bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of lytic viral infection and IL‐6 in early versus late passage lymphoblastoid cell lines and EBV‐associated lymphoproliferative disease
Authors: Jones Richard J. , Seaman William T. , Feng Wen‐Hei , Barlow Elizabeth , Dickerson Sarah , Delecluse Henri‐Jacque , Kenney Shannon C.
Published: May 2007

Lytically infected EBV‐positive lymphoblastoid cells enhance the growth of early‐passage, but not late‐passage, EBV‐immortalized lymphoblastoid cell lines (LCLs) in SCID mice and have enhanced IL‐6 secretion. Here, we have examined the importance of IL‐6 for the growth of early‐passage LCLs (EPL) in SCID mice, identified lytic EBV proteins that activate IL‐6 production and compared viral and cellular differences between early versus late passage LCLs (LPL). IL‐6 was required for efficient growth of EPL in SCID mice. The EBV immediate‐early (IE) proteins, BRLF1 and BZLF1, each induced IL‐6 secretion when transfected into 293 and BJAB cells. Interestingly, the combination of BZLF1 and the latent EBV protein, LMP‐1, induced much more IL‐6 expression in both 293 and BJAB cells than either protein alone. Both BZLF1 and BRLF1 also enhanced IL‐10 production in 293 cells. In comparison to the EPL, LPL had much reduced expression of early lytic viral proteins and cellular IL‐6. In contrast, expression of cellular IL‐10 was similar in EPL versus LPL, while VEGF secretion was increased in late‐passage LCLs. These results suggest that both BRLF1 and BZLF1 contribute to IL‐6 secretion in lytically infected cells and that lytically infected cells may promote early lymphoproliferative disease in patients through enhanced IL‐6 production. © 2007 Wiley‐Liss, Inc.
Towards development of a water quality monitoring strategy for Mashreq and Maghreb countries
Authors: Sayed A. El , Fahmy Hussam
Published: May 2007

There is an immanent need to develop accustomed regional guidelines for Mashreq countries (Egypt, Jordan, Lebanon, Syria, West Bank and Gaza) and Maghreb countries (Morocco, Algeria and Tunisia) to enhance water quality monitoring (WQM) and information dissemination to institutions and decision makers.

The purpose of this paper is to provide an outline for the developed WQM strategy for Mashreq and Maghreb countries. The implementation of these WQM guidelines is expected to achieve accurate and reliable water quality data coverage; improved access and dissemination of water quality information and its guidelines by stakeholders in forms that are suitable for influencing policy dialogue.

Based on the needs of decision‐making and policy development issues, the WQM guidelines can be tailored for countries in the region. In addition, several international WQM guidelines and information dissemination strategies are considered as suitable modules that can be adopted to build regional guidelines for WQM. The final guidelines address namely three issues: monitoring program design, monitoring procedures, and information dissemination.

Reviewing the available information proved that it would be impossible to come up with a generic WQM program or a network design that can be applied for the entire region (Egypt is considered as a guide case study). Copyright © 2007 John Wiley & Sons, Ltd.
Aglycone geniposidic acid, a naturally occurring crosslinking agent, and its application for the fixation of collagenous tissues
Authors: Mi Fwu‐Long , Huang Chin‐Tsung , Chiu Ya‐Ling , Chen Mei‐Chin , Liang Hsiang‐Fa , Sung Hsing‐Wen
Published: May 2007

A natural compound, aglycone geniposidic acid (aGSA), originated from the fruits of Gardenia jasminoides ELLIS was used for the fixation of collagenous tissues. The presumed crosslinking reaction mechanism of collagenous tissues with aGSA was inferred by reacting aGSA with a bifunctional amine, 1,6‐hexanediamine, using a series of 1H NMR, FT‐IR, and UV/Vis spectra analyses. aGSA reacted with 1,6‐hexanediamine by a nucleophilic attack on the olefinic carbon atom at C‐2 of deoxyloganin aglycone, followed by opening the dihydropyran ring to form heterocyclic amine compounds. It is inferred that aGSA may form intramolecular and intermolecular crosslinks with a heterocyclic structure within collagen fibers in tissues. The degrees of tissue fixation by aGSA at different pH values were investigated by examining the fixation indices and denaturation temperatures of test samples. It was found that the fixation indices and denaturation temperatures of test samples fixed at neutral or basic pH (pH 7.4 or pH 8.5) were significantly greater than at acidic pH (pH 4.0). The results obtained in this study may be used to elucidate the crosslinking mechanism and optimize the fixation process for developing bioprostheses fixed by aGSA. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007
Processing and in vitro behavior of hydroxyapatite coatings prepared by electrostatic spray assisted vapor deposition method
Authors: Hou Xianghui , Choy Kwang‐Leong , Leach Sian Ellen
Published: May 2007

Hydroxyapatite (HA) bioactive coatings are often used to improve bone attachment and reduce corrosion of metal prosthesis implants. This paper reports the preparation of HA coatings onto titanium substrates using a novel electrostatic spray assisted vapor deposition (ESAVD)‐based method. The deposited coatings are characterized using a combination of Fourier transform infrared spectrometer, X‐ray diffraction, Raman spectroscopy, scanning electron microscopy, and scratch test. The results confirm that well‐crystallized HA coatings can be produced directly via the single‐step ESAVD process, without further heat‐treatment. The in vitro behavior of the as‐deposited HA coating in simulated body fluid (SBF) is also presented. After 14‐day immersion in SBF, the adhesion of the HA coating to the substrate increases significantly. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007
Synthesis and characterization ofin situchitosan‐based hydrogel via grafting of carboxyethyl acrylate
Authors: Kim Mi‐Sook , Choi Yoon‐Jeong , Noh Insup , Tae Giyoong
Published: May 2007

We developed and characterized a novelin situchitosan‐poly(ethylene oxide) (PEO) hydrogel via two steps: 2‐carboxyethyl acrylate molecules were grafted to the primary amine functional groups in chitosan in the first step and then Michael type addition reaction was processed between the grafted acrylate end groups and the thiol end groups in the PEO. Grafting of acrylate molecules to the amine groups in the deacetylated water soluble chitosan was confirmed by observing the new acrylate peaks by the FTIR and NMR spectra of the acrylated chitosan samples, as well as changes in relative viscosities of chitosan and acrylated chitosan. Formation of the chitosan‐PEO hydrogel was visually observed with digital images after both gelation and hydration. Rheological analyses of the hydrogel formation were performed to detect its gelation time, phase angle changes, and visco‐elastic properties over frequency and strain percentage. Their results indicated that the gelation process was completed within 10 min after mixing the precursor liquid solutions. An extent of water swelling, mechanical strength against compression and the morphologies of the hydrogel surface and cross sections after dehydration process were analyzed by microbalance measurement, texture analyzer, and scanning electron microscopy observation, respectively. Biological activities of the hydrogels were evaluated by observing smooth muscle cell behaviors such as cell adhesion and viability as well as by measuring the number of adhered cells on their surfaces. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007
Temperature‐responsive polymer–gold nanocomposites as intelligent therapeutic systems
Authors: Owens Donald E. , Eby Jackson K. , Jian Yicun , Peppas Nicholas A.
Published: May 2007

The objective of this study was to synthesize and characterize a thermally responsive polymer‐metal nanocomposite system comprised of a solid gold nanoparticle core and thermally responsive interpenetrating polymer network (IPN) shell, which was surface functionalized or PEGylated with a covalently bound linear poly(ethylene glycol) chain layer. Gold nanoparticles (50 nm diameter) were prepared using standard gold chloride and citrate reduction method. These particles were then encapsulated inside of a polyacrylamide (PAAm)/poly(acrylic acid) (PAA) IPN shell via an in situ inverse emulsion polymerization. The surface of the nanocomposite system was then PEGylated via covalent grafting of a linear methoxy‐PEG‐N‐hydroxysuccinimide (M.W. 3400) to the primary amine groups of the PAAm network. Scanning and transmission electron microscopy were used to confirm the successful synthesis and encapsulation of gold nanoparticles within the IPN shell. Dynamic light scattering was used to examine the temperature swelling response of the IPN particles. Zeta‐potential analysis was used to confirm the successful PEGylation of the final nanocomposite system. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007
Tissue response in the rat and the mouse to degradable dextran hydrogels
Authors: De Jong Wim H. , Dormans Jan A.M.A. , Van Steenbergen Mies J. , Verharen Henny W. , Hennink Wim E.
Published: May 2007

Two types of hydroxyethyl‐methacrylated dextran (dex‐HEMA) hydrogels differing in crosslink density were compared for local tissue responses and degradation characteristics in mice and rats. Implants (1 mm thick, rat: 10 mm diameter, mouse: 6 mm diameter) varying in degree of HEMA substitution (DS5 and DS13, meaning 5 or 13 HEMA groups per 100 glucose units of dextran) were subcutaneously implanted and tissue responses were evaluated at week 2, 6, and 13 after implantation. In the rat after 2 weeks a slight fibrous capsule was formed composed of macrophages and fibroblasts sometimes accompanied by a minimal infiltrate. Small fragments, surrounded by macrophages and giant cells indicated hydrogel degradation. After 13 weeks DS5 implants were resorbed while parts of the DS13 implants were still present. In the mouse a moderate to strong capsule formation was present at 2 weeks accompanied by inflammatory cells (macrophages and polymorphonuclear granulocytes) and debris. Draining lymph node activation was observed. Skin ulceration was present irrespective of the type of implant. Clear differences in the tissue responses between the rat and mouse were noted, as well as between implants of different degree of substitution. Mice showed a more pronounced early inflammatory response compared with rats, whereas the degradation was more complete in rats than in mice. The differences in histology between the hydrogels disappeared over time at 13 weeks after implantation and similar responses were noted for both types of hydrogels. Both in mice and rats the DS5 hydrogels showed a faster degradation rate than the DS13 hydrogels. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007
Fabrication and in vitro drug release of drug‐loaded star oligo/poly(DL‐lactide) microspheres made by novel ultrasonic‐dispersion method
Authors: Zou Tao , Li Song‐Lin , Cheng Si‐Xue , Zhang Xian‐Zheng , Zhuo Ren‐Xi
Published: May 2007

Cholic acid functionalized star oligo/poly(DL‐lactide)s with different molecular weights were synthesized through the ring‐opening polymerization of DL‐lactide initiated by cholic acid. On the basis of the specific physicochemical properties of the star oligo/poly(DL‐lactide)s, submicron sized drug‐delivery systems were fabricated using a very convenient “ultrasonic dispersion method,” which did not involve toxic organic solvents. The drug‐loaded microspheres had a regular spherical shape with a narrow size distribution. The effects of ultrasonic power and the molecular weight of polymers on the microsphere fabrication were investigated. The in vitro drug release was studied. The release profiles were fitted by the classical empirical exponential expression. The fitting result indicated that the drug release was controlled by combined degradation and diffusion mechanism. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007
Poly(butyl methacrylate‐co‐methacrylic acid) tissue engineering scaffold with pro‐angiogenic potential in vivo
Authors: Butler Mark J. , Sefton Michael V.
Published: May 2007

A poly(butyl methacrylate‐co‐methacrylic acid) (BMA‐MAA) scaffold was fabricated by an in situ polymerization solvent casting/particulate leaching technique. It displayed high porosity (85–90%), pore interconnectivity, and a pore size range of 100–650 μm. Compression testing of the scaffolds demonstrated a dependence of the compressive stiffness on several fabrication variables including the ratio of monomer to salt used during the polymerization, the degree of salt fusion, and the choice of alternative comonomers to BMA. Subcutaneous implantation of BMA‐MAA scaffolds in mice revealed an increased level of histological angiogenesis in tissue invading the pores of the scaffold compared to a BMA control, consistent with the prediction that methacrylic acid (MAA) containing copolymer beads are angiogenic in a wound healing context. At postoperative day 21, the capillary density in the BMA‐MAA scaffolds was 56 ± 13/mm2 as compared to 32 ± 8/mm2 for the BMA scaffolds. With further investigation, it is expected that this biomaterial capable of eliciting an angiogenic response will have widespread application in tissue engineering. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007
Modified polycarbonate urethane: Synthesis, properties and biological investigation in vitro
Authors: Szelest‐Lewandowska A. , Masiulanis B. , Szymonowicz M. , Pielka S. , Paluch D.
Published: May 2007

A new polycarbonate urethane (PCU‐I) was synthesized from aliphatic monomers, i.e. polyhexamethylene carbonate diol and 4,4′‐methylene‐bis cyclohexane diisocyanate, a mixture of low molecular diols, and castor oil (containing mainly the triglyceride of 12‐hydroxyoleic acid). The second synthesized polymer (PCU‐II) did not contain castor oil. Both PCUs had good tensile strength, i.e. 32.5 and 27.8 MPa for PCU‐I and PCU‐II, respectively. Modification by castor oil led to a decrease in glass transition temperature (Tg = −14°C for PCU‐I and −6°C for PCU‐II) and an increase in the softening temperature (135 and 125°C for PCU‐I and PCU‐II, respectively). Partial crosslinking of PCU‐I increased the storage modulus of elasticity and provided better resistance to sterilization by ETO and γ radiation. Both PCUs displayed good stability when subjected to sterilization by hydrogen peroxide plasma. Neither PCU caused cytotoxic effect in mouse fibroblasts (3T3 Balb C). They also had no toxic effects on the morphotic components and did not influence changes in the hematologic parameters or plasmatic coagulation system of human blood. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2007
Nanogel‐based delivery system enhances PGE2 effects on bone formation
Authors: Kato Norihiko , Hasegawa Urara , Morimoto Nobuyuki , Saita Yoshitomo , Nakashima Kazuhisa , Ezura Yoichi , Kurosawa Hisashi , Akiyoshi Kazunari , Noda Masaki
Published: May 2007

Recovery of bone loss is one of the active research issues in bone medicine due to the need for efficient measures for bone gain. We examined here a novel drug delivery system using a nanogel of cholesterol‐bearing pullulan (CHP) in combination with prostaglandin E2 (PGE2). PGE2 or PGE2/CHP, vehicle (saline containing 0.06% ethanol and 0.02% Tween 80) or CHP were injected on to the calvariae of mice once every day for 5 days per week for 4 weeks. Low dosage of PGE2 (0.6 µg) alone or CHP alone did not induce new bone formation in this system. In contrast, PGE2 (0.6 µg)/CHP induced new bone formation. Bone formation activities of PGE2 was enhanced by CHP nanogels only at the site of injection (calvaria) but not in the distant sites of the skeleton, showing that PGE2/CHP could avoid systemic effects. In spite of the fact that previously reported animal models of bone formation by PGE2 were associated with loss of body weight, bone formation based on PGE2/CHP did not associate with loss of body weight. Furthermore, only a single application of PGE2 in combination with nanogel cross‐linking hydrogel sphere (PGE2/CHP‐PEO) induced new bone formation. Thus, nanogel‐based delivery system is an efficient delivery system of bone anabolic agent, PGE2. J. Cell. Biochem. 101:1063–1070, 2007. © 2007 Wiley‐Liss, Inc.
Estrogen receptor α/β isoforms, but not βcx, modulate unique patterns of gene expression and cell proliferation in Hs578T cells
Authors: Secreto Frank J. , Monroe David G. , Dutta Shamit , Ingle James N. , Spelsberg Thomas C.
Published: May 2007

The actions of 17β‐estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor‐α (ERα) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERβ, along with multiple estrogen receptor (ER) splice variants such as ERβcx, an ERβ splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERα, ERβ, or ERβcx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH‐tamoxifen (4HT) treated Hs578T ERα and ERβ cells revealed distinct ligand and receptor‐dependent patterns of gene regulation, while the induction of ERβcx did not alter gene expression patterns. E2 stimulation of Hs578T ERβ cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERα or ERβ cells to 4HT. Expression of ERβcx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2‐stimulated Hs578T ERβ cells entering S‐phase, along with a 17% increase in G0/G1 cell‐cycle arrest. We demonstrate here that ERα and ERβ regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform‐specific changes in cell proliferation. Hs578T ER expressing cell‐lines provide a unique BC model system, permitting the comparison of ERα, ERβ, and ERβcx actions in the same cell‐line. J. Cell. Biochem. 101:1125–1147, 2007. © 2007 Wiley‐Liss, Inc.
Proteomic analysis of cardiomyocytes differentiation in mouse embryonic carcinoma P19CL6 cells
Authors: Wen Jianyan , Xia Qing , Lu Cailing , Yin Lina , Hu Juan , Gong Yanhua , Yin Bin , Monzen Koshiro , Yuan Jiangang , Qiang Boqin , Zhang Xuemin , Peng Xiaozhong
Published: May 2007

A clonal derivative named P19CL6 has been isolated from pluripotent P19 mouse embryonic carcinoma cells, and this subline efficiently differentiates into beating cardiomyocytes when treated with 1% dimethyl sulfoxide (DMSO). It offers a valuable model to study cardiomyocytes differentiation in vitro. In this study, comparative proteomic analysis was used to characterize the protein profiles associated with the DMSO‐induced cardiomyocytes differentiation of P19CL6 cells. We demonstrated that P19CL6 cells indeed differentiated into cardiomyocytes after DMSO inducement as they expressed sarcomeric myosin heavy chain (MHC) as well as three cardiac‐specific transcription factors (Csx/Nkx‐2.5, GATA‐4, and MEF2C). Image analysis of silver‐stained two‐dimensional gels was used to find protein spots that exhibited an at least 1.5‐fold change in abundance after successful differentiation. Seventeen protein spots were selected for further analysis by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF‐MS) and/or nano‐electrospray ionization MS/MS (ESI‐MS/MS), and 16 protein spots were identified. The identified proteins are involved in different cellular functions such as metabolism, signal transduction, and cellular organization. To confirm the expression changes of the identified proteins during differentiation, the mRNA levels of six identified proteins (including seven protein spots) were assessed by the real‐time polymerase chain reaction and three showed a correlation between mRNA level and protein abundance. As an initial step toward identifying proteins involved in maintaining the differentiated state of cardiomyocytes derived from P19CL6 cells, our data provide some helpful information that may lead to a better understanding of the molecular mechanisms by which P19CL6 cells differentiate into cardiomyocytes after treatment with DMSO. J. Cell. Biochem. 102: 149–160, 2007. © 2007 Wiley‐Liss, Inc.
The effect of simulated microgravity on osteoblasts is independent of the induction of apoptosis
Authors: Bucaro M.A. , Zahm A.M. , Risbud M.V. , Ayyaswamy P.S. , Mukundakrishnan K. , Steinbeck M.J. , Shapiro I.M. , Adams C.S.
Published: May 2007

Bone loss during spaceflight has been attributed, in part, to a reduction in osteoblast number, altered gene expression, and an increase in cell death. To test the hypothesis that microgravity induces osteoblast apoptosis and suppresses the mature phenotype, we created a novel system to simulate spaceflight microgravity combining control and experimental cells within the same in vitro environment. Cells were encapsulated into two types of alginate carriers: non‐rotationally stabilized (simulated microgravity) and rotationally stabilized (normal gravity). Using these specialized carriers, we were able to culture MC3T3‐E1 osteoblast‐like cells for 1–14 days in simulated microgravity and normal gravity in the same rotating wall vessel (RWV). The viability of cells was not affected by simulated microgravity, nor was the reductive reserve. To determine if simulated microgravity sensitized the osteoblasts to apoptogens, cells were challenged with staurosporine or sodium nitroprusside and the cell death was measured. Simulated microgravity did not alter the sensitivity of C3H10T‐1/2 stem cells, MC3T3‐E1 osteoblast‐like cells, or MLO‐A5 osteocyte‐like cells to the action of these agents. RT‐PCR analysis indicated that MC3T3‐E1 osteoblasts maintained expression of RUNX2, osteocalcin, and collagen type I, but alkaline phosphatase expression was decreased in cells subjected to simulated microgravity for 5 days. We conclude that osteoblast apoptosis is not induced by vector‐averaged gravity, thus suggesting that microgravity does not directly induce osteoblast death. J. Cell. Biochem. 102: 483–495, 2007. © 2007 Wiley‐Liss, Inc.
Changes in Osteoblast, Chondrocyte, and Adipocyte Lineages Mediate the Bone Anabolic Actions of PTH and Small Molecule GSK‐3 Inhibitor
Authors: Kulkarni Nalini H. , Wei Tao , Kumar Amar , Dow Ernst R. , Stewart Trent R. , Shou Jianyong , N'cho Mathias , Sterchi Diane L. , Gitter Bruce D. , Higgs Richard E. , Halladay David L. , Engler Thomas A. , Martin T. John , Bryant Henry U. , Ma Yanfei L. , Onyia Jude E.
Published: May 2007

Parathyroid hormone (PTH) and glycogen synthase kinase‐3 (GSK‐3) inhibitor 603281‐31‐8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281‐31‐8 in rat osteopenia model. Female 6‐month‐old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1–38) at 10 µg/kg/day s.c. or 603281‐31‐8 at 3 mg/kg/day p.o. for 60 days. Twenty‐four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P < 0.05) were subjected to pathway analysis to delineate relevant bio‐processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK‐3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three‐fold increase in marrow adiposity. PTH and GSK‐3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK‐3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU‐fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK‐3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes. J. Cell. Biochem. 102: 1504–1518, 2007. © 2007 Wiley‐Liss, Inc.
Effect of hyperthermia and chemotherapeutic agents on TRAIL‐induced cell death in human colon cancer cells
Authors: Yoo Jinsang , Lee Yong J.
Published: May 2007

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In the previous study [Yoo and Lee, 2007], we have reported that hyperthermia could enhance the cytotoxicity of TRAIL‐induced apoptosis. We observed in human colorectal cancer cell line CX‐1 that TRAIL‐induced apoptotic death and also that mild hyperthermia promoted TRAIL‐induced apoptotic death through caspase activation and cytochrome‐c release. Although its effects in vivo are not clear, hyperthermia has been used as an adjunctive therapy for cancer. Hyperthermia is often accompanied by chemotherapy to enhance its effect. In this study, CX‐1 colorectal adenocarcinoma cells were treated with TRAIL concurrently with hyperthermia and oxaliplatin or melphalan. To evaluate the cell death effects on tumor cells via hyperthermia and TRAIL and chemotherapeutic agents, FACS analysis, DNA fragmentation, and immunoblottings for PARP‐1 and several caspases and antiapoptotic proteins were performed. Activities of casapse‐8, caspase‐9, and caspase‐3 were also measured in hyperthermic condition. Interestingly, when analyzed with Western blot, we detected little change in the intracellular levels of proteins related to apoptosis. Clonogenic assay shows, however, that chemotherapeutic agents will trigger cancer cell death, either apoptotic or non‐apoptotic, more efficiently. We demonstrate here that CX‐1 cells exposed to 42°C and chemotherapeutic agents were sensitized and died by apoptotic and non‐apoptotic cell death even in low concentration (10 ng/ml) of TRAIL. J. Cell. Biochem. 103: 98–109, 2008. © 2007 Wiley‐Liss, Inc.
Characterization of voltage‐and Ca2+‐activated K+ channels in rat dorsal root ganglion neurons
Authors: Li Wei , Gao Shang‐Bang , Lv Cai‐Xia , Wu Ying , Guo Zhao‐Hua , Ding Jiu‐Ping , Xu Tao
Published: May 2007

Auxiliary β‐subunits associated with pore‐forming Slo1 α‐subunits play an essential role in regulating functional properties of large‐conductance, voltage‐ and Ca2+‐activated K+ channels commonly termed BK channels. Even though both noninactivating and inactivating BK channels are thought to be regulated by β‐subunits (β1, β2, β3, or β4), the molecular determinants underlying inactivating BK channels in native cells have not been extensively demonstrated. In this study, rβ2 (but not rβ3‐subunit) was identified as a molecular component in rat lumbar L4‐6 dorsal root ganglia (DRG) by RT‐PCR responsible for inactivating large‐conductance Ca2+‐dependent K+ currents (BKi currents) in small sensory neurons. The properties of native BKi currents obtained from both whole‐cell and inside‐out patches are very similar to inactivating BK channels produced by co‐expressing mSlo1 α‐ and hβ2‐subunits in Xenopus oocytes. Intracellular application of 0.5 mg/ml trypsin removes inactivation of BKi channels, and the specific blockers of BK channels, charybdotoxin (ChTX) and iberiotoxin (IbTX), inhibit these BKi currents. Single BKi channel currents derived from inside‐out patches revealed that one BKi channel contained three rβ2‐subunits (on average), with a single‐channel conductance about 217 pS under 160 K+ symmetrical recording conditions. Blockade of BKi channels by 100 nM IbTX augmented firing frequency, broadened action potential waveform and reduced after‐hyperpolarization. We propose that the BKi channels in small diameter DRG sensory neurons might play an important role in regulating nociceptive input to the central nervous system (CNS). J. Cell. Physiol. 212: 348–357, 2007. © 2007 Wiley‐Liss, Inc.
Morphogenetic signals from chondrocytes promote chondrogenic and osteogenic differentiation of mesenchymal stem cells
Authors: Hwang Nathaniel S. , Varghese Shyni , Puleo Christopher , Zhang Zijun , Elisseeff Jennifer
Published: May 2007

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well‐defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte‐conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte‐secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte‐conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo. J. Cell. Physiol. 212: 281–284, 2007. © 2007 Wiley‐Liss, Inc.
IGF‐1 activates hEAG K+ channels through an Akt‐dependent signaling pathway in breast cancer cells: Role in cell proliferation
Authors: Borowiec Anne‐Sophie , Hague Frédéric , Harir Noria , Guénin Stéphanie , Guerineau François , Gouilleux Fabrice , Roudbaraki Morad , Lassoued Kaiss , Ouadid‐Ahidouch Halima
Published: May 2007

Previous work from our laboratory has shown that human ether à go‐go (hEAG) K+ channels are crucial for breast cancer cell proliferation and cell cycle progression. In this study, we investigated the regulation of hEAG channels by an insulin‐like growth factor‐1 (IGF‐1), which is known to stimulate cell proliferation. Acute applications of IGF‐1 increased K+ current‐density and hyperpolarized MCF‐7 cells. The effects of IGF‐1 were inhibited by hEAG inhibitors. Moreover, IGF‐1 increased mRNA expression of hEAG in a time‐dependent manner in parallel with an enhancement of cell proliferation. The MCF‐7 cell proliferation induced by IGF‐1 is inhibited pharmacologically by Astemizole or Quinidine or more specifically using siRNA against hEAG channel. Either mitogen‐activated protein kinase (MAPK) or phosphatidylinositol 3‐kinase (PI3K) are known to mediate IGF‐1 cell proliferative signals through the activation of extracellular signal‐regulated kinase 1/2 (Erk 1/2) and Akt, respectively. In MCF‐7 cells, IGF‐1 rapidly stimulated Akt phosphorylation, whereas IGF‐1 had little stimulating effect on Erk 1/2 which seems to be constitutively activated. The application of wortmannin was found to block the effects of IGF‐1 on K+ current. Moreover, the inhibition of Akt phosphorylation by the application of wortmannin or by a specific reduction of Akt kinase activity reduced the hEAG mRNA levels. Taken together, our results show, for the first time, that IGF‐1 increases both the activity and the expression of hEAG channels through an Akt‐dependent pathway. Since a hEAG channel is necessary for cell proliferation, its regulation by IGF‐1 may thus play an important role in IGF‐1 signaling to promote a mitogenic effect in breast cancer cells. J. Cell. Physiol. 212:690–701, 2007. © 2007 Wiley‐Liss, Inc.
Increased Smad1 expression and transcriptional activity enhances trans‐differentiation of hepatic stellate cells
Authors: Shen Hong , Fan Jianghong , Burczynski Frank , Minuk Gerald Y. , Cattini Peter , Gong Yuewen
Published: May 2007

Smad1 is a receptor‐activated intracellular signaling protein, which mediates signal transduction of bone morphogenetic proteins. Current study investigated the expression and transcriptional activity of Smad1 during hepatic stellate cell (HSC) activation. Rat HSCs were isolated from rats at 1, 2, 3 and 4 days after gavaged with carbon tetrachloride (CCl4) or corn oil. RT‐PCR, Western blot, gel‐shift assay and luciferase assay were employed to examine Smad1 expression and transcriptional activity, respectively. CCl4‐cirrhotic liver fat‐storing cells‐8B (CFSC‐8B) cells were infected with recombinant adenoviruses of Smad1 and/or Smad1 shRNA. Both mRNA and protein levels of Smad1 were significantly increased at 48 h after gavage of CCl4. Gel shift assays demonstrated a significant increase in nuclear Smad1 in day 9 HSCs. Transfection of HSCs with Smad1 responsible luciferase indicated an increase in Smad1 transcriptional activity in day 6 HSCs (1.563 ± 0.229 in day 6 versus 0.785 ± 0.192 in day 3). When CFSC‐8B cells were infected with adenoviruses with Smad1 or Smad1 short hairpin RNA (shRNA), there was an increase or decrease in Smad1 mRNA and protein, respectively. Smooth muscle α‐actin expression was increased or decreased according to induction or reduction of Smad1. In conclusion, there were significantly increases in Smad1 expression and transcriptional activity during in vivo activation of hepatic stellate cells. J. Cell. Physiol. 212:764–770, 2007. © 2007 Wiley‐Liss, Inc.
Importance of several cysteine residues for the chloride conductance of trout anion exchanger 1 (tAE1)
Authors: Martial Sonia , Guizouarn Hélène , Gabillat Nicole , Pellissier Bernard , Borgese Franck
Published: May 2007

In this study, we devised a cysteine‐focused point mutation analysis of the chloride channel function of trout anion exchanger 1 (tAE1) expressed in X. lævis oocytes. Seven cysteines, belonging to the transmembrane domain of tAE1, were mutated into serines (either individually or in groups) and the effects of these mutations on the chloride conductance of injected oocytes were measured. We showed that three cysteines were essential for the functional expression of tAE1. Namely, mutations C462S, C583S and C588S reduced Cl− conductance by 68%, 52% and 83%, respectively, when compared to wild type tAE1. These residual conductances were still inhibited by 0.5 mM niflumic acid. Western blot experiments demonstrated that C462 was involved in protein expression onto the plasma membrane. A mutant devoid of this residue was unable to express onto the plasma membrane, especially if several other cysteines were missing: consequently, the cysteine‐less mutant of tAE1 was not functional. C583 and C588 were involved in the channel function of tAE1 as shown by anion substitution experiments proving that selectivity of the mutated pore differs from the wild type one. On the contrary, they were not involved in the Cl−/HCO exchange function of tAE1, as demonstrated by intracellular pH measurements. These and several complementary mutations allow us to conclude that a mutant of tAE1 containing the sole C462 can drive a marginal Cl− current; however, the minimal configuration necessary to get optimal functional expression of the tAE1 chloride channel is that of a mutant containing unaffected residues C462, C583 and C588. J. Cell. Physiol. 213: 70–78, 2007. © 2007 Wiley‐Liss, Inc.
Stimulation by ghrelin of p42/p44 mitogen‐activated protein kinase through the GHS‐R1a receptor: Role of G‐proteins and β‐arrestins
Authors: Camiña Jesus P. , Lodeiro Maria , Ischenko Olga , Martini Ana C. , Casanueva Felipe F.
Published: May 2007

Results presented in this study indicate that in human embryonic kidney 293 cells (HEK 293), the ghrelin receptor growth hormone secretagogue receptor type 1a (GHS‐R1a) activates the extracellular signal‐related kinases 1 and 2 (ERK 1/2) via three pathways. One pathway is mediated by the β‐arrestins 1 and 2, and requires entry of the receptor into a multiprotein complex with the β‐arrestins, Src, Raf‐1, and ERK 1/2. A second pathway is Gq/11‐dependent and involves a Ca2+‐dependent PKC (PKCα/β) and Src. A third pathway is Gi‐dependent and involves phosphoinositide 3‐kinase (PI3K), PKCε, and Src. Our current study reveals that Gi/o‐ and Gq/11‐proteins are crucially involved in the β‐arrestin‐mediated ERK 1/2 activation. These results thus support the view that the β‐arrestins act as both scaffolding proteins and signal transducers in ERK 1/2 activation, as reported for other receptors. The different pathways of ERK 1/2 activation suggest that binding to GHS‐R1a activates ERK 1/2 pools at different locations within the cell, and thus probably with different physiological consequences. J. Cell. Physiol. 213: 187–200, 2007. © 2007 Wiley‐Liss, Inc.
Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220NPAT in human embryonic stem cells
Authors: Ghule Prachi N. , Becker Klaus A. , Harper J. Wade , Lian Jane B. , Stein Janet L. , van Wijnen Andre J. , Stein Gary S.
Published: May 2007

Human embryonic stem (ES) cells have an expedited cell cycle (∼15 h) due to an abbreviated G1 phase (∼2.5 h) relative to somatic cells. One principal regulatory event during cell cycle progression is the G1/S phase induction of histone biosynthesis to package newly replicated DNA. In somatic cells, histone H4 gene expression is controlled by CDK2 phosphorylation of p220NPAT and localization of HiNF‐P/p220NPAT complexes with histone genes at Cajal body related subnuclear foci. Here we show that this ‘S point’ pathway is operative in situ in human ES cells (H9 cells; NIH‐designated WA09). Immunofluorescence microscopy shows an increase in p220NPAT foci in G1 reflecting the assembly of histone gene regulatory complexes in situ. In contrast to somatic cells where duplication of p220NPAT foci is evident in S phase, the increase in the number of p220NPAT foci in ES cells appears to precede the onset of DNA synthesis as measured by BrdU incorporation. Phosphorylation of p220NPAT at CDK dependent epitopes is most pronounced in S phase when cells exhibit elevated levels of cyclins E and A. Our data indicate that subnuclear organization of the HiNF‐P/p220NPAT pathway is rapidly established as ES cells emerge from mitosis and that p220NPAT is subsequently phosphorylated in situ. Our findings establish that the HiNF‐P/p220NPAT gene regulatory pathway operates in a cell cycle dependent microenvironment that supports expression of DNA replication‐linked histone genes and chromatin assembly to accommodate human stem cell self‐renewal. J. Cell. Physiol. 213: 9–17, 2007. © 2007 Wiley‐Liss, Inc.
Complement 3 is involved in changing the phenotype of human glomerular mesangial cells
Authors: Wan Jian‐Xin , Fukuda Noboru , Endo Morito , Tahira Yoshiko , Yao En‐Hui , Matsuda Hiroyuki , Ueno Takahiro , Matsumoto Koichi
Published: May 2007

Complement activation contributes to tissue injury in various forms of glomerulopathy and is characterized by deposition of complement components, which accelerates the progression of chronic renal damage. We recently reported that complement 3 (C3), a critical component of the complement system, is associated with the synthetic phenotype of vascular smooth muscle cells. It is possible that C3 stimulates mesangial cells to assume the synthetic phenotype to, in turn, induce glomerular injury and sclerosis. We investigated the role of C3 in the growth and phenotype of mesangial cells. Cultured human mesangial cells (HMCs) expressed C3 mRNA and protein, and levels were increased in response to IFN‐γ and TNF‐α. HMCs also expressed C3a receptor mRNA and protein. Exogenous C3a stimulated DNA synthesis in HMCs in a dose‐dependent manner. C3a decreased expression h‐caldesmon mRNA, a marker of the contractile phenotype, and increased the expression of osteopontin, matrix Gla, and collagen type1 α1 (collagen IV) mRNAs, which are markers of the synthetic phenotype. C3a decreased expression of α‐smooth muscle actin in HMCs. Small interfering RNA (siRNA) targeting C3 reduced the DNA synthesis and proliferation of HMCs, increased expression of h‐caldesmon mRNA, and decreased expression of osteopontin, matrix Gla, and collagen IV mRNAs in HMCs. These results indicate that C3 causes HMCs to convert to the synthetic phenotype and stimulates growth of mesangial cells, suggesting that C3 may play an important role in phenotypic regulation of mesangial cells in renal diseases. J. Cell. Physiol. 213: 495–501, 2007. © 2007 Wiley‐Liss, Inc.
AFAP‐110 is required for actin stress fiber formation and cell adhesion in MDA‐MB‐231 breast cancer cells
Authors: Dorfleutner Andrea , Stehlik Christian , Zhang Jing , Gallick Gary E. , Flynn Daniel C.
Published: May 2007

Regulation of actin organization and dynamics is a highly complex process that involves a number of actin‐binding proteins, including capping, branching, severing, sequestering, and cross‐linking proteins. The actin‐binding and cross‐linking protein AFAP‐110 is expressed in normal myoepithelial cells. Screening of different breast epithelial cell lines revealed high expression levels of AFAP‐110 in the human breast cancer cell lines MDA‐MB‐231 and MDA‐MB‐435. Knockdown of AFAP‐110 expression in MDA‐MB‐231 cells does not result in any changes in cell proliferation but did result in a loss of actin stress fiber cross‐linking and decreased adhesion to fibronectin. An inducible knockdown approach confirms that MDA‐MB‐231 breast cancer cells require AFAP‐110 expression for stress fiber formation and adhesion. Thus, AFAP‐110 may provide cytoskeletal tension through stress fiber formation, which is required for focal adhesion formation. Indeed, we could not detect any focal contacts or focal adhesions in AFAP‐110 knockdown cells after adhesion to fibronectin. Although expression levels of crucial focal adhesion components were not influenced by AFAP‐110 expression levels, treatment of AFAP‐110 knockdown cells with LPA did not result in induction of actin stress fibers and focal adhesions. In summary, AFAP‐110 plays an important role in MDA‐MB‐231 breast cancer cell adhesion possibly by regulating stress filament cross‐linking which would promote focal adhesion formation. J. Cell. Physiol. 213:740–749. © 2007 Wiley‐Liss, Inc.
Suppression by glutamate of proliferative activity through glutathione depletion mediated by the cystine/glutamate antiporter in mesenchymal C3H10T1/2 stem cells
Authors: Iemata Mika , Takarada Takeshi , Hinoi Eiichi , Taniura Hideo , Yoneda Yukio
Published: May 2007

Although previous studies including ours have demonstrated the functional expression of different glutamate (Glu) signaling machineries such as Glu receptors (GluRs) and transporters in osteoblasts and chondrocytes, little attention has been paid to the role of Glu in their ancestral mesenchymal stem cells to date. In the present study, we have evaluated the possible functionality of Glu in cultured mouse mesenchymal stem cell line C3H10T1/2 cells endowed to proliferate for the self‐renewal and to differentiate toward osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits, in addition to particular excitatory amino acid transporter (EAAT) isoforms and ionotropic GluRs, in undifferentiated C3H10T1/2 cells. Glu significantly suppressed the proliferation activity at a concentration over 500 µM without inducing cell death or differentiation, while the suppression occurred in a manner sensitive to the prevention by cystine and reduced glutathione (GSH), but not by EAAT inhibitors. A significant decrease was seen in intracellular GSH levels in C3H10T1/2 cells cultured with Glu, whereas the cellular proliferation activity was drastically decreased by the addition of the GSH depleter cyclohexene‐1‐one and the GSH biosynthesis inhibitor L‐buthionine‐[S,R]‐sulfoximine, respectively. Transient overexpression of both xCT and 4F2hc subunits led to an increased basal proliferative activity in C3H10T1/2 cells. These results suggest that Glu could suppress the cellular proliferation toward self‐renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the cystine/Glu antiporter in C3H10T1/2 cells. J. Cell. Physiol. 213:721–729. © 2007 Wiley‐Liss, Inc.
Resistance to 1,25D‐induced differentiation in human acute myeloid leukemia HL60‐40AF cells is associated with reduced transcriptional activity and nuclear localization of the vitamin D receptor
Authors: Garay Edward , Donnelly Robert , Wang Xuening , Studzinski George P.
Published: May 2007

The anti‐neoplastic effects of 1,25‐dihydroxyvitamin D3 (1,25D) are well documented in numerous tumor cell systems and animal models of cancer. However, despite this pre‐clinical success, the clinical use of 1,25D is currently impeded by the dose‐limiting hypercalcemia, and the risk of development of resistance to 1,25D. In this study, we investigated the mechanism of resistance to 1,25D of HL60‐40AF cells, a model of drug‐resistant acute myeloid leukemia, derived from HL60 cells by cultivation in the presence of 1,25D. The data indicate that transcriptional activity of vitamin D receptor (VDR) in 40AF cells increases only briefly when the cells are treated with 1,25D, despite greater basal cellular levels of VDR protein in the resistant than in the 1,25D‐sensitive cells. Analysis of the 40AF VDR mRNA sequence indicated alterations in the 5′ untranslated region (UTR), but coding domain variations were not observed. When resistance to 1,25D‐induced differentiation of 40AF cells was reversed by a combination of 1,25D with potentiators of differentiation (plant derived antioxidants and a p38MAPK inhibitor), an increase in the level of nuclear VDR, as well as an increase in CYP24 mRNA expression was observed. These data suggest that decreased ability of 1,25D to induce VDR nuclear localization and the consequent VDR target gene transcription may be an important reason for the resistance of 40AF cells to 1,25D. Further, our data show that VDR localization and phosphorylation can be increased by combining 1,25D with potentiators of differentiation. Analysis of the mechanisms that underlie the reduction and potentiation of 1,25D‐mediated changes in VDR activity may lead to the identification of new cellular targets that enhance 1,25D‐induced monocytic differentiation. J. Cell. Physiol. 213:816–825. © 2007 Wiley‐Liss, Inc.
Antioxidants slow photoreceptor cell death in mouse models of retinitis pigmentosa
Authors: Komeima Keiichi , Rogers Brian S. , Campochiaro Peter A.
Published: May 2007

Retinitis pigmentosa (RP) is a heterogeneous group of diseases in which one of a wide variety of mutations selectively causes rod photoreceptor cell death. After rods die, cone photoreceptors gradually die resulting in blindness. Antioxidants reduce cone cell death in rd1/rd1 mice indicating that cones die from oxidative damage in that model of rapidly progressive RP. In this study, we sought to determine if this observation could be generalized to models of other types of RP, rd10/rd10 mice, a model of more slowly progressive recessive RP, and Q344ter mice, a model of rapidly progressive dominant RP. Compared to appropriate vehicle‐treated controls, rd10/rd10 and Q344ter mice treated between P18 and P35 with a mixture of antioxidants previously found to be effective in rd1/rd1 mice showed significantly greater cone survival. Antioxidant‐treated rd10/rd10 mice showed preservation of cone function as shown by a significant increase in photopic ERG b‐wave amplitudes, and surprisingly showed temporary preservation of scotopic a‐wave amplitudes, prolonged rod survival, and slowed depletion of rhodopsin mRNA. These data suggest that oxidative damage contributes to cone cell death regardless of the disease causing mutation that leads to the demise of rods, and that in more slowly progressive rod degenerations, oxidative damage may also contribute to rod cell death. Protection from oxidative damage may be a broadly applicable treatment strategy in RP. J. Cell. Physiol. 213:809–815. © 2007 Wiley‐Liss, Inc.