bluuebunny/crossref_metadata_2025 huggingface dataset:
Determining the degree of cortical bone asymmetry in bilateral, nonpathological, human femur pairs
Authors: Rosenbaum Teri G. , Hamblin Tracy , Bloebaum Roy D.
Published: November 2005

When testing the effects of a femoral component on cortical bone following total hip arthroplasty, the patient's implanted femur is often compared with his/her contralateral nonimplanted femur, with differences attributed to the femoral component. However, if normal anatomical differences exist between bilateral femurs, they need to be quantified in order to validate whether the differences between implanted and nonimplanted bilateral femurs are due to the implant or possibility due to intrinsic differences before implantation. This study quantified the geometric properties of cortical bone shape between seven pairs of bilateral, cadaveric, human femurs. The null hypothesis tested stated that the bilateral femurs would not be significantly different in cortical bone geometry. Digitized images of cortical bone cross‐sections taken at percent biomechanical lengths (levels 1–8) were used to calculate bone geometry measurements. The paired t‐test showed that the only significant difference was in the location of principal axes at the most proximal location, level 1 (p = 0.015). All other measurements and levels were not significant with percent differences less than 6.6%. In conclusion, the data supports attributing cortical bone shape differences between implanted and contralateral nonimplanted femurs in levels 2–8 to the presence of the implant when the significant differences are greater than 6.6%. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Osteoblast response to zirconia‐hybridized pyrophosphate‐stabilized amorphous calcium phosphate
Authors: Whited Bryce M. , Skrtic Drago , Love Brian J. , Goldstein Aaron S.
Published: November 2005

Calcium phosphate bioceramics, such as hydroxyapatite, have long been used as bone substitutes because of their proven biocompatibility and bone binding properties in vivo. Recently, a zirconia‐hybridized pyrophosphate‐stabilized amorphous calcium phosphate (Zr‐ACP) has been synthesized, which is more soluble than hydroxyapatite and allows for controlled release of calcium and phosphate ions. These ions have been postulated to increase osteoblast differentiation and mineralization in vitro. The focus of this work is to elucidate the physicochemical properties of Zr‐ACP and to measure cell response to Zr‐ACP in vitro using a MC3T3‐E1 mouse calvarial‐derived osteoprogenitor cell line. Cells were cultured in osteogenic medium and mineral was added to culture at different stages in cell maturation. Culture in the presence of Zr‐ACP showed significant increases in cell proliferation, alkaline phosphatase activity (ALP), and osteopontin (OPN) synthesis, whereas collagen synthesis was unaffected. In addition, calcium and phosphate ion concentrations and medium pH were found to transiently increase with the addition of Zr‐ACP, and are hypothesized to be responsible for the osteogenic effect of Zr‐ACP. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Dissolution behavior and in vitro evaluation of sputtered hydroxyapatite films subject to a low temperature hydrothermal treatment
Authors: Ozeki K. , Aoki H. , Fukui Y.
Published: November 2005

Hydroxyapatite (HA) was coated onto titanium substrates using radio frequency sputtering. Some of the as‐sputtered films were hydrothermally recrystallized at 110°C. In immersion tests, the as‐sputtered film completely dissolved after 2 days in a culture medium, whereas the thickness of hydrothermally treated films increased with an increase in immersion period, reaching a thickness of 127% after a period of 4 weeks. The proliferation and alkaline phosphatase (ALP) activity of MC3T3‐E1 osteoblast‐like cells on the as‐sputtered and hydrothermally treated films were investigated, and the cell morphology was also observed using scanning electron microscopy. The proliferation of MC3T3‐E1 cells on the as‐sputtered films was suppressed, whereas proliferation on the hydrothermally treated films was comparable to that on control and titanium substrate. The suppression of cell proliferation is associated with an increase in pH of the culture medium caused by dissolution of the as‐sputtered film. After a 96‐h culture time, the ALP activity of the cells on the hydrothermally treated film was higher than that on the control, titanium substrate, and as‐sputtered film samples. From scanning electron microscopic observations, it was found that the MC3T3‐E1 cells on the hydrothermally treated films were elongated and had established more intricate filopodia networks with each other, which were also observed for MC3T3‐E1 cells on the as‐sputtered films after a period of 24 h. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
The effects of short‐term stimulation on fibroblast spreading in an in vitro 3D system
Authors: Puk C. K. , Miller D. J. , Gamradt S. , Wu B. M. , McAllister D. R.
Published: November 2005

Although the short‐term effects of mechanical and biochemical stimulation on cell protein and DNA production have recently begun to be evaluated using 3D models, the effects that such stimulation have on cell morphology and adaptation remains unclear. Using a custom‐built bioreactor, we were able to create a systematic model to examine the short‐term effects of stimulation on cell morphology in a 3D model, specifically by evaluating cell spreading as the short‐term indicator of cell adaptation. Fibroblasts were seeded on a porous poly(L‐lactic acid) scaffold and cultured in a computerized bioreactor for 24 h under various uniaxial strains (0, 0.6, 3, 6%) and frequencies (0.0125, 0.125 Hz). Also, the effects of transforming growth factor (TGF‐β1) (1, 10, 100 ng/mL) were examined on static, nonstimulated cells‐scaffold constructs after 24 h. Fibroblasts that had been subjected to mechanical stretching were found to exhibit significantly more spreading than the static control group. Conversely, TGF‐β1 between 1 and 100 ng/mL did not produce any significant difference in fibroblast spreading from the control groups after 24 h. Collectively, the findings suggest that cell morphology and adaptation may be affected by short‐term mechanical stimulation, as seen by increased cell spreading by the fibroblasts under these experimental conditions. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Nanofiber generation of hydroxyapatite and fluor‐hydroxyapatite bioceramics
Authors: Kim Hae‐Won , Kim Hyoun‐Ee
Published: November 2005

In this study, we produced hydroxyapatite (HA) and fluor‐hydroxyapatite (FHA) bioceramics as a novel geometrical form, the nanoscale fiber, for the biomedical applications. Based on the sol‐gel precursors of the apatites, an electrospinning technique was introduced to generate nanoscale fibers. The diameter of the fibers was exploited in the range of a few micrometers to hundreds of nanometers (1.55 μm–240 nm) by means of adjusting the concentration of the sols. Through the fluoridation of apatite, the solubility of the fiber was tailored and the fluorine ions were well released from the FHA. The HA and FHA nanofibers produced in this study are considered to find potential applications in the biomaterials and tissue engineering fields. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006
A novel processing method for injection‐molded polyether–urethane scaffolds. Part 2: Cellular interactions
Authors: Haugen H. , Aigner J. , Brunner M. , Wintermantel E.
Published: November 2005

A large‐scale scaffold processing method with injection molding has been successfully developed. Water was used as a foaming agent for the new technique. NaCl was used as a porogen to achieve an open‐cell structure. Organic solvents, which are common foaming agents for polyurethane, where not used. Toxic remains in the polymer were therefore prevented. Biocompatibility tested gave a mean optical density of 81% from WST‐1 proliferation assay. In comparison to the previously study processing method, hot pressing (Haugen H, Ried V, Brunner M, Will J, Wintermantel E. J Mater Sci: Mater Med2004;15:343–346), the current scaffolds had an increase of 20% of the mean optical density. Cell seeding showed that human fibroblasts adhered to the surface and proliferated. The spread of the adhered fibroblasts was uniform on the surface. A quantitative MTT analysis proved that there was a significant (p < 0.01) increase in the OD level after 7 and 14 days of incubation. This cell layer thickened with increased incubation time from 7 to 14 days (p < 0.05) and had typical fibroblast morphology. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006
Characterization of human primary osteoblast response on bioactive glass (BaG 13–93)‐ coated poly‐L,DL‐lactide (SR‐PLA70) surface in vitro
Authors: Ruuttila P. , Niiranen H. , Kellomäki M. , Törmälä P. , Konttinen Y. T. , Hukkanen M.
Published: November 2005

Bioabsorbable polylactide‐based polymers are commonly used for bone reconstruction. Although these polymers have proven successful in many applications, they do not have the capacity to induce osteoconduction. Therefore, several strategies have been developed to manufacture osteoconductive polylactide‐based composites. In this study, we have investigated in vitro response of human primary osteoblasts for self‐reinforced poly‐L,DL‐lactide 70/30 (SR‐PLA70) plates coated with spheres of bioactive glass 13–93 (SR‐PLA70 + BaG). Osteoblasts were cultured on SR‐PLA70 and SR‐PLA70 + BaG plates for 2, 7, or 14 days. By day 7, both materials induced a reduction in total cell population. However, by day 14 the proliferative response of osteoblasts on SR‐PLA70 + BaG surface was such that the cell population had regained similar levels as that of day 2 controls. Alkaline phosphatase activity was higher on SR‐PLA70 at day 7 but declined to control levels by day 14. There were no significant time‐dependent variations in alkaline phosphatase activity on SR‐PLA70 + BaG. After in vitro hydrolysis for 7 days, the elemental analysis of SR‐PLA70 + BaG surface showed the presence of mineral precipitates that were confirmed as crystalline hydroxyapatite. This was accompanied by osteoblast spreading, protrusions of microvilli adhered to BaG 19–39 surface, cuboidal phenotype and cell surface associated formation of hydroxyapatite microspheres. In conclusion, the SR‐PLA70 + BaG composite is capable of inducing a proliferative response of human primary osteoblasts, and appears to support the development of mature osteoblast phenotype. Therefore, the SR‐PLA70 + BaG composites appear as promising osteoconductive scaffold candidates for reconstruction and regeneration of bone matrix. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006
PLP1 alternative splicing in differentiating oligodendrocytes: Characterization of an exonic splicing enhancer
Authors: Wang Erming , Huang Zhong , Hobson Grace M. , Dimova Neviana , Sperle Karen , McCullough Andrew , Cambi Franca
Published: November 2005

Proteolipid protein (PLP) and DM20 are generated by alternative splicing of exon 3B of PLP1 transcript in differentiating oligodendrocytes. We investigated the role of exonic splicing enhancers (ESE) in the selection of PLP 5′ donor site, focusing on putative ASF/SF2, and SC35 binding motifs in exon 3B on the basis of mutations that cause disease in humans. Mutations in a putative ASF/SF2 binding motif (nucleotides 406–412) reduced PLP 5′ donor site selection, whereas a mutation in a putative SC35 binding motif (nucleotides 382–389) had no effect. UV crosslinking and immunoprecipitation (IP) assays using an antibody to ASF/SF2 showed that the ASF/SF2 protein specifically binds to the ESE (nucleotides 406–412). The single nucleotide mutations that reduced PLP splice site selection greatly diminished ASF/SF2 protein binding to this motif. We next tested the effect of overexpressed ASF/SF2 on PLP 5′splice selection in differentiating oligodendrocytes. ASF/SF2 positively regulates PLP splice site selection in a concentration‐dependent manner. Disruption of the putative ASF/SF2 binding site in exon 3B reduced the positive effect of ASF/SF2 on PLP splicing. We conclude that an ESE in exon3B regulates PLP 5′ donor site selection and that ASF/SF2 protein participates in the regulation of PLP alternative splicing in oligodendrocytes. J. Cell. Biochem. 97: 999–1016, 2006. © 2005 Wiley‐Liss, Inc.
Curcumin blocks activation of pancreatic stellate cells
Authors: Masamune Atsushi , Suzuki Noriaki , Kikuta Kazuhiro , Satoh Masahiro , Satoh Kennichi , Shimosegawa Tooru
Published: November 2005

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis and inflammation. Inhibition of activation and cell functions of PSCs is a potential target for the treatment of pancreatic fibrosis and inflammation. The polyphenol compound curcumin is the yellow pigment in curry, and has anti‐inflammatory and anti‐fibrotic properties. We here evaluated the effects of curcumin on the activation and cell functions of PSCs. PSCs were isolated from rat pancreas tissue and used in their culture‐activated, myofibroblast‐like phenotype unless otherwise stated. The effects of curcumin on proliferation, α‐smooth muscle actin gene expression, monocyte chemoattractant protein (MCP)‐1 production, and collagen expression were examined. The effect of curcumin on the activation of freshly isolated cells in culture was also assessed. Curcumin inhibited platelet‐derived growth factor (PDGF)‐induced proliferation, α‐smooth muscle actin gene expression, interleukin‐1β‐ and tumor necrosis factor (TNF)‐α‐induced MCP‐1 production, type I collagen production, and expression of type I and type III collagen genes. Curcumin inhibited PDGF‐BB‐induced cyclin D1 expression and activation of extracellular signal‐regulated kinase (ERK). Curcumin inhibited interleukin‐1β‐ and TNF‐α‐induced activation of activator protein‐1 (AP‐1) and mitogen‐activated protein (MAP) kinases (ERK, c‐Jun N‐terminal kinase (JNK), and p38 MAP kinase), but not of nuclear factor‐κB (NF‐κB). In addition, curcumin inhibited transformation of freshly isolated cells to myofibroblast‐like phenotype. In conclusion, curcumin inhibited key cell functions and activation of PSCs. J. Cell. Biochem. 97: 1080–1093, 2006. © 2005 Wiley‐Liss, Inc.
Binding and functional characteristics of two E‐box motifs within the S100A6 (calcyclin) gene promoter
Authors: Leśniak Wiesława , Kuźnicki Jacek
Published: November 2005

S100A6 (calcyclin) is a small calcium‐binding protein of the S100 family often associated with cancer and metastasis. We have previously shown that the E‐box sequence at position −283/−278 of the S100A6 gene promoter interacts with USF transcription factor and contributes to promoter transcriptional activity. We now present evidence that a second E‐box motif at position −593/−588 of the promoter also binds USF and that the USF1/USF2 heterodimer is the prevailing dimeric form of the transcription factor bound. Using the chromatin immunoprecipitation assay (ChIP), we show that USF is bound in vivo to the E‐box regulatory element(s). Depletion of the endogenous USF pool by means of a decoy oligonucleotide evokes a severe inhibition of S100A6 gene promoter activity. Furthermore, we show that S100A6 gene promoter activity can be stimulated by palmitate and that mutation of the −283/−278 E‐box sequence completely blocks this stimulation. J. Cell. Biochem. 97: 1017–1024, 2006. © 2005 Wiley‐Liss, Inc.
Effect of chlorpromazine on bone sialoprotein (BSP) gene transcription
Authors: Nakajima Yu , Kato Naoko , Nakayama Youhei , Kim Dong‐Soon , Takai Hideki , Arai Masato , Saito Ryoichiro , Samoto Hiroshi , Shimizu Emi , Ogata Yorimasa
Published: November 2005

Bone sialoprotein (BSP), an early marker of osteoblast differentiation. Whereas physical forces may play an important role in the regulation of bone cell function, little is known about how cells are able to sense mechanical loads. Chlorpromazine, a tranquilizing agent for treatments of psychiatric disorders, mimics hypotonic stress and causes membrane deformation. Application of 10 µg/ml of chlorpromazine suppressed BSP mRNA levels after 12 and 24 h in osteoblast‐like ROS17/2.8 cells and rat stromal bone marrow cells (SBMC‐D8). Chlorpromazine (10 µg/ml) decreased luciferase activity of the construct (pLUC3; −116 to +60 of the rat BSP gene promoter) after 12 h, the effect was inhibited by the tyrosine kinase inhibitor herbimycin A (HA) and MAP kinase kinase inhibitor U0126. Introduction of 2‐bp mutation in the pLUC3 construct showed that the chlorpromazine effects were mediated by cAMP response element (CRE) and FGF2 response element (FRE). In gel shift assays, using radiolabeled double‐stranded CRE and FRE oligonucleotides, which revealed decreased binding of nuclear proteins from chlorpromazine‐stimulated cells. These studies, therefore, show that chlorpromazine suppresses BSP gene transcription through tyrosine and MAP kinases‐dependent pathways and that the chlorpromazine effects are mediated by CRE and FRE elements in the proximal promoter of the BSP gene. J. Cell. Biochem. 97: 1198–1206, 2006. © 2005 Wiley‐Liss, Inc.
Microarray and phosphokinase screenings leading to studies on ERK and JNK regulation of connective tissue growth factor expression by angiotensin II 1a and bradykinin B2 receptors in Rat1 fibroblasts
Authors: Liu B. , Yu J. , Taylor L. , Zhou X. , Polgar P.
Published: November 2005

Rat1 fibroblasts stably transfected with the rat angiotensin II (AngII) AT1a and bradykinin (BK) B2 receptor cDNAs gained the ability to bind Ang II and BK. Wild‐type Rat1 cells bound neither ligand. Exposure to either effector led to characteristic Gαi and Gαq signal cascades, the release of arachidonic acid (ARA), and the intracellular accumulation of inositol phosphates (IP). Microarray analyses in response to BK or AngII showed that both receptors markedly induce the CCN family genes, CTGF (CCN2) and Cyr61 (CCN1), as well as the vasculature‐related genes, Cnn1 and Egr1. Real time PCR confirmed the increased expression of connective tissue growth factor (CTGF) mRNA. Combined sequence‐based analysis of gene promoter regions with statistical prevalence analyses identified CREB, SRF, and ATF‐1, downstream targets of ERK, and JNK, as prominent products of genes that are regulated by ligand binding to the BK or AngII receptors. The binding of AngII or BK markedly stimulated the phosphorylation and thus the activation of ERK2, JNK, and p38MAPK. A BKB2R and an AT1aR chimera which displayed only negligible G‐protein‐related signaling were constructed. Both mutant receptors continued to activate these kinases and stimulate CTGF expression. Inhibitors of ERK1/2 and JNK but not p38MAPK inhibited the BK‐ and AngII‐stimulated expression of CTGF in cells expressing either the WT or mutant receptors, illustrating that ERK and JNK participate in the control of CTGF expression in a manner that appears to be independent of G‐protein. Conversely, addition of BK or AngII to the cell line expressing WT AT1aR and BKB2R downregulated the expression of collagen α1(I) (COL1A1) mRNA. However, these effectors did not have this effect in cells expressing the mutant receptors. Thus, a robust G‐protein related response is necessary for BK or AngII to affect COL1A1 expression. J. Cell. Biochem. 97: 1104–1120, 2006. © 2005 Wiley‐Liss, Inc.
Heparanase mechanisms of melanoma metastasis to the brain: Development and use of a brain slice model
Authors: Murry Brian P. , Blust Bryan E. , Singh Amandip , Foster Timothy P. , Marchetti Dario
Published: November 2005

Heparanase (HPSE‐1) is an endo‐β‐D‐glucuronidase that cleaves heparan sulfate (HS) chains of proteoglycans (HSPG), and its expression has been associated with increased cell growth, invasion, and angiogenesis of tumors as well as with embryogenesis and tissue development. Since metastatic cancer cells express HPSE‐1, we have developed an orthotopic brain slice model to study HPSE‐1 involvement in brain‐metastatic melanoma. This model allows for the characterization of tumor cell invasion at both quantitative and qualitative levels. Brain‐metastatic melanoma cells (B16B15b) showed augmenting levels of HPSE‐1 protein expression in a time‐dependent manner. Secondly, B16B15b cells pre‐treated with HPSE‐1 showed a significant increase in the number of cells that invaded into the brain tissue. Finally, HPSE‐1 exposure‐augmented invasion depth in brain sections by brain‐metastatic melanoma cells. We concluded that applying this brain slice model can be beneficial to investigate HPSE‐1‐ related in vivo modalities in brain‐metastatic melanoma and brain invasion in general. These results also further emphasize the potential relevance of using this model to design therapies for controlling this type of cancer by blocking HPSE‐1 functionality. © 2005 Wiley‐Liss, Inc.
Computation of vertical excitation energies of retinal and analogs: Scope and limitations
Authors: López Carlos Silva , Faza Olalla Nieto , Estévez Susana López , de Lera Angel R.
Published: November 2005

A comprehensive survey of computational methods: semiempirical (ZINDO/S), Time‐Dependent Hartree–Fock (TD‐HF), Configuration Interaction Singles (CIS), and several approximate functionals within the Time‐Dependent Density Functional Theory (TD‐DFT) has been carried out for the description of vertical excitation energies and oscillator strengths of retinal and related polyenals. ZINDO and TD‐DFT computations showed the best agreement with the experimental data. In particular, hybrid functionals including approximately 25% of exact exchange (B3LYP, B3P86, and PBE0) were found to perform best with these highly conjugated polyenes. A systematic average error of 0.18–0.22 eV has been found after a simple one‐parameter correction. Thus, 0.18 eV might be considered the upper limit of accuracy for current one‐determinant methods in the computation of vertical excitation energies. The consideration of adiabatic excitations, conformational sampling, solvation, and nondynamic correlation should describe this processes more accurately, but this leads to highly demanding methods beyond feasibility for these large polyenes. The trends observed, particularly the good performance of the ZINDO/S method, should pave the way for the prediction of excited states properties in natural and artificial photoreceptor proteins, thus advancing towards the description of their light‐transducing biological role in Nature. © 2005 Wiley Periodicals, Inc. J Comput Chem 27: 116–123, 2006
Optimal holey packings OHP4(2, 4, n ,3)'s
Authors: Wang Jianmin , Yan J.
Published: November 2005

An optimal holey packing OHPd(2, k, n, g) is equivalent to a maximal (g + 1)‐ary (n, k, d) constant weight code. In this paper, we provide some recursive constructions for OHPd(2, k, n, g)'s and use them to investigate the existence of an OHP4(2, 4, n, 3) for n ≡ 2, 3 (mod 4). Combining this with Wu's result (18), we prove that the necessary condition for the existence of an OHP4(2, 4, n, 3), namely, n ≥ 5 is also sufficient, except for n ∈ {6, 7} and except possibly for n = 26. © 2005 Wiley Periodicals, Inc. J Combin Designs 14: 111–123, 2006
Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells
Authors: Lele Tanmay P. , Pendse Jay , Kumar Sanjay , Salanga Matthew , Karavitis John , Ingber Donald E.
Published: November 2005

The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant kOFF of individual GFP‐labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques—chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices—increases the kOFF of the focal adhesion protein zyxin. In contrast, the kOFF of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force‐dependent increases in zyxin's kOFF that occur over seconds are sufficient to quantitatively predict large‐scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force‐dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells. J. Cell. Physiol. 207: 187–194, 2006. © 2005 Wiley‐Liss, Inc.
Slow virus disease: Deciphering conflicting data on the transmissible spongiform encephalopathies (TSE) also called prion diseases
Authors: Bastian Frank O. , Fermin Cesar D.
Published: November 2005

The transmissible spongiform encephalopathies (TSE) that manifest as Creutzfeldt‐Jakob disease in humans, as scrapie in sheep and goats, mad cow disease in cattle, or chronic wasting disease in cervids (deer) represent a serious human health crisis and a significant economical problem. Despite much research, the nature of the elusive pathogen directly involved with TSE is currently unresolved. This article reviews current pathogen‐cell plasma membrane properties, showing that the primary biochemical marker of the prion disease is used as a receptor by the intracellular bacterium Brucella abortus. Such observation makes plausible the role for the prion in the pathogenesis of TSE, and supports the concept that Spiroplasma, a wall‐less bacterium, may be a transmissible agent of TSE. Over the past three decades, we have published convincing evidence that Spiroplasma infection is associated with TSE. The bacterial‐prion‐receptor concept by other laboratories support a model for TSE wherein a Spiroplasma bacterium can bind to prion receptors (alone or with anchors) on the cell surface lipid raft, allowing entry of the microbe into the cell to initiate infection. The relevance of this new concept is that it offers a new window for future research involving a bacterium in the pathogenesis of TSE. Data from the bacterial‐prion‐receptor model will aid in the development diagnostic tests and/or treatment protocols for TSE. Microsc. Res. Tech. 68:239–246, 2005. © 2005 Wiley‐Liss, Inc.
Coexpression of major histocompatibility complex class I and LMP1 in Epstein‐Barr virus‐positive Hodgkin's Lymphoma in a pediatric age group
Authors: Aybar Argelia , Krause John R.
Published: November 2005

The Epstein‐Barr virus (EBV)‐encoded latent membrane proteins (LMP1 and LMP2) are consistently expressed by malignant Hodgkin Reed‐Sternberg (HRS) cells of EBV‐associated Hodgkin's disease (HD). The EBV LMP1 has been implicated in tumorogenesis. Cytotoxic T lymphocyte (CTL) responses to both of these proteins have been shown in the blood of EBV‐seropositive individuals, and in HD, the apparent failure of the CTL response to eliminate HRS cells expressing LMP1 in vivo has given rise to the suggestion that HD may be characterized by the presence of defects in antigen CTL‐presentation or in CTL dysfunction. This study examined 28 cases seen at the Regional Children's Hospital Arturo Grullón in Santiago, the Dominican Republic, by immunohistochemistry. High level of expression of major histocompatibility complex (MHC) class I molecules in HRS cells of EBV‐associated HD was recorded. These results suggest that deregulation of MHC class I molecules does not account for the apparent ability of EBV to infect HRS cells and to evade CTL protection. Therefore, the result from our work and similar studies will help to determine whether, or which, immunotherapy should be used in positive and negative cases of HD related to EBV. Microsc. Res. Tech. 68:247–249, 2005. © 2005 Wiley‐Liss, Inc.
Of mice, cats, and men: Is human breast cancer a Zoonosis?
Authors: Szabo Sara , Haislip Allyson M. , Garry Robert F.
Published: November 2005

Mouse mammary tumor virus (MMTV), a member of the betaretroviridae, is the most common cause of breast cancer (BC) in mice. MMTV is transmitted in mice both in the germline as endogenous proviruses and exogenously as infectious virions. Here, we review a variety of evidence accumulated for six decades that has suggested that a human homologue of MMTV may exist. The findings include recent studies from several independent laboratories that have detected sequences very closely related to MMTV in DNA isolated from human BC tumors. Other laboratories, however, have failed to detect the MMTV‐related sequences in human DNA samples, and conclusive evidence for a human mammary tumor virus has been elusive. We also reviewed additional studies, suggesting that betaretroviruses are present in a much wider range of species than previously known, including rodents, felines, and primates. The observation that a subset of cats may be infected with a close homologue of MMTV may be of epidemiological significance for human BC. Cats may become infected by MMTV from mice, and in turn may transmit the virus to humans, possibly after selection for variants with an expanded host range. Microsc. Res. Tech. 68:197–208, 2005. © 2005 Wiley‐Liss, Inc.
Human, rhesus macaque, and feline sequences highly similar to mouse mammary tumor virus sequences
Authors: Szabo Sara , Haislip Allyson M. , Traina‐Dorge Vicki , Costin Joshua M. , Crawford Byron E. , Wilson Russell B. , Garry Robert F.
Published: November 2005

Sequences highly similar (>95%) to the mouse mammary tumor virus (MMTV) env gene have been amplified from human DNA samples, including DNA samples from patients with breast cancer (BC) and persons who did not have BC. The sequences from human DNA were distinct from the MMTV sequences used as controls in these PCR reactions, indicating that these results are not simply due to contamination. In addition to both, mouse and human‐related sequences were also amplified from some monkey and cat genomic DNA samples. These products were shown to be distinct from, but highly related to, the MMTV env gene, whereas, testing of other sources (lambda phage, snake, cockroach, sea urchin, chicken, or dog) demonstrated no specific amplification. A sequence ˜90% similar to the MMTV group antigen gene (gag) was amplified from cat DNA. These results indicate that DNA from vertebrate species other than rodents, including some but not all humans, monkeys, and cats, can contain sequences closely related to MMTV. Microsc. Res. Tech. 68:209–221, 2005. © 2005 Wiley‐Liss, Inc.
Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk‐1) following lentiviral‐mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model
Authors: Kyriakou Chara A. , Yong Kwee L. , Benjamin Reuben , Pizzey Arnold , Dogan Ahmet , Singh Nalini , Davidoff Andrew M. , Nathwani Amit C.
Published: November 2005

Background

Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma.

Methods

HIV‐based lentiviral vectors were pseudotyped with four different envelope proteins; amphotropic murine leukaemia virus (ampho), murine leukaemia virus (10A1), feline endogenous virus (RD114), and the vesicular stomatitis virus glycoprotein (VSVG). These pseudotypes were examined for transduction efficiency in human bone marrow derived MSC. The effect of lentiviral expression of truncated soluble vascular endothelial growth factor decoy receptor (tsFlk‐1) in MSC on growth of Raji cells was determined, both in vitro and in vivo.

Results

All lentiviral vectors produced significant levels of transduction at an multiplicity of infection (MOI) of 1, those bearing the RD114 envelope glycoprotein consistently produced higher transduction levels (mean 70 ± 6%) compared with the other pseudotyped lentiviral vectors, although there was significant inter‐donor variation. Stable transgene expression was achieved after multiple rounds of transduction with VSVG‐pseudotyped particles, without alteration in the differentiative capacity of transduced cells. Co‐injection of MSC stably expressing tsFlk‐1 with Raji Burkitt's lymphoma cells significantly impaired subcutaneous tumour growth in immunodeficient mice when compared to controls where either unmanipulated MSC or GFP‐expressing MSC were used.

Conclusions

Human MSC are easily transduced by pseudotyped lentiviral particles but there is inter‐donor variation in transduction efficiency. Gene‐modified MSC expressing a gene of therapeutic potential can moderate growth of haematological malignancies. Copyright © 2005 John Wiley & Sons, Ltd.
Polybrene improves transfection efficacy of recombinant replication‐deficient adenovirus in cutaneous cells and burned skin
Authors: Jacobsen F. , Hirsch T. , Mittler D. , Schulte M. , Lehnhardt M. , Druecke D. , Homann H. H. , Steinau H. U. , Steinstraesser L.
Published: November 2005

Background

The hostile environment found in acute and chronic wounds decreases the physiological half‐life of purified synthetic or recombinant peptides dramatically. Gene therapy, on the other hand, may be a viable option since it relies on the cellular machinery of the host to locally manufacture the proteins of interest. The aim of this study was to evaluate and optimize the local administration of transient cutaneous adenoviral gene delivery in wounds.

Methods

Primary human keratinocytes (HKC) and HaCaT cells were transfected with replication‐deficient adenovirus (Ad5) containing the reporter gene for β‐galactosidase (LacZ). The vector was used alone or precoated with either (1) Lipofectamine™ 2000, (2) FuGENE™ 6, or (3) Polybrene. For in vivo testing a rat burn model was used. Animals were randomized into three groups: (1) Ad5‐LacZ alone; (2) Ad5‐LacZ precoated with Polybrene, or (3) carrier control (phosphate‐buffered saline (PBS)). Samples were harvested from burned and unburned tissue sections after either 48 h or 7 days. Transgene expression was quantified by bioluminometric assay and localized using immunohistochemistry. A BrdU assay was performed to determine the influence of the used transfection reagents on cell proliferation.

Results

Transfection efficacy was significantly improved in vitro (p < 0.001) as well as in partial thickness burned (p = 0.015) and unburned skin (p > 0.001) after precoating Ad5 with Polybrene compared to Ad5 alone. Transgene expression was 10‐fold higher in burned skin (9305 pg/mg protein) compared to unburned skin (859 pg/mg protein).

Conclusions

It is feasible to improve transfection efficacy in vitro and in vivo by precoating the adenovirus with Polybrene. Copyright © 2005 John Wiley & Sons, Ltd.
Polyethylenimine‐based antisense oligodeoxynucleotides of IL‐4 suppress the production of IL‐4 in a murine model of airway inflammation
Authors: Seong Ji‐Hye , Lee Kyung‐Mi , Kim Sung Tae , Jin Su‐Eon , Kim Chong‐Kook
Published: November 2005

Background

Interleukin‐4 (IL‐4) plays a crucial role as an inflammatory mediator in allergic asthma via inducing Th2 inflammation and IgE synthesis. To develop an effective therapeutic agent which specifically inhibits production of IL‐4, antisense oligodeoxynucleotides (AS‐ODNs) against murine IL‐4 mRNA were generated and complexed with polyethylenimine (PEI) to improve intracellular delivery.

Methods

AS‐ODNs were generated against the translation initiation region of murine IL‐4 mRNA, and complexed with linear PEI. In vitro efficacy of AS‐ODNs/PEI complexes was tested by measuring IL‐4 production in the D10.G4.1 cell line, and cytotoxicity was tested by XTT assay. Physicochemical properties of polyplexes were examined using atomic force microscopy (AFM) and DNase I protection assay. In vivo effects of IL‐4 AS‐ODNs/PEI complexes were tested in a murine model of airway inflammation. IL‐4 concentrations in the bronchoalveolar lavage (BAL) fluid and circulating IgE levels were measured by ELISA, and histological analysis of lung tissues was performed.

Results

IL‐4 AS‐ODNs/PEI complexes were spheres with an average diameter of 98 nm and resistant to DNase I‐mediated degradation. IL‐4 AS‐ODNs/PEI complexes showed up to 35% inhibition of IL‐4 production in D10.G4.1 cells without causing any toxicity, while naked ODNs gave less than 1% reduction. Furthermore, IL‐4 AS‐ODNs/PEI complexes were effective in suppressing secretion of IL‐4 (up to 30% reduction) in the BAL fluid in an ovalbumin‐sensitized murine model of airway inflammation. Circulating IgE levels were decreased, and airway inflammation was alleviated by treatment with IL‐4 AS‐ODNs polyplexes.

Conclusions

These data demonstrate that complexation of IL‐4 AS‐ODNs with PEI provides a potential therapeutic tool in controlling inflammation associated with allergic asthma, and further presents an opportunity to the development of clinical therapy based on combination of multiple AS‐ODNs of cytokines and/or signaling effectors involved in Th2 inflammation and eosinophilia. Copyright © 2005 John Wiley & Sons, Ltd.
Stimulatory effect of polyethylene glycol (PEG) on gene expression in mouse liver following hydrodynamics‐based transfection
Authors: Ishida Tatsuhiro , Li Wenhao , Liu Zhihui , Kiwada Hiroshi
Published: November 2005

Background

Rapid intravenous injection of a large volume of plasmid DNA (pDNA), i.e. a transfection procedure based on hydrodynamics, is known to be an efficient and liver‐specific method ofin vivogene delivery. However, the gene expression is transient.

Methods

We investigated the effect of addition of polyethylene glycol (PEG) to a solution of naked pDNA (luciferase) on the expression of the gene in mouse liver following transfection by the hydrodynamics‐based technique. In addition, the mechanism leading to the enhancement of the gene expression was studied.

Results

The addition of 1% (w/v) PEG2000 to the pDNA solution enhanced the resulting gene expression in the liver. Increasing the PEG2000 concentration to more than 1 and up to 10% (w/v) rather diminished the gene expression level. By contrast, increasing the molecular weight of PEG to over 2000 up to 10 000 did not affect the level of gene expression. Histopathological and serum‐chemistry examinations indicated that hydrostatic or osmotic pressure increased tissue and hepatocellular damage in a PEG‐concentration‐dependent manner, and resulted in a decrease in gene expression. Quantitative evaluation showed that the enhanced gene expression resulted from stabilization of the pDNA introduced into the hepatocytes and an enhancement of the transport ofintactpDNA to the nucleus.

Conclusions

For most gene therapy applications and gene function studies, sustained expression of the introduced gene(s) is necessary. This simple method to achieve enhanced gene expression in liver may have a great potential for a wide variety of laboratory studies in molecular and cellular biology as well as possibly for future clinical applications in humans. Copyright © 2005 John Wiley & Sons, Ltd.
Development of a gas chromatographic/mass spectrometric method to quantify R(–)‐apomorphine, R(–)‐apocodeine and R(–)‐norapomorphine in human plasma and urine
Authors: Libert Frédéric , Coudoré François , Richard Damien , Durif Franck , Eschalier Alain
Published: November 2005

A method was developed and validated for the analysis of R(−)‐apomorphine, (R−)‐apocodeine and R(−)‐norapomorphine in human plasma and urine with N‐propylnorapomorphine as internal standard using gas chromatography/mass spectrometry (GC/MS) and single‐ion monitoring after a single liquid–liquid extraction and silylation of compounds. The quantification limits were 1 ng/ml for apomorphine and apocodeine and 25 ng/ml for norapomorphine. Calibration curves were linear, within the range 1–100 ng/ml. Variation in intraday and interday precision was below 10%. This method was applied to study apomorphine bioavailability in nine patients with Parkinson's disease before and after coadministration of a catechol‐O‐methyl transferase inhibitor. Copyright © 2005 John Wiley & Sons, Ltd.
Isotope dilution SPME GC/MS for the determination of methylmercury in tuna fish samples
Authors: Centineo Giuseppe , González Elisa Blanco , García Alonso J. Ignacio , Sanz‐Medel Alfredo
Published: November 2005

The development of a rapid, precise and accurate analytical method for the determination of methylmercury in tuna fish samples is described. The method is based on the use of isotope dilution GC/MS with electron impact ionization, a widespread technique in routine testing laboratories. A certified spike containing 202Hg‐enriched methylmercury was used for the isotope dilution of the samples. After extraction of the methylmercury from the sample, methylmercury was propylated using sodium tetrapropyl borate in SPME vials and the analytes were sampled from the headspace for 15 min. For isotope measurements, the molecular ion (MePrHg+) was used in the SIM mode. Five molecular ions were monitored, corresponding to the 198Hg, 199Hg, 200Hg, 201Hg and 202Hg isotopes. The detection at masses corresponding to 198Hg was used to correct for m + 1 contributions of 13C from the organic groups attached to the mercury atom on the 199Hg, 200Hg, 201Hg and 202Hg masses with simple mathematical equations, and the concentration of methylmercury was calculated on the basis of the corrected 200Hg/202Hg isotope ratio. The 202Hg‐enriched methylmercury spike was applied, with satisfactory results, to the determination of methylmercury in the certified reference material BCR 464. The method was successfully applied to the determination of methylmercury in tuna fish samples, and the obtained results were included in the CCQM‐P39 interlaboratory exercise, organized by the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) with excellent agreement between our results and the average obtained by the other participants. Copyright © 2005 John Wiley & Sons, Ltd.
Cultured astrocytes from heme oxygenase‐1 knockout mice are more vulnerable to heme‐mediated oxidative injury
Authors: Chen‐Roetling Jing , Benvenisti‐Zarom Luna , Regan Raymond F.
Published: November 2005

Hemin, the oxidized form of heme, is released from hemoglobin after CNS hemorrhage and may contribute to injury to surrounding tissue. The heme oxygenase (HO) enzymes catalyze the breakdown of hemin to biliverdin, carbon monoxide, and ferric iron. Although HO‐2, the isoform expressed predominantly in neurons, accelerates heme‐mediated neuronal injury, inhibitor studies suggest that HO‐1 induction has a protective effect on astrocytes. In the present study, we directly compared the vulnerability of cultured HO‐1 knockout and wild‐type astrocytes to hemin. Consistent with prior observations, exposure of wild‐type cultures to hemin for 24 hr resulted in protein carbonylation and concentration‐dependent cell death between 10 and 60 μM, as determined by MTT and lactate dehydrogenase release assays. In cultures prepared from mice lacking the HO‐1 gene, oxidative cell injury was approximately doubled. Both protein oxidation and cell death in HO‐1 knockout astrocytes were significantly reduced by pretreating cultures with an adenovirus encoding the HO‐1 gene prior to hemin exposure. HO‐2 expression was observed in both knockout and wild‐type cultures and was not altered by HO‐1 gene deletion. Cell hemin accumulation after 20 hr hemin exposure was 4.7‐fold higher in knockout cells. These results support the hypothesis that HO‐1 protects astrocytes from heme‐mediated oxidative injury. Selectively increasing its expression in astrocytes may be beneficial after hemorrhagic CNS injuries. © 2005 Wiley‐Liss, Inc.
Blockade of nicotinic acetylcholine receptors suppresses hippocampal long‐term potentiation in wild‐type but not ApoE4 targeted replacement mice
Authors: Yun Sung Hwan , Park Kyung A. , Sullivan Patrick , Pasternak Joseph F. , LaDu Mary Jo , Trommer Barbara L.
Published: November 2005

Both impaired nicotinic neurotransmission and the inheritance of apoE4 are associated with increased risk for Alzheimer disease (AD) as well as other deficiencies in memory‐related behavior. Long‐term potentiation (LTP), a cellular model of memory, is known to be altered by nicotinic agents. Recent studies also support an emergent role for apoE in LTP. We compared the effects of mecamylamine, a nonspecific antagonist of nicotinic acetylcholine receptors (nAChRs), on basal synaptic transmission and LTP in hippocampal slices from wild‐type (wt) mice and targeted replacement mice expressing human apoE4 (apoE4‐TR). Field excitatory postsynaptic potentials (EPSPs) were recorded in the dentate gyrus (DG) in response to medial perforant path activation, and theta burst stimulation was used to induce LTP. Bath application of mecamylamine (3 μM) did not alter input–output relationships or paired pulse depression in either mouse strain. Under control conditions, apoE4‐TR mice showed significantly less LTP than wt mice (17.5% ± 3.2%, n = 9, vs. 30.1% ± 3.9%, n = 11, P < 0.02). Mecamylamine reduced LTP in wt mice to a level that was similar to control levels for apoE4‐TR mice (15.7% ± 3.4%, n = 9), whereas apoE4‐TR showed no further reduction of LTP (16.6% ± 3.7%, n = 8) by mecamylamine. Thus mice expressing human apoE4 differ from wt mice both in their capacity for LTP and in the effect on LTP of nicotinic cholinergic blockade. It is possible that nicotinic neurotransmission is already compromised in apoE4‐TR mice and, hence, that interference with the integrity of this cholinergic system represents a mechanism by which inheritance of the apoE4 allele contributes to cognitive risk. © 2005 Wiley‐Liss, Inc.
Activation and circuitry of uterine‐cervix‐related neurons in the lumbosacral dorsal root ganglia and spinal cord at parturition
Authors: Puder B.A. , Papka R.E.
Published: November 2005

Stimulation of the uterine cervix at parturition activates neural circuits involving primary sensory nerves and supraspinally projecting neurons of the lumbosacral spinal cord, resulting in output of hypothalamic neurohormones. Dorsal root ganglia (DRG) and spinal neurons of these circuits are not well‐characterized. The objectives of this study were to detail the activation of DRG and spinal neurons of the L6/S1 levels that are stimulated at late pregnancy, verify hypothalamic projections of activated spinal neurons, and determine whether activated neurons express estrogen receptor‐α (ERα). Expression of phosphorylated cyclic‐AMP response element‐binding protein (PCREB) and Fos immunohistochemistry were used to “mark” activated DRG and spinal neurons, respectively. Retrograde tracing identified uterine‐cervix‐related and spinohypothalamic neurons. Baseline PCREB expression in the DRG increased during pregnancy and peaked during the last trimester. Some PCREB‐expressing neurons contained retrograde tracer identifying them as cervix‐related neurons. Fos‐expressing neurons were few in spinal cords of nonpregnant and day 22 pregnant rats but were numerous in parturient animals. Some Fos‐expressing neurons located in the dorsal half of the spinal cord contained retrograde tracer identifying them as spinohypothalamic neurons. Some DRG neurons expressing PCREB also expressed ERα, and some spinal neurons activated at parturition projected axons to the hypothalamus and expressed ERα. These results indicate that DRG and spinal cord neurons are activated at parturition; that those in the spinal cord are present in areas involved in autonomic and sensory processing; that some spinal neurons project axons to the hypothalamus, ostensibly part of a neuroendocrine reflex; and that sensory and spinal neurons can respond to estrogens. Moreover, some activated sensory neurons may be involved in the animal's perception of labor pain. © 2005 Wiley‐Liss, Inc.