh year−1. This article investigates the impact of wind power integration in Norway on the damping of interarea mode oscillations in the Nordic grid. The impact on the interarea mode oscillations is tested for various types of generators. Results from computer analyses of a simplified yet realistic equivalent of the Nordic power system are used to compute the interarea mode oscillations. Copyright © 2005 John Wiley & Sons, Ltd.
Ecosystem structure and soil‐surface conditions drive the variability in the foliar δ13C and δ15N ofStipa tenacissimain semiarid Mediterranean steppes
Authors: Maestre Fernando T. , Cortina Jordi
Published: July 2005

We evaluated the effects of ecosystem composition and structure (species richness and diversity, cover and spatial attributes of vegetation), abiotic factors (climate and topographical features) and the condition of the bare‐ground areas (evaluated using soil‐surface indicators) on the performance ofStipa tenacissima[evaluated using foliar δ13C, δ15N, nitrogen concentration and the carbon‐to‐nitrogen (C:N) ratio] in 15 steppes of SE Spain. Foliar δ13C values ofS. tenacissimashowed a low degree of variation in the studied steppes, with average values ranging from −24.1 to −22.9‰. Higher variation was found in the δ15N values, which ranged from −5.5 to −2.4‰. The nitrogen concentration and the C:N ratio varied between 5.0 and 8.0 mg g−1, and between 55.4 and 85.3, respectively. The δ13C values became less negative with increasing spatial aggregation of perennial vegetation, while the C:N values increased with increasing perennial vegetation cover. The δ15N values became more negative with increasing infiltration in the bare‐ground areas, but the nitrogen concentration was not related to any of the environmental variables measured. Our results suggest that the relative importance of ecosystem structure and soil‐surface conditions in the bare ground areas was higher than that of abiotic factors as determinants of the performance ofS. tenacissima. The results also show that even subtle changes in these ecosystem features may lead to modifications in plant performance in semiaridS. tenacissimasteppes, and thus to modifications in the associated ecosystem functions in the mid‐ to long‐term.
Effect of angiotensin‐converting enzyme inhibition on functional class in patients with left ventricular systolic dysfunction—a meta‐analysis
Authors: Abdulla Jawdat , Pogue Janice , Abildstrøm Steen Z. , Køber Lars , Christensen Erik , Pfeffer Marc A. , Yusuf Salim , Torp‐Pedersen Christian
Published: July 2005

Background:

The effect of angiotensin converting enzyme (ACE) inhibitors on symptoms in patients with left ventricular systolic dysfunction (LVSD) is controversial.

Aims:

To perform a meta‐analysis of studies evaluating effect of ACE inhibitors on New York Heart Association (NYHA) class in patients with LVSD.

Methods:

Individual data from 10389 patients in NYHA classes I–IV from four large long‐term studies (2–4‐year follow‐up) and summary data from 2302 patients in NYHA classes II–IV from 16 short‐term studies (3 months follow‐up) were meta‐analysed to assess changes in NYHA class.

Results:

The large long‐term studies showed a significant improvement in the worst NYHA classes (classes II–IV compared to class I) in the ACE inhibitor arm versus placebo, odds ratio (OR)=0.875 (0.811–0.943) p=0.0005. This effect was only present in studies which included patients with chronic heart failure and was particularly pronounced on deterioration to the worst NYHA class IV, OR=0.66 (0.52–0.84) p=0.001. There was no effect in the studies which included patients after myocardial infarction. The short‐term chronic heart failure studies showed a significant improvement in NYHA class; OR for improvement of at least one NYHA class was 2.11 (1.48–2.98, 95% CI) p<0.0001.

Conclusion:

ACE inhibition significantly improves symptomatic status measured as NYHA classification in patients with chronic heart failure.
Effects of combined administration of low dose atorvastatin and vitamin E on inflammatory markers and endothelial function in patients with heart failure
Authors: Tousoulis Dimitris , Antoniades Charalambos , Vassiliadou Carmen , Toutouza Marina , Pitsavos Christos , Tentolouris Costas , Trikas Athanasios , Stefanadis Christodoulos
Published: July 2005

Background:

Heart failure has been associated with impaired endothelial function, increased inflammatory process and elevated oxidative stress status. Both statins and vitamin E separately improve endothelial function in patients with hypercholesterolemia and/or advanced atherosclerosis.

Aim:

To evaluate the effect of atorvastatin alone or in combination with vitamin E on endothelial function and serum levels of interleukin‐6 (IL‐6), tumor necrosis factor alpha (TNF‐α) and vascular cells adhesion molecule (sVCAM‐1) in patients with ischemic heart failure.

Methods:

Thirty‐eight male patients with ischemic cardiomyopathy were randomly divided into three groups and received either atorvastatin 10 mg/day (n=14), a combination of atorvastatin 10 mg/day plus vitamin E 400 IU/day (n=12), or no statin or antioxidant treatment (n=12), controls) for 4 weeks. Forearm blood flow (FBF) was measured using venous occlusion strain‐gauge plethysmography. Forearm vasodilatory response to reactive hyperemia (RH%) or to nitrate (NTG%) was defined as the percent change of FBF from rest to the maximum flow during reactive hyperemia or after nitrate administration, respectively.

Results:

RH% was significantly improved in both the atorvastatin‐treated (p<0.01) and atorvastatin plus vitamin E groups (p<0.05), but the increase was significantly higher in the atorvastatin‐treated group (p<0.05). Serum levels of IL‐6, TNF‐α and sVCAM‐1 were decreased in the atorvastatin‐treated group (p<0.05 for all), but remained unaffected in the other two groups (p=NS for all).

Conclusions:

Low dose atorvastatin treatment improves endothelial function and reduces the expression of proinflammatory cytokines and adhesion molecules in patients with ischemic heart failure, an effect partly depressed by vitamin E.
Clinical trials update from the European Society of Cardiology Heart Failure meeting and the American College of Cardiology: Darbepoetin alfa study, ECHOS, and ASCOT‐BPLA
Authors: Cleland John G.F. , Coletta Alison P. , Clark Andrew L. , Velavan Periaswamy , Ingle Lee
Published: July 2005

This article provides information and a commentary on landmark trials presented at the European Society of Cardiology Heart Failure meeting held in June 2005, relevant to the pathophysiology, prevention and treatment of heart failure. All reports should be considered as preliminary data, as analyses may change in the final publication. The erythropoiesis stimulating protein, darbepoetin alfa, increased haemoglobin levels, improved quality of life and showed a trend for improved exercise duration in anaemic patients with symptomatic chronic heart failure. In the ECHOS study, the selective dopamine agonist nolomirole (CHF1035) showed no benefit in heart failure patients. Preliminary results of the ASCOT‐BPLA study, which were reported at the American College of Cardiology meeting in March 2005, showed that in hypertensive patients, treatment with a calcium antagonist plus an ACE inhibitor was more effective at reducing cardiovascular outcomes than atenolol plus a diuretic.
Reliability and validity of a modified Brief Pain Inventory short form in patients with osteoarthritis
Authors: Mendoza Tito , Mayne Tracy , Rublee Dale , Cleeland Charles
Published: July 2005

The Brief Pain Inventory short form (BPI‐sf) is a validated, widely used, self‐administered questionnaire developed to assess the severity of pain and the impact of pain on daily functions. A modified version was used daily in randomised control trials of patients with arthritis undergoing treatment with cyclooxygenase‐2 specific inhibitors and non‐steroidal anti‐inflammatory drugs. Results indicate that the modified BPI‐sf, much like the original scale, was internally reliable, consistent over time, and had good construct, as well as convergent and predictive validity in assessment of patients suffering from conditions of chronic pain. Each scale and individual pain intensity item refers to changes in osteoarthritis pain associated with medication use. The modified BPI‐sf, like the parent scale, is a valid and reliable tool for situations in which pain is assessed daily and minimises the burden placed on patients to record information necessary for scientific investigations.