bluuebunny/crossref_metadata_2025 huggingface dataset:
Aging‐related alterations in the distribution of Ca2+‐dependent PKC isoforms in rabbit hippocampus
Authors: Van der Zee E.A. , Palm I.F. , O'Connor M. , Maizels E.T. , Hunzicker‐Dunn M. , Disterhoft J.F.
Published: March 2004

The immunocytochemical and subcellular localization of the Ca2+‐dependent protein kinase C (cPKC) isoforms (PKCα, β1, β2, and γ) was examined in rabbit hippocampus of young (3 months of age; n = 11) and aging (36 months of age; n = 14) subjects. Detailed immunocytochemical analyses revealed a significant increase in PKCβ1, β2, and γ immunoreactivity in principal cell bodies and associated dendrites, and interneurons of the hilar region in the aging rabbits. The number of PKCα‐ and γ‐positive interneurons in the aging stratum oriens declined significantly. PKCα was least affected in principal cells, showing an increase in immunostaining in granule cells only. Weakly PKC‐positive principal cells intermingled between densely stained ones were seen in parts of the hippocampus in most of the aging rabbits, showing that the degree of aging‐related alterations in PKC‐immunoreactivity varies between neurons. Changes in PKC expression in the molecular and subgranular layer of the aging dentate gyrus suggested a reorganization of PKC‐positive afferents to this region. Western blot analysis revealed a significant loss of PKC in the pellet fraction for all isoforms, and a tendency for increased levels of cytosolic PKC. However, no significant changes were found in total PKC content for any PKC isoform. A concurrent dramatic loss of the PKC anchoring protein receptor for activated C kinase (RACK1) in the pellet fraction was shown by Western blotting. These findings suggest that the loss of RACK1 contributes to the dysregulation of the PKC system in the aging rabbit hippocampus. The enhanced PKC‐immunoreactivity might relate to reduced protein‐protein interactions of PKC with the anchoring protein RACK1 leading to increased access of the antibodies to the antigenic site. In conclusion, the results suggest that memory deficits in aging rabbits are (in part) caused by dysregulation of subcellular PKC localization in hippocampal neurons. © 2004 Wiley‐Liss, Inc.
Drug induced encephalopathy in six epileptic patients: topiramate? valproate? or both?
Authors: Latour Patrick , Biraben Arnaud , Polard Elisabeth , Bentué‐Ferrer Danièle , Beauplet Anne , Tribut Olivier , Allain Hervé
Published: March 2004

Six severe epileptic patients developed stuporous encephalopathy with marked cognitive impairment when topiramate (TPM) and sodium valproate (VPA) were coprescribed for five patients, and when monotherapy with TPM was introduced for one patient. In four patients, ammonaemia increased and then returned to normal after TPM or VPA withdrawal. This severe potential side effect must be recognized. Moreover two distinct mechanisms might explain this toxicity: (1) a pharmacokinetic interaction between VPA and TPM, leading to hyperammonaemia, (2) a pharmacodynamic mechanism due to a direct toxicity of TPM in at‐risk epileptic patients. Copyright © 2004 John Wiley & Sons, Ltd.
Combination of mood stabilizers with quetiapine for treatment of acute bipolar disorder: an open label study
Authors: Bahk Won‐Myong , Yoon Bo‐Hyun , Lee Kyoung‐Uk , Chae Jeong‐Ho
Published: March 2004

Objectives

The atypical antipsychotics are being increasingly used to control acute manic episodes, and data are emerging to support their mood‐stabilizing and antidepressant properties. This study investigated the short‐term efficacy of quetiapine as an add‐on therapy in the treatment of acute mania.

Method

This study was a 4‐week, open‐label, add‐on, prospective investigation using quetiapine in addition to mood stabilizers. Data on 18 patients fulfilling DSM‐IV diagnostic criteria for bipolar I disorder were analysed. The Young mania rating scale (YMRS), the Hamilton scale for depression (HDRS), the brief psychiatric rating scale (BPRS) and extrapyramidal symptom rating scale (ESRS) were applied at baseline and at weeks 1, 2 and 4. The clinical global impression scale (CGI) was evaluated at baseline and week 4.

Results

The addition of quetiapine produced a statistically significant improvement on the YMRS, HDRS, BPRS and CGI score at week 4 from baseline (p<0.005). Quetiapine was well tolerated, with no subjects discontinuing because of side effects.

Conclusions

The combination of quetiapine was associated with a substantial symptomatic improvement in patients with acute mania. Randomized placebo‐controlled prospective studies are needed. Copyright © 2004 John Wiley & Sons, Ltd.
Ziprasidone‐related neuroleptic malignant syndrome in a patient with Parkinson's disease: a diagnostic challenge
Authors: Gray Nicola S.
Published: March 2004

A case of neuroleptic malignant syndrome (NMS) in a patient with Parkinson's disease (PD) is presented. The syndrome was precipitated by the atypical antipsychotic, ziprasidone. The challenge of recognizing NMS in a patient with underlying parkinsonian symptoms, where prominent symptom overlap can occur, is discussed. Copyright © 2004 John Wiley & Sons, Ltd.
Ex vivo generation and expansion of anti‐tumor cytotoxic T‐cell lines derived from patients or their HLA‐identical sibling
Authors: Montagna Daniela , Schiavo Roberta , Gibelli Nadia , Pedrazzoli Paolo , Tonelli Roberto , Pagani Sara , Assirelli Elisa , Locatelli Franco , Pession Andrea , Fregoni Vittorio , Montini Elisa , Da Prada Gian Antonio , Siena Salvatore , Maccario Rita
Published: March 2004

Successful ex‐vivo priming and long‐term maintenance of anti‐tumor cytotoxic T‐cell (CTL) lines are preliminary conditions for their use in approaches of adoptive immunotherapy for patients with cancer. We describe the results of a novel procedure for generating in vitro anti‐tumor CTL using CD8‐enriched peripheral blood mononuclear cells (PBMC) and dendritic cells (DC), pulsed with irradiated tumor cells (TC) as source of tumor antigen. Eight patients were enrolled in our study: 4 sarcoma, 2 renal cell carcinoma, 1 ovarian carcinoma and 1 breast carcinoma. Ten anti‐tumor CTL‐lines cytotoxic towards patient TC were generated. Five CTL‐lines were obtained using both DC and PBMC from the patients (autologous setting). For 5 CTL‐lines, DC derived from an HLA‐identical sibling were employed (allogeneic setting): patients or siblings PBMC were used to generate CTL‐lines in 2 and 3 cases, respectively,. After tumor‐specific rounds of stimulation, followed by antigen‐independent cycle of expansion, CTL‐lines obtained in both autologous and allogeneic setting showed an expansion of the absolute number of cultured cells. In 6 of 10 CTL‐lines, the majority of effector cells (>70%) were CD3+/CD8+, while in the remaining 4, 40–70% of effector cells were CD3+/CD4+. Both CD8+ and CD4+ T cells displayed anti‐tumor cytotoxic activity. Spectratyping analysis of the TCR‐Vβ subfamilies revealed a preferential expansion of oligoclonal populations in 18 of 24Vβ subfamily. Altogether these results demonstrate that our experimental approach is suitable for efficiently generating and expanding anti‐solid tumor CTL to be used for adoptive immunotherapy. © 2004 Wiley‐Liss, Inc.
Bikunin plus paclitaxel markedly reduces tumor burden and ascites in mouse model of ovarian cancer
Authors: Kobayashi Hiroshi , Yagyu Tatsuo , Inagaki Kiyokazu , Kondo Toshiharu , Suzuki Mika , Kanayama Naohiro , Terao Toshihiko
Published: March 2004

Our previous studies of intraperitoneal ovarian carcinoma in a mouse model demonstrated that bikunin gene transfection could prevent ascites formation and intraperitoneal disseminated metastasis. Although ascites was almost completely inhibited, tumor burden was variably reduced. Several reports have indicated that bikunin may be involved in tumor survival. In the present study, the effectiveness of exogenous bikunin and the biodistribution characteristics of 125I‐bikunin were initially examined in a mouse model of human ovarian cancer HRA cells. The once‐daily i.p. administration of bikunin significantly decreased progressive growth of HRA tumors and ascites formation in a dose‐dependent manner. Maximal radioisotope tumor uptake peaked at 7.4% injected dose/g at 3 hr. Bikunin binding specificity was demonstrated by reduced tumor uptake after coinjection of excess nonradioactive bikunin. Bikunin was rapidly excreted renally. The bikunin therapy produced the significant inhibition in expression of the proteolysis (uPA and uPAR) and angiogenesis‐related molecules (VEGF and bFGF). The second purpose of our study was to optimize the antimetastatic activity of bikunin in combination with paclitaxel against HRA cells growing orthotopically in mice. The once‐daily i.p. administration of bikunin (25 μg/g body weight/day) in combination with paclitaxel (i.p., 100 μg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. In conclusion, combination therapy with bikunin plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian carcinoma possibly through suppression of uPA, uPAR, VEGF and bFGF expression. © 2004 Wiley‐Liss, Inc.
Nanoindentation of soft hydrated materials for application to vascular tissues
Authors: Ebenstein D. M. , Pruitt L. A.
Published: March 2004

Soft hydrated materials, such as vascular tissues and other biomaterials, provide a number of challenges in the field of nanoindentation. However, the ability of nanoindentation to probe local, nanoscale mechanical properties of heterogeneous materials makes it desirable to adapt this technique for application to biologic tissues. To develop the field of nanoindentation for the analysis of soft hydrated materials, the goals of this study were fourfold: develop a sample hydration system, select an appropriate tip for soft material indentation, identify a substrate to be used for blunt tip alignment, and determine an appropriate control material for the development of future indentation protocols. A hydration system was developed that maintained sample hydration for over 8 h without completely submerging the sample. Further, a 100‐μm radius of curvature conospherical tip was shown to be a suitable tip for indenting a variety of soft hydrated materials and back‐illuminated agarose gel was found to be an effective material for use in tip alignment. Finally, agarose gel demonstrated similar qualitative and quantitative nanomechanical behavior to vascular tissue, suggesting that it will be an appropriate control material for the development of future indentation protocols for soft biologic tissues. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 222–232, 2004
Self‐hardening calcium phosphate cement–mesh composite: Reinforcement, macropores, and cell response
Authors: Xu Hockin H. K. , Simon Carl G.
Published: March 2004

Calcium phosphate cement (CPC) self‐hardens to form hydroxyapatite, has excellent osteoconductivity and bone‐replacement ability, and is promising for craniofacial and orthopedic repair. However, its low strength limits CPC to only nonstress repairs. This study aimed to reinforce CPC with meshes to increase strength, and to form macropores in CPC for bone ingrowth after mesh dissolution. A related aim was to evaluate the biocompatibility of the new CPC–mesh composite. Absorbable polyglactin meshes, a copolymer of poly(glycolic) and poly(lactic) acids, were incorporated into CPC to provide strength and then form interconnected cylindrical macropores suitable for vascular ingrowth. The composite flexural strength, work‐of‐fracture, and elastic modulus were measured as a function of the number of mesh sheets in CPC ranging from 1 (a mesh on the tensile side of the specimen) up to 13 (mesh sheets throughout the entire specimen), and as a function of immersion time in a physiological solution from 1 to 84 days. Cell culture was performed with osteoblast‐like cells and the cell viability was quantified using an enzymatic assay. The strengths (mean ± SD; n = 6) of CPC containing 13 or 6 meshes were 24.5 ± 7.8 and 19.7 ± 4.3 MPa, respectively, not significantly different from each other; both were significantly higher than 8.8 ± 1.9 MPa of CPC without mesh (Tukey's at 0.95). The work‐of‐fracture of CPC with 13 or 6 meshes was 3.35 ± 0.80 and 2.95 ± 0.58 kJ/m2, respectively, two orders of magnitude higher than 0.021 ± 0.006 kJ/m2 of CPC without mesh. Interconnected macropores were formed in CPC at 84 days' immersion. The new CPC–mesh formulation supported the adhesion, spreading, proliferation, and viability of osteoblast‐like cells in vitro. In conclusion, absorbable meshes in CPC increased the implant strength by three‐fold and work‐of‐fracture by 150 times; interconnected macropores suitable for bone ingrowth were created in CPC after mesh dissolution. The higher strength may help extend the use of CPC to larger stress‐bearing repairs, and the macropores may facilitate tissue ingrowth and integration of CPC with adjacent bone. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 267–278, 2004
Micro‐Raman spectroscopic investigation of dental calcified tissues
Authors: Schulze K. A. , Balooch M. , Balooch G. , Marshall G. W. , Marshall S. J.
Published: March 2004

The purpose of this study was to determine if dental calcified junctions (DEJs/CDJs) in human teeth contain different compositional phases compared to the adjacent dental calcified tissues. Peak positions and intensities were determined from micro‐Raman spectra for PO and the CH modes and compared among the mineralized tissues and their junctions. Values of width were determined from the intersections of intensity regression lines through the junctions and in the adjacent tissues. The peaks were measured in 1‐μm steps along a l00‐μm line across the junction. High‐resolution analysis revealed that PO band peaks for dentin, the DEJ, enamel, the CDJ, and cementum were at the same position (959 cm−1), while for the CH stretching mode a significant shift of 4.6 cm−1 was found between enamel, the DEJ, and dentin. The mean width of the DEJ was 7.6 (± 2.8) μm using the PO band and 8.6 (± 3.6) μm using the CH stretching mode. Across the DEJ, the mineral content monotonically decreased from enamel to dentin while the organic component monotonically increased. The DEJ width was in agreement with prior nanoindentation studies. No width estimate was possible for the CDJ because the compositional differences between cementum and dentin were small. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 286–293, 2004
Evaluation of thermoreversible polymers containing fibroblast growth factor 9 (FGF‐9) for chondrocyte culture
Authors: Au Angela , Polotsky Anna , Krzyminski Karol , Gutowska Anna , Hungerford David S. , Frondoza Carmelita G.
Published: March 2004

We previously evaluated a thermoreversible polymer gel composed of N‐isopropylacrylamide and acrylic acid as a cell culture substrate and cell‐delivery vehicle. The copolymer promoted phenotype expression and amplification of chondrocytes. In this study, we determined whether addition of fibroblast growth factor 9 (FGF‐9), which is mitogenic for chondrocytes, would further enhance cell proliferation and phenotype expression in the polymer. We tested the hypothesis that the thermoreversible polymer containing FGF‐9 would promote increased chondrocyte proliferation and phenotype expression. Articular chondrocytes (1 × 105/150 μL) were plated onto control (without gel) and gel containing 24‐well plates. The gels were prepared in media alone or in media containing heparin (100 μg/mL) and FGF‐9 (5 μg/mL). The cultures were incubated at 37°C in 5% CO2 for 3 days. Cells remained viable in the thermoreversible polymer in the presence or absence of FGF‐9. Addition of FGF‐9 to the copolymer did not induce proliferation and the cell numbers did not increase. Reverse transcription polymerase chain reaction (RT‐PCR)‐determined expression of chondrocyte markers collagen type II and aggrecan. FGF‐9 did not enhance chondrocyte proliferation nor alter the phenotype after 3 days in culture. These findings suggest the poly(NiPA‐co‐AAc) gel alone may provide the optimal 3D environment for propagation of chondrocytes. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 367–372, 2004
Wheat germ lectin induces G2/M arrest in mouse L929 fibroblasts
Authors: Liu W.K. , Sze S.C.W. , Ho J.C.K. , Liu B.P.L. , Yu M.C.
Published: March 2004

Wheat germ lectin (WGA) is a cytotoxic lectin for many cell lines [Wang et al., 2000], but its underlying mechanism is not clear. In this report, we found that incubation of synchronized mouse L929 fibroblasts with WGA resulted in a dose‐dependent reduction of intracellular incorporation of 3H‐thymidine and MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide)‐conversion activity (IC50 ≅ 0.4 μM). Fluorescein‐conjugated WGA was demonstrated to transport from the cell surface into the paranuclear region of cultured L929 cells within 30 min, and subsequently evoked lipid peroxidation of plasma membrane and vacuolation in the cytoplasm of these cells. Studies with tritiated thymidine incorporation, immunofluorescence microscopy, immunoblotting analysis and flow cytometry revealed that WGA inhibited cell cycle progression after one replication, resulting in G2/M arrest and alteration of cell cycle regulatory proteins, particularly activation of p21Cip1/WAF1 and suppression of cyclin B and cdc 2. Although there was an increase of cytosolic caspase 3 and bax protein expression, no apoptotic bodies were observed by both fluorescence and transmission electron microscopy. These results suggest that WGA arrested L929 proliferation after one cell cycle in the G2/M phase through activation of the p21Cip1/WAF1 and suppression of Cyclin B–Cdc2. © 2004 Wiley‐Liss, Inc.
Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors
Authors: Annabi Borhane , Naud Emmanuelle , Lee Ying‐Ta , Eliopoulos Nicoletta , Galipeau Jacques
Published: March 2004

Bone marrow‐derived stromal cells (BMSC) possess a population of vascular progenitor cells that enable them to acquire a histology and immunophenotype coherent with endothelial cells (EC). Recent evidence indicates that a hypoxic environment such as that encountered in tumor masses regulates BMSC angiogenic properties by pathways that remain to be defined. It is also unclear as to what extent these marrow‐derived precursor cells could contribute to the growth of endothelium‐lined vessels at the vicinity of tumor masses. In this study, we found that BMSC exhibited the ability to generate three‐dimensional capillary‐like networks on Matrigel, and that this property was up‐regulated by growth factors‐enriched conditioned media isolated from several tumor‐derived cell lines. In particular, basic fibroblast growth factor, a key mediator of angiogenesis, was found to be the most potent growth factor for inducing BMSC proliferation, migration, and tubulogenesis. The setup of a new two‐dimensional in vitro co‐culture assay further showed that BMSC were massively recruited when cultured in the presence of either cancerous or differentiated EC lines. In vivo, subcutaneous co‐injection of BMSC with U‐87 glioma cells in nude mice resulted in the formation of highly vascularized tumors, where BMSC differentiated into CD31‐positive cells and localized at the lumen of vascular structures. Our data suggest that BMSC could be recruited at the sites of active tumor neovascularization through paracrine regulation of their angiogenic properties. These observations may have crucial implications in the development of novel therapies using BMSC engineered to secrete anti‐cancerous agents and to antagonize tumor progression. © 2004 Wiley‐Liss, Inc.
Role of protein kinase C in arginine vasopressin‐stimulated ERK and p70S6 kinase phosphorylation
Authors: Ghosh Paramita M. , Bedolla Roble , Thomas Charles A. , Kreisberg Jeffrey I.
Published: March 2004

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)‐stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF‐R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972–F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP‐stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0–60 min stimulated the serine/threonine phosphorylation of PKC isoforms α, δ, ε, and ζ. The activation of PKC was dependent on EGF‐R and phosphatidylinositol 3‐kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12‐myristate 13‐acetate (PMA) inhibited AVP‐induced activation of ERK and p70S6 kinase as well as EGF‐R phosphorylation. Rottlerin, a specific inhibitor of PKCδ, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCε translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCζ (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, Gö6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCδ plays an important role in AVP‐stimulated ERK and p70S6 kinase activation and cell proliferation. © 2004 Wiley‐Liss, Inc.
Static magnetic fields‐induced bone sialoprotein (BSP) expression is mediated through FGF2 response element and pituitary‐specific transcription factor‐1 motif
Authors: Shimizu Emi , Matsuda‐Honjyo Yuko , Samoto Hiroshi , Saito Ryoichiro , Nakajima Yu , Nakayama Youhei , Kato Naoko , Yamazaki Muneyoshi , Ogata Yorimasa
Published: March 2004

Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein found almost exclusively in mineralized connective tissues. Recent studies on the developmental expression of BSP mRNA and temporo‐spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts, and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. Physical forces may play a fundamental role in the regulation of cell function in bone, but little is known about how cells are able to sense mechanical loads and signal transduction. Magnetic fields of sufficient magnitude have been shown to affect various biologic systems at organ, tissue, cellular, and subcellular levels. In the present study, rat osteosarcoma‐derived osteoblast‐like cells, UMR 106, were used to assess the effect of static magnetic fields (SMF) on gene transcription of BSP. In our culture system, application of 300 and 800 Gauss SMF increased BSP mRNA levels after 24 h stimulation. To determine the molecular basis of the transcriptional regulation of BSP gene transcription by SMF, we conducted transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase (LUC) reporter gene. SMF (300 and 800 Gauss) increased expression of the construct (pLUC3; −116 to +60) after 24 h treatment. Further deletion analysis of the BSP promoter showed that a region within nt −116 to −84 was targeted by SMF, the effect of which was inhibited by the tyrosine kinase inhibitor herbimycin A (HA). Mutations (2 bp) were made in an inverted CCAAT box between nt −50 and −46, a cyclicAMP response element (CRE; between nt −75 and −68), a fibroblast growth factor‐2 response element (FRE; −92 to −85), and a pituitary‐specific transcription factor‐1 motif (Pit‐1; nt −111 to −105) within the pLUC3 construct. Transcriptional stimulation by SMF was almost completely abrogated in constructs that included 2‐bp mutations in the FRE and Pit‐1. Binding of nuclear proteins to a radiolabeled FRE was increased and that to a Pit‐1 was decreased in nuclear extracts prepared from SMF‐stimulated UMR 106 cells. Further, the stimulatory and inhibitory effects of SMF on FRE and Pit‐1 DNA‐protein complexes were completely abolished by HA treatment. These studies, therefore, show that SMF increases BSP transcription through a tyrosine kinase‐dependent pathway and that the SMF effects are mediated through juxtaposed FRE and Pit‐1elements in the proximal promoter of the BSP gene. © 2004 Wiley‐Liss, Inc.
ATP‐dependent nucleosome remodeling complexes: Enzymes tailored to deal with chromatin
Authors: Sif Saïd
Published: March 2004

Chromatin remodeling complexes play a central role in the control of nuclear processes that utilize genomic DNA as a template including transcription, replication, recombination, and repair. Modulation of chromatin structure is mediated by a wide variety of enzymes which can affect nucleosome stability by either disrupting histone‐DNA contacts or by covalently modifying histones and/or DNA. Although the biochemical properties of most chromatin‐modifying enzymes have been well characterized and links between histone and DNA‐modifying enzymes and ATP‐dependent chromatin remodeling complexes have been established, the importance of their concerted action has just begun to emerge. As more and more genes are examined, new rules are being established about their transcriptional regulation, and it is becoming clear that diverse mechanisms are used to modify chromatin and either promote or hinder accessibility to DNA and histones. Moreover, the involvement of ATP‐dependent chromatin remodelers in transcriptional regulation of cyclin genes and the association of misregulated expression of chromatin remodeling subunits with many cancers highlight the importance of chromatin remodeling complexes in the control of cell growth and proliferation. © 2004 Wiley‐Liss, Inc.
Tranferrins receptor association and endosomal localization of soluble HFE are not sufficient for regulation of cellular iron homeostasis
Authors: Laham Nihay , Rotem‐Yehudar Rinat , Shechter Chana , Coligan John E. , Ehrlich Rachel
Published: March 2004

Iron uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. A non‐classical class I MHC molecule, the hemochromatosis factor HFE, has been shown to regulate iron metabolism, potentially via its direct interaction with the transferrin receptor (TfR). In this study, we demonstrate that a soluble β2microglobulin‐HFE monochain (sHFE) folds with β2microglobulin (β2m) and associates with the TfR, indicating that the transmembrane and cytoplasmic domains are not necessary for assembly and trafficking through the ER‐Golgi network. We also demonstrate human TfR‐specific uptake and accumulation of extracellular sHFE by treated cells. The sHFE localized to the endosomal compartment albeit we observed variation in the time taken for endosomal trafficking between different cell types. The sHFE monochain was effective in reducing Tf uptake into cells, however this did not correlate to any changes in TfR or ferritin synthesis, in contrast to the HFE‐induced increase and decrease of TfR and ferritin, respectively. These findings of incongruent sHFE activity suggest that either variation in affinity binding of sHFE to TfR prevents efficient modulation of iron‐regulated proteins or that HFE has multiple functions some of which may be independent of TfR but dependent on interactions within the endosomal compartment for effective modulation of iron metabolism. © 2004 Wiley‐Liss, Inc.
Proliferation marker pKi‐67 occurs in different isoforms with various cellular effects
Authors: Schmidt Mirko H.H. , Broll Rainer , Bruch Hans‐Peter , Finniss Susan , Bögler Oliver , Duchrow Michael
Published: March 2004

The Ki‐67 antigen, pKi‐67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi‐67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi‐67 N‐terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi‐67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi‐67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi‐67 N‐terminus is differentially spliced resulting in at least five different isoforms with different functions. © 2004 Wiley‐Liss, Inc.
Promoter activity and regulation of the corneal CYP4B1 gene by hypoxia
Authors: Mastyugin Vladimir , Mezentsev Alexandre , Zhang Wen‐Xiang , Ashkar Silvia , Dunn Michael W. , Laniado‐Schwartzman Michal
Published: March 2004

Hypoxic injury to the ocular surface provokes an inflammatory response that is mediated, in part, by corneal epithelial‐derived 12‐hydroxyeicosanoids. Recent studies indicate that a cytochrome P450 (CYP) monooxygenase, identified as CYP4B1, is involved in the production of these eicosanoids which exhibit potent inflammatory and angiogenic properties. We have isolated and cloned a corneal epithelial CYP4B1 full‐length cDNA and demonstrated that the CYP4B1 mRNA is induced by hypoxia in vitro and in vivo. To further understand the molecular regulation that underlies the synthesis of these potent inflammatory eicosanoids in response to hypoxic injury, we isolated and cloned the CYP4B1 promoter region. GenomeWalker libraries constructed from rabbit corneal epithelial genomic DNA were used as templates for primary and nested PCR amplifications with gene‐ and adaptor‐specific primers. A 3.41‐kb DNA fragment of the 5′‐flanking region of the CYP4B1 promoter was isolated, cloned, sequenced, and analyzed by computer software for the presence of knowncis‐acting elements. Analysis of the promoter sequence revealed the presence of consensus DNA binding sequences for factors known to activate gene transcription in response to hypoxia including HIF‐1, NFκB, and AP‐1. Transient transfection of luciferase reporter (pGL3‐Basic) vectors containing different lengths of the CYP4B1 promoter fragment demonstrated hypoxia‐induced transcription in rabbit corneal epithelial (RCE) cells. Electrophoretic mobility shift assay (EMSA) revealed a marked induction of nuclear binding activity for the labeled HIF‐1 probe from the CYP4B1 promoter in nuclear extracts of cells exposed to hypoxia. This binding activity was due to sequence‐specific binding to the HIF‐1 oligonucleotide probe as shown by competition with excess unlabeled probe for the HIF‐1 but not with unlabeled NFκB probe. The nuclear binding activity of AP‐1 and NFκB probes from the CYP4B1 promoter was also enhanced in response to hypoxia suggesting that these transcription factors contribute to the hypoxic induction of CYP4B1 expression. The results of this study provide the first molecular mechanistic explanation for the induction of CYP4B1 and, thereby, the production of inflammatory eicosanoids in response to hypoxic injury. Further studies are needed to fully evaluate the molecular regulation of this gene during inflammation. © 2004 Wiley‐Liss, Inc.
Distinct functions of BMP4 and GDF5 in the regulation of chondrogenesis
Authors: Hatakeyama Yuji , Tuan Rocky S. , Shum Lillian
Published: March 2004

Bone morphogenetic protein 4 (BMP4) and growth/differentiation factor 5 (GDF5) are closely related protein family members and regulate early cartilage patterning and differentiation. In this study, we compared the functional outcome of their actions systematically at various stages of chondrogenesis in mouse embryonic limb bud mesenchyme grown in micromass cultures. Overall, both growth factors enhanced cartilage growth and differentiation in these cultures. Uniquely, BMP4 not only accelerated the formation and maturation of cartilaginous nodules, but also induced internodular mesenchymal cells to express cartilage differentiation markers. On the other hand, GDF5 increased the number of prechondrogenic mesenchymal cell condensation and cartilaginous nodules, without altering the overall pattern of differentiation. In addition, GDF5 caused a more sustained elevated expression level of Sox9 relative to that associated with BMP4. BMP4 accelerated chondrocyte maturation throughout the cultures and sustained an elevated level of Col10 expression, whereas GDF5 caused a transient increase in Col10 expression. Taken together, we conclude that BMP4 is instructive to chondrogenesis and induces mesenchymal cells toward the chondrogenic lineage. Furthermore, BMP4 accelerates the progression of cartilage differentiation to maturation. GDF5 enhances cartilage formation by promoting chondroprogenitor cell aggregation, and amplifying the responses of cartilage differentiation markers. These differences may serve to fine‐tune the normal cartilage differentiation program, and can be exploited for the molecular manipulation in biomimetics. Published 2004 Wiley‐Liss, Inc.
Sp1‐dependent regulation of the tissue inhibitor of metalloproteinases‐1 promoter
Authors: Lee Minhyung , Song Sun U. , Ryu Ji Kan , Suh Jun Kyu
Published: March 2004

Extracellular matrix (ECM) remodeling is involved in many cellular properties such as division, migration, differentiation, and death. The turnover of ECM is regulated by matrix metalloproteinases (MMPs) and the MMPs are inhibited by the tissue inhibitors of metalloproteinases (TIMPs). In this study, the transcriptional regulation of the TIMP‐1 promoter was investigated. The 5′‐deletion assay showed that the region between −1,200 and −1,101 was responsible for the TIMP‐1 promoter activity. The mutations of the two Sp1 sites in this region reduced the transcription activity. In addition, the co‐transfection with antisense Sp1 oligonucleotide decreased the promoter activity, suggesting that the transcription of the TIMP‐1 promoter is mediated by Sp1. Previously, it was reported that the TIMP‐1 expression was enhanced under hypoxia. Therefore, the TIMP‐1 promoter activity was investigated with or without cobalt ion, which elicits the same physiological effect as hypoxia. The results showed that the TIMP‐1 promoter was induced in the presence of cobalt ion and that the promoter activity was regulated by Sp1 as well as HIF‐1. Therefore, this study suggests that Sp1 is involved in the regulation of the TIMP‐1 promoter in the presence of cobalt ion as well as in the basal level transcription. © 2004 Wiley‐Liss, Inc.
Nuclear matrix protein expressions in hepatocytes of normal and cirrhotic rat livers under normal and regenerating conditions
Authors: Yun Jing‐Ping , Liew Choong‐Tsek , Chew Eng Ching , Yin Xiao‐Yu , Lai Paul Bo San , Fai Yam Hin , Li H.K. Richard , Jin Mei‐Lin , Ding Ming‐Xiao , Li Ming‐Tao , Lin Han‐Liang , Lau Wan Yee
Published: March 2004

We explored the feasibility of studying nuclear matrix protein (NMP) expressions of the hepatocytes in normal and cirrhotic rat livers with liver regeneration after partial hepatectomy. Sixteen Wistar healthy rats were studied with experimental liver regeneration and/or liver cirrhosis. Two‐dimensional (2‐D) gel electrophoresis was used to generate these NMP compositions from these rat liver samples. Several antibodies against cytokeratin, vimentin, actin, B23, HNF4alpha, and heat shock protein 70 were used for identification by Western blot. Totally, 41 strongly stained protein spots were characterized on the 2‐D gels. Thirty‐four protein spots were detected in all of these rat livers, of which, cytokeratin, vimentin, actin, HNF4alpha, and heat shock protein 70 were identified. B23 was detected in the regenerated livers. Three protein spots (s33, s34, and s35) were detectable only in NMP preparation extracted from the regenerating rat livers after hepatectomy. Another three protein spots (s36, s37, and s38) were detectable only in NMP preparation extracted from thioacetamide‐induced cirrhotic rat livers. Under these conditions including experimental liver regeneration and/or liver cirrhosis, Over thirty higher abundance NMPs of hepatocytes were consistently expressed and considered as common and basic NMPs. Some of the NMPs are specific for liver regeneration and may play a critical role in cell proliferation and cell cycle, and some are specific for liver cirrhosis. © 2004 Wiley‐Liss, Inc.
TOR kinase and ran are downstream from PI3K/Akt in H2O2‐induced mitosis
Authors: Radisavljevic Ziv Manasija , González‐Flecha Beatriz
Published: March 2004

Hydrogen peroxide (H2O2) activates signaling cascades essential for cell proliferation via phosphatidylinositol‐3‐kinase (PI3K) and Akt. Here we show that induction of mitogenic signaling by H2O2 activates sequentially PI3K, Akt, mammalian target of rapamycin (mTOR), and Ran protein. Akt activation is followed by signaling through the mTOR kinase and upregulation of Ran in primary type II pneumocytes, a cell type implicated in the development of lung adenocarcinoma. Pretreatment of the cells with wortmannin, a specific inhibitor of PI3K, or rapamycin, a specific inhibitor of mTOR kinase, prevented H2O2‐increased mitosis. H2O2‐induced Akt ser‐473 phosphorylation and upregulation of Ran protein were prevented by wortmannin but not by rapamycin, indicating that PI3K is upstream of Akt and mTOR is downstream from Akt. Overexpression of myr‐Akt or Ran‐wt in type II pneumocytes increased Akt ser‐473 phosphorylation and mitosis in a catalase‐dependent manner, indicating that H2O2 is essential for Akt and Ran signaling. These results indicate that H2O2‐induced mitogenic signaling in primary type II pneumocytes is mediated by PI3K, Akt, mTOR‐kinase, and Ran protein. © 2004 Wiley‐Liss, Inc.
Establishment and characterization of a stable cell line to evaluate cellular Runx2 activity
Authors: Kim Hyun‐Jung , Park Hee‐Dae , Kim Jung‐Hwan , Cho Je‐Yoel , Choi Je‐Yong , Kim Jung‐Keun , Kim Hyun‐Jung , Shin Hong‐In , Ryoo Hyun‐Mo
Published: March 2004

Runx2 is an essential transcription factor for osteoblast differentiation from early commitment step to final differentiation. Based on its crucial role in osteoblast differentiation, the transcriptional activity of Runx2 protein implies more valuable information for osteoblast differentiation than any other parameters, such as Runx2 mRNA or protein level. Thus, a sensitive, specific, and consistent method to determine the Runx2 transcriptional activity has long been expected. Here we suggest a stable cell line that carries 6xOSE2‐Luciferase reporter vector would be a good evaluation system to determine biological Runx2 transcriptional activity. The proliferation rate, cell shape, and the myogenic differentiation potential of the cloned cell line were similar to those of parental premyoblastic C2C12 cells. The cells specifically responded to Runx2 modulating agent such as FGF2. The stable cell line responded 5–6 folds more sensitively than the transiently transfected cells with Runx2. Though overexpression of any Runx gene stimulated the luciferase activity, Runx2 enhanced the reporter activity the highest. Collectively, the 6xOSE2‐luc stable cells would be a good biological evaluation system to assess the activity of extracellular Runx2 modulating stimulations as well as the signal transduction pathways involved in the stimulations. © 2004 Wiley‐Liss, Inc.
The phenomenon of quantum change
Authors: Miller William R.
Published: March 2004

Quantum change—the subject of this issue of In Session—refers to sudden, dramatic, and enduring transformations that affect a broad range of personal emotion, cognition, and behavior. This phenomenon has been described since the beginnings of psychology, most notably by William James in Varieties of Religious Experience. Quantum changes occur both within and (mostly) outside the context of psychotherapy and show certain common features in both process and content. This introduction provides an overview of the phenomenon of quantum change, describing its essential features, two qualitative subtypes, antecedents, course, and the nature of the enduring changes that ensue. © 2004 Wiley Periodicals, Inc. J Clin Psychol/In Session.
Epiphany in autobiography: The quantum changes of Dostoevsky and Tolstoy
Authors: Bidney Martin
Published: March 2004

The quantum changes of Dostoevsky and Tolstoy are clarified by the partly overlapping concept of a literary epiphany. The qualities of an epiphany—intensity, resonance, and mystery—are much like the vividness and surprise of a quantum change (which also is felt to be beneficent and permanent). Epiphanic intensity and mystery reinforce the quantum attributes of love and lasting transformation in Dostoevsky's sudden memory of the motherlike tenderness of the peasant Marey. Tolstoy underlines the quantum effects of the embracing light seen inA Confession, but this epiphany is more abstract and attenuated than Dostoevsky'sDiaryepisode. Tolstoy's earlier memoir shows how the shock of identity questioning introduces a complicating factor: the trauma of remorse. Clinical implications of the two authors' quantum‐change accounts are discussed. © 2004 Wiley Periodicals, Inc. J Clin Psychol/In Session.
Quantum change and psychotherapy
Authors: Bien Thomas H.
Published: March 2004

Deep change in psychotherapy more typically comes slowly rather than suddenly, but this difference between therapeutic change and quantum change may be one of perspective rather than substance. Psychotherapy may be understood as a kind of mindfulness practice similar to working with koans in that the client presents a life dilemma incapable of rational solution. While quantum change cannot be engineered, the psychotherapist can create an environment conducive to such transformation by producing true presence and modeling calm, concerned, sustained attention to the dilemma that precipitated treatment. Psychotherapists who also maintain a sense of their work as a high art and a way of being, and who in consequence cultivate their own emotional and spiritual development, may be more likely to create such an environment. © 2004 Wiley Periodicals, Inc. J Clin Psychol/In Session.
De profundis: Spiritual transformations in Alcoholics Anonymous
Authors: Forcehimes Alyssa A.
Published: March 2004

The mechanism of change in Alcoholics Anonymous is described as “spiritual transformation.” A.A. acknowledges that such transformation can occur gradually; however, nearly all of the examples presented in the Big Book of A.A. involve discrete and sudden experiences that resemble the phenomenon of quantum change. The sequence offered describes how spiritual transformations transpire. The sequence begins with hitting bottom, recognition of inability to control the problem. A feeling of contrition follows, describing not only sorrow for the present state, but also desire for a new way. The final step is the act of surrendering one's will to a higher power. The de profundis sequence sets the process of spiritual transformation in motion, offering stabilization to sobriety. © 2004 Wiley Periodicals, Inc. J Clin Psychol/In Session.
Evil by default: The origins of dark visions
Authors: Nowinski Joseph
Published: March 2004

The growing body of literature on transformational change reflects an increasing interest among professionals in understanding epiphany and visionary change. However, we typically assume that such quantum changes are positive and pro‐social ones. It is equally possible for an individual to lead a life that is organized around a motivating vision that is destructive to self, others, or both. The author examines the phenomenon of “dark visions,” offering some hypotheses as to their origins. The article also offers strategies to minimize the number of individuals who choose dark visions, with all their unfortunate consequences. © 2004 Wiley Periodicals, Inc. J Clin Psychol/In Session.
Sacred changes: Spiritual conversion and transformation
Authors: Mahoney Annette , Pargament Kenneth I.
Published: March 2004

We use Pargament's (1997) definition of religion—“the search for significance in ways related to the sacred”—as a framework to understand spiritual conversion. Like other life‐changing transformations, spiritual conversion alters the destinations that clients perceive to be of greatest importance in life (significance) and the pathways by which clients discover what is most significant in life (search). Unlike other transformative experiences, however, spiritual conversion incorporates the third element of religion, “the sacred,” into the content of change. To illustrate these points, we discuss two theological models of spiritual conversion rooted in Christianity: a traditional model based on classic western theology and an alternative model based on feminist theology. We then compare processes of spiritual conversion to nonreligious models of transformation. We also highlight the importance for clinical work of the fit between the context of a client's life and the type of spiritual conversion experienced. © 2004 Wiley Periodicals, Inc. J Clin Psychol/In Session.
Exclusion of repetitive DNA elements from gnathostome Hox clusters
Authors: Fried Claudia , Prohaska Sonja J. , Stadler Peter F.
Published: March 2004

Despite their homology and analogous function, the Hox gene clusters of vertebrates and invertebrates are subject to different constraints on their structural organization. This is demonstrated by a drastically different distribution of repetitive DNA elements in the Hox cluster regions. While gnathostomes have a strong tendency to exclude repetitive DNA elements from the inside of their Hox clusters, no such trend can be detected in the Hox gene clusters of protostomes. Repeats “invade” the gnathostome Hox clusters from the 5′ and 3′ ends while the core of the clusters remains virtually free of repetitive DNA. This invasion appears to be correlated with relaxed constraints associated with gene loss after cluster duplications. J. Exp. Zool. (Mol.Dev.Evol.) 302B:165–173, 2004. © 2004 Wiley‐Liss, Inc.
Molecular cloning and expression of the osteoclast‐stimulating‐factor‐like gene from the rice field eel
Authors: Xia Laixin , Cheng Hanhua , Yu Hongshi , Guo Yiqing , Zhou Rongjia
Published: March 2004

Sexual development in vertebrates is a complex process. Vertebrates use several mechanisms to determine the development of a male or female organism. The genes for determination of sequential hermaphrodite sex are unknown. We identified a homologue of human osteoclast‐stimulating factor (OSF) in the rice field eel, a teleost that undergoes natural sex transformation from female, via intersex, to male during its lifetime. The rice field eel OSF‐like gene cDNA encoded a peptide of 214 amino acids that contains a c‐Src homology 3 domain, proline‐rich region, and ankyrin repeats, suggesting potential involvement in cell signaling. The gene was clustered into the OSF gene group of all the other vertebrates. Although expressed in the three kinds of gonads and in other tissues, OSF‐like gene expression in gonads of all the three sexes was restricted to the gonadal germinal epithelium, from where bipotential gonia (oogonia or spermatogonia) will differentiate, suggesting that the OSF‐like gene may be involved in sexual differentiation, in addition to its other roles as a regulator in development. J. Exp. Zool. (Mol. Dev. Evol.) 302B:174‐181, 2004. © 2004 Wiley‐Liss, Inc.