bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and regulation of the insulin‐like growth factor axis components in rat liver myofibroblasts
Authors: Novosyadlyy Ruslan , Tron Kyrylo , Dudas Jozsef , Ramadori Giuliano , Scharf Jens‐Gerd 
Published: March 2004

AbstractApart from hepatic stellate cells (HSC), liver myofibroblasts (MF) represent a second mesenchymal cell population involved in hepatic fibrogenesis. The IGF system including the insulin‐like growth factors I and II (IGF‐I, ‐II), their receptors (IGF‐I receptor, IGF‐IR; IGF‐II/mannose 6‐phosphate receptor, IGF‐II/M6‐PR), and six high affinity IGF binding proteins (IGFBPs) participate in the regulation of growth and differentiation of cells of the fibroblast lineage, possibly contributing to the fibrogenic process. The aim of this work was to study the expression and regulation of the IGF axis components in rat liver MF. Methods: Cultures of MF from passages 1 to 4 (P1–4) were studied. IGFBP secretion was analyzed by [125I]‐IGF‐I ligand and immunoblotting. IGF‐I, IGF‐IR, IGF‐II/M6‐PR, and IGFBP messenger RNA (mRNA) expression was assessed by Northern blot hybridization. DNA synthesis was evaluated by 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay. Results: MF from P1 to 4 constitutively expressed mRNA transcripts specific for IGF‐I, IGF‐IR, and IGF‐II/M6‐PR. In MF, biosynthesis of IGFBP‐3 and ‐2 was observed that was stimulated by IGF‐I, insulin, and transforming growth factor β (TGF‐β), whereas platelet‐derived growth factor (PDGF‐BB) revealed inhibitory effects. IGF‐I and to a lesser extent insulin increased DNA synthesis of MF. Simultaneous addition of recombinant human IGFBP‐2 or ‐3 with IGF‐I diminished the mitogenic effect of IGF‐I on MF whereas preincubation of MF with IGFBP‐2 or ‐3 further potentiated the IGF‐I stimulated DNA synthesis. In conclusion, the present study demonstrates that the IGF axis may play a role in the regulation of MF proliferation in vitro which might be relevant in vivo for the process of fibrogenesis during acute and chronic liver injury. © 2004 Wiley‐Liss, Inc.
Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathways
Authors: Quinlan Margaret P. 
Published: March 2004

AbstractTransformation progression of epithelial cells involves alterations in their morphology, polarity, and adhesive characteristics, all of which are associated with the loss and/or reorganization of actin structures. To identify the underlying mechanism of formation of the adhesion‐dependent, circumferential actin network, the expression and localization of the actin binding and regulating proteins (ABPs), vinculin, VASP, and profilin were evaluated. Experimental depolarization of epithelial cells results in the loss of normal F‐actin structures and the transient upregulation of vinculin, VASP, and profilin. This response is due to the loss of cell–cell, and not cell–substrate interactions, since cells that no longer express focal adhesions or stress fibers are still sensitive to changes in adhesion and manifest this in the altered profile of expression of these ABPs. Transient upregulation is dependent upon de novo protein synthesis, and protein kinase‐, but not phosphatase‐sensitive signal transduction pathway(s). Inhibition of the synthesis of these proteins is accompanied by dephosphorylation of the ribosomal S6 protein, but does not involve inhibition of the PI3‐kinase‐Akt‐mTOR pathway. Constitutive expression of VASP results in altered cell morphology and adhesion and F‐actin and vinculin structures. V12rac1 expressing epithelial cells are constitutively nonadhesive, malignantly transformed, and constitutively express high levels of these ABPs, with altered subcellular localizations. Transformation suppression is accompanied by the restoration of normal levels of the three ABPs, actin structures, adhesion, and epithelial morphology. Thus, vinculin, VASP, and profilin are coordinately regulated by signal transduction pathways that effect a translational response. Additionally, their expression profile maybe indicative of the adhesion and transformation status of
epithelial cells. J. Cell. Physiol. 200: 277–290, 2004. © 2004 Wiley‐Liss, Inc.
Significance of N‐methyl‐d‐aspartate (NMDA) receptor‐mediated signaling in human keratinocytes
Authors: Nahm Walter K. , Philpot Benjamin D. , Adams Michelle M. , Badiavas Evangelos V. , Zhou Linda H. , Butmarc Janet , Bear Mark F. , Falanga Vincent 
Published: March 2004

AbstractIncreasing data suggest that glutamate might act as a cell‐signaling molecule in non‐neuronal tissues such as the skin. Here we demonstrate the presence of functional N‐methyl‐d‐aspartate (NMDA)‐type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell‐to‐cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re‐epithelialization, and the process of re‐epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact‐mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders. © 2003 Wiley‐Liss, Inc.
Role of RB and RB2/P130 genes in marrow stromal stem cells plasticity
Authors: Jori Francesco P. , Napolitano Marco A. , Melone Mariarosa A.B. , Cipollaro Marilena , Cascino Antonino , Giordano Antonio , Galderisi Umberto 
Published: March 2004

AbstractMarrow stromal cells (MSCs) are stem‐like cells having a striking somatic plasticity. In fact, besides differentiating into mesenchymal lineages (bone, cartilage, and fat), they are capable of differentiating into neurons and astrocytes in vitro and in vivo. The RB and RB2/P130 genes, belonging to the retinoblastoma gene family, play a key role in neurogenesis, and for this reason, we investigated their role in neural commitment and differentiation of MSCs. In MSCs that were either uncommitted or committed toward neural differentiation, we ectopically expressed RB and RB2/P130 genes and analyzed their role in regulating the cell cycle, apoptosis and differentiation. In uncommitted MSCs, the activity of RB and RB2/P130 appeared limited to negatively regulating cell cycle progression, having no role in apoptosis and differentiation (toward either mesenchymal or neural lineages). On the other hand, in MSCs committed toward the neural phenotype, both RB and RB2/P130 reduced cell proliferation rate and affected the apoptotic process. RB protected differentiating cells from programmed cell death. On the contrary, RB2/P130 increased the percentage of cells in apoptosis. All of these activities were accomplished mainly in an HDAC‐independent way. The retinoblastoma genes also influenced differentiation in neural committed MSCs. RB2/P130 contributes mainly to the induction of generic neural properties, while RB triggers cholinergic differentiation. These differentiating activities are HDAC‐dependent. Our research shows that there is a critical temporal requirement for the RB genes during neuronal differentiation of MSCs: they are not required for cell commitment but play a role in the maturation process. For the above reasons, RB and RB2/P130 may have a role in neural differentiation but not in neural determination. © 2004 Wiley‐Liss, Inc.
Temperature‐sensitive polymer‐conjugated IFN‐γ induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG)
Authors: Sarkhosh Kourosh , Tredget Edward E. , Uludag Hasan , Kilani Ruhangiz T. , Karami Ali , Li Yunyuan , Iwashina Takashi , Ghahary Aziz 
Published: March 2004

AbstractIndoleamine 2,3‐dioxygenase (IDO) is an intracellular tryptophan‐catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co‐cultured with IDO‐expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon‐gamma (IFN‐γ) conjugated with a temperature‐sensitive polymer to induce the expression of IDO mRNA and protein. SDS–PAGE showed successful conjugation of IFN‐γ with the temperature‐sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer‐conjugated IFN‐γ. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN‐γ‐treated fibroblasts than in controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer‐conjugated IFN‐γ was significantly longer than in those treated with free (non‐conjugated) IFN‐γ (P < 0.001). IFN‐γ radiolabeling showed a prolonged retention of IFN‐γ within collagen gel in its polymer‐conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN‐γ). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO‐expressing FPCG treated with polymer‐conjugated IFN‐γ were co‐cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co‐cultured with IFN‐γ‐treated IDO‐expressing fibroblasts, compared to those co‐cultured with non‐IDO‐expressing fibroblasts (P < 0.001). The addition of an IDO inhibitor (1‐methyl‐D‐tryptophan) reversed the suppressive effects of IDO on PBMC proliferation. In conclusion, IDO expression in FPCG suppresses the proliferation of immune cells in vitro. The use of a temperature‐sensitive polymer further prolongs the effect of IFN‐γ on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts. J. Cell. Physiol. 201: 146–154, 2004. © 2004 Wiley‐Liss, Inc.
Uncoupling protein‐3 sensitizes cells to mitochondrial‐dependent stimulus of apoptosis
Authors: Dejean Laurent , Cámara Yolanda , Sibille Brigitte , Solanes Gemma , Villarroya Francesc 
Published: March 2004

AbstractThe mitochondrial uncoupling protein‐3 is a member of the mitochondrial carrier protein family. As a homologue of the thermogenic brown fat uncoupling protein‐1, it possesses a mitochondrial uncoupling activity and thus can influence cell energy metabolism but its exact biological function remains unclear. In the present study, uncoupling protein‐3 was expressed in 293 cells using the tetracycline‐inducible system and its impact on cell bioenergetics and responsiveness to the apoptotic stimulus was determined. The induction of uncoupling protein‐3 expression in mitochondria did not lead to uncontrolled respiratory uncoupling in intact cells. However, it caused a GDP‐inhibition of state 4 respiration and a GDP‐induced re‐polarization of the inner mitochondrial membrane in the presence of fatty acids, in agreement with its expected physiological behavior as an uncoupling protein (UCP). Uncoupling protein‐3 expression did not cause apoptosis per se but increased the responsiveness of the cells to a mitochondrial apoptotic stimulus (i.e., addition of staurosporine in the culture medium). It enhanced caspase 3 and caspase 9 activation and favored cytochrome c release. Moreover, cells in which uncoupling protein‐3 expression had been induced showed a higher mitochondrial Bax/Bcl‐2 ratio essentially due to enhanced translocation of Bax from cytosol to mitochondria. Finally, the induction of uncoupling protein‐3 also increased the sensitivity of mitochondria to open the permeability transition pore in response to calcium. It is concluded that the presence of uncoupling protein‐3 in mitochondria sensitizes cells to apoptotic stimuli involving mitochondrial pathways. © 2004 Wiley‐Liss, Inc.
Mitogenic signalling by B2 bradykinin receptor in epithelial breast cells
Authors: Greco S. , Muscella A. , Elia M.G. , Romano S. , Storelli C. , Marsigliante Santo 
Published: March 2004

AbstractThe kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain, and oedema. A role in the modulation or induction of healthy breast tissue growth has been postulated for tissue kallikrein present in human milk. Moreover, tissue kallikrein was found in malignant human breast tissue and bradykinin (BK) stimulates the proliferation of immortalised breast cancer cells. Aim of the present article was to investigate whether BK also exerts mitogenic activity in normal breast epithelial cells and partially characterise the signalling machinery involved. Results show that BK increased up to 2‐fold the 24 h proliferation of breast epithelial cells in primary culture, and that the BK B2 receptor (not B1) inhibitor alone fully blocked the BK response. Intracellular effects of B2 stimulation were the following: (a) the increase of free intracellular Ca2+ concentration by a mechanism dependent upon the phospholipase C (PLC) activity; (b) the cytosol‐to‐membrane translocation of conventional (PKC)‐α and ‐β isozymes, novel PKC‐δ, ‐ε, and ‐η isozymes; (c) the phosphorylation of the extracellular‐regulated kinase 1 and 2 (ERK1/2); and (d) the stimulation of the expression of c‐Fos protein. EGF, a well known stimulator of cell proliferation, regulated the proliferative response in human epithelial breast cells to the same extent of BK. The effects of BK on proliferation, ERK1/2 phosphorylation, and c‐Fos expression were abolished by GF109203X, which inhibits PKC‐δ isozyme. Conversely, Gö6976, an inhibitor of PKC‐α and ‐β isozymes, and the 18‐h treatment of cells with PMA, that led to the complete down‐regulation of PKC‐α, ‐β, ‐ε, and ‐η, but not of PKC‐δ, did not have any effect, thereby indicating that the PKC‐δ mediates the mitogenic signalling of BK. Phosphoinositide 3‐kinase (PI3K), tyrosine kinase of the epidermal growth factor receptor (EGFR), and mitogen activated protein kinase kinases (MEK) inhibitors were also tested. The results suggest that EGFR, PI3K, and ERK are required for the proliferative effects of BK. In addition, the BK induced cytosol‐to‐membrane translocation of PKC‐δ was blocked by PI3K inhibition, suggesting that PI3K is upstream to PKC‐δ. In conclusion, BK has mitogenic actions in cultured human epithelial breast cells; the activation of PKC‐δ through B2 receptor acts in concert with ERK and PI3K pathways to induce cell proliferation. J. Cell. Physiol. 201: 84–96, 2004. © 2004 Wiley‐Liss, Inc.
Impact of novel histone deacetylase inhibitors, CHAP31 and FR901228 (FK228), on adenovirus‐mediated transgene expression
Authors: Taura Kojiro , Yamamoto Yuzo , Nakajima Akio , Hata Koichiro , Uchinami Hiroshi , Yonezawa Kei , Hatano Etsuro , Nishino Norikazu , Yamaoka Yoshio 
Published: March 2004

AbstractBackgroundHistone deacetylase inhibitors (HDIs) are known to enhance adenovirus (Ad)‐mediated transgene expression. Recently, novel HDIs, including cyclic hydroxamic‐acid‐containing peptide 31 (CHAP31) and FR901228 (FK228), have been developed.MethodsThe effects of these two novel HDIs on Ad‐transduced or endogenous gene expression were investigated. Acetylation of core histones and the expression of the coxsackie and adenovirus receptor (CAR) in HDI‐treated cells were examined using Western blot and a quantitative reverse transcription polymerase chain reaction (TaqMan RT‐PCR), respectively. Their in vivo effect on adenoviral gene expression was investigated in BALB/c mice.ResultsBoth compounds enhanced and prolonged Ad‐mediated β‐galactosidase expression more effectively than did trichostatin A, a classic HDI. The same effect was observed in Ad‐transduced heat shock protein 72 (HSP72), but not in hyperthermia‐induced endogenous expression of HSP72, suggesting that the effect is specific for transduced gene expression. Hyperacetylation of core histones induced by HDIs was considered responsible for the augmentative effects of gene expression. Intravenous administration of either CHAP31 or FR901228 enhanced β‐galactosidase expression in mice infected with AdLacZ.ConclusionsCHAP31 and FR901228 amplified Ad‐mediated transgene expression. The enhancement of transgene expression by HDIs may result in fewer vector doses for necessary gene expression, helping to alleviate disadvantages caused by Ad vectors. This could be a useful tool in overcoming current limitations of gene therapy using adenovirus vectors. Copyright © 2004 John Wiley & Sons, Ltd.
Association of albumin or protamine to lipoplexes: enhancement of transfection and resistance to serum
Authors: Faneca H. , Simões S. , Pedroso de Lima M. C. 
Published: March 2004

AbstractBackgroundThe successful application of gene therapy depends on the availability of carriers to efficiently deliver genetic material into target cells. Such efficacy is strongly related to key parameters including serum resistance and protection of DNA.MethodsThe complexes were tested in terms of their biological activity, in the absence or presence of serum, by following transfection activity. Interaction with plasma proteins was evaluated by immunoblotting, while cytotoxicity was assessed by the Alamar Blue assay. Extent of DNA protection was determined both by using ethidium bromide intercalation and DNase I digestion assays.ResultsOur results show that, depending on the charge ratio and on the lipid composition, albumin and protamine can be used (either individually or co‐associated) to generate cationic liposome/DNA complexes fulfilling in vivo requirements, while exhibiting high levels of transfection activity. In the present work a novel cationic lipid was tested. It was demonstrated that 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐ethylphosphocholine (EPOPC):cholesterol (Chol) liposomes constitute a very promising carrier for gene delivery as illustrated by their enhancing effect on transfection, as compared with DOTAP‐containing liposomes. Moreover, the biological activity of EPOPC‐containing complexes is significantly improved upon association of albumin, even in the presence of 60% serum (namely for the 4/1 lipid/DNA charge ratio). Nevertheless, our studies also show that transfection activity mediated by DOTAP‐containing complexes can be significantly enhanced upon pre‐condensation of DNA with protamine.ConclusionsCo‐association of HSA and protamine to lipoplexes ensures a high degree of DNA protection and results in high levels of transfection activity even in the presence of serum. Copyright © 2004 John Wiley & Sons, Ltd.
Gene therapy with novel adeno‐associated virus vectors substantially diminishes atherosclerosis in a murine model of familial hypercholesterolemia
Authors: Lebherz Corinna , Gao Guangping , Louboutin Jean‐Pierre , Millar John , Rader Daniel , Wilson James M. 
Published: March 2004

AbstractBackgroundFamilial hypercholesterolemia is an inherited disease caused by mutations in the LDL receptor gene leading to severe hypercholesterolemia and atherosclerosis. The LDL receptor is predominantly expressed in the liver, making it a preferred target organ for somatic gene therapy. We recently isolated a new family of vectors based on adeno‐associated viruses (AAVs) isolated from nonhuman primates, which enable efficient and stable transgene expression following in vivo gene delivery to liver.MethodsTraditional vectors based on AAV serotype 2 and two novel AAVs from nonhuman primates, serotypes AAV7 and AAV8, were produced encoding for the human LDL receptor. Vectors were injected into the portal veins of LDL receptor deficient mice that were fed a high‐fat diet to achieve severe pretreatment hypercholesterolemia.ResultsAnimals receiving the novel AAV vectors realized nearly complete normalization of serum lipids and failed to develop the severe atherosclerosis that characterized the untreated animals; the AAV2 vector constructs demonstrated partial lipid correction and only a modest improvement in atherosclerosis.ConclusionsUsing vectors based on novel nonhuman primate AAVs, which provide advantages in terms of efficiency, we were able to achieve a long‐term correction of the metabolic defect in LDL receptor deficient mice. Copyright © 2004 John Wiley & Sons, Ltd.
Optimization of an siRNA‐expression system with an improved hairpin and its significant suppressive effects in mammalian cells
Authors: Miyagishi Makoto , Sumimoto Hidetoshi , Miyoshi Hiroyuki , Kawakami Yutaka , Taira Kazunari 
Published: March 2004

AbstractBackgroundRNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double‐stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector‐based siRNA‐expression systems that can induce RNAi in living cells.MethodsWe describe here a comparative analysis of various siRNA‐expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors.ResultsAs a result of the comparative analysis, we determined the following optimized siRNA‐expression system: U6 promoter‐driven hairpin‐type dsRNA with 21‐nt stem length, three to four mutations in the sense strand only, and the optimized 9‐nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA‐expression system with a tetracycline‐regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector‐mediated transfer of an siRNA‐expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five.ConclusionThe mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi. Copyright © 2004 John Wiley & Sons, Ltd.