bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering bone‐like tissue in vitro using human bone marrow stem cells and silk scaffolds
Authors: Meinel Lorenz , Karageorgiou Vassilis , Hofmann Sandra , Fajardo Robert , Snyder Brian , Li Chunmei , Zichner Ludwig , Langer Robert , Vunjak‐Novakovic Gordana , Kaplan David L. 
Published: August 2004

AbstractPorous biodegradable silk scaffolds and human bone marrow derived mesenchymal stem cells (hMSCs) were used to engineer bone‐like tissue in vitro. Two different scaffolds with the same microstructure were studied: collagen (to assess the effects of fast degradation) and silk with covalently bound RGD sequences (to assess the effects of enhanced cell attachment and slow degradation). The hMSCs were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, seeded on scaffolds, and cultured for up to 4 weeks. Histological analysis and microcomputer tomography showed the development of up to 1.2‐mm‐long interconnected and organized bonelike trabeculae with cuboid cells on the silk‐RGD scaffolds, features still present but to a lesser extent on silk scaffolds and absent on the collagen scaffolds. The X‐ray diffraction pattern of the deposited bone corresponded to hydroxyapatite present in the native bone. Biochemical analysis showed increased mineralization on silk‐RGD scaffolds compared with either silk or collagen scaffolds after 4 weeks. Expression of bone sialoprotein, osteopontin, and bone morphogenetic protein 2 was significantly higher for hMSCs cultured in osteogenic than control medium both after 2 and 4 weeks in culture. The results suggest that RGD‐silk scaffolds are particularly suitable for autologous bone tissue engineering, presumably because of their stable macroporous structure, tailorable mechanical properties matching those of native bone, and slow degradation. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 25–34, 2004
Increased GADD gene expression in human colon epithelial cells exposed to deoxycholate
Authors: Scott David W. , Mutamba Sophia , Hopkins Robin G. , Loo George 
Published: August 2004

AbstractThe colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress‐response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage‐inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis‐related genes in DOC‐treated HCT‐116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene‐specific relative RT‐PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC‐treated HCT‐116 colonocytes, which express wild‐type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT‐15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin‐D (Act‐D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC‐induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre‐existing, and not just newly synthesized, mRNA. © 2005 Wiley‐Liss, Inc.
Caspase‐3 inhibits the growth of breast cancer cells independent of protease activity
Authors: Faraglia Beatrice , Bonsignore Alessia , Scaldaferri Franco , Boninsegna Alma , Cittadini Achille , Mancuso Cesare , Sgambato Alessandro 
Published: August 2004

AbstractIn this study, the MCF‐7 breast cancer cells that lack caspase‐3 were transfected with a wild type (WT) or mutant caspase‐3 cDNA. Expression of the WT, but not of the mutant, caspase‐3 was associated with increased caspase activity and susceptibility to staurosporine (STS)‐induced apoptosis. Both derivatives displayed inhibition of cell growth compared with vector control cells. Growth inhibition was associated with increased expression of the cyclin dependent kinase (CDK) inhibitor p27Kip1 in the WT, but not in the mutant caspase‐3 expressing cells. Cyclin D1 expression level was not affected by caspase‐3 expression. Phosphorylation of the Akt protein was decreased in both WT and mutant caspase transfected cells, although Akt expression level remained unchanged. These results suggest that caspase‐3 might have biological functions independent of its protease activity and that its loss might contribute to tumor development by increasing the growth potential of cancer cells. © 2004 Wiley‐Liss, Inc.
PKC mediates cyclic stretch‐induced cardiac hypertrophy through Rho family GTPases and mitogen‐activated protein kinases in cardiomyocytes
Authors: Pan Jing , Singh Ugra S. , Takahashi Toshiyuki , Oka Yoshitomo , Palm‐Leis Ants , Herbelin Bradley S. , Baker Kenneth M. 
Published: August 2004

AbstractSignaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch‐induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho‐guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12‐myristate 13‐acetate, indicating that phorbol ester‐sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral‐mediated gene transfer of wild‐type (WT) and dominant‐negative (DN) mutants of PKCα and δ, we found that stretch‐induced activation of Rho GTPases and phosphorylation of Rho‐GDI were mainly regulated by PKCα. PKCδ was involved in regulation of the activation of Rac1. Stretch‐induced increases in [3H]‐leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCα and δ, suggesting that PKCα and δ are both required in stretch‐induced hypertrophy, through Rho GTPases‐mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen‐activated protein (MAP) kinases. Stretch‐stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCα and δ, and that of MKK3/p38 inhibited by DN PKCδ. The phosphorylation of ERK and JNK induced by overexpression of WT PKCα, and the phosphorylation of p38 induced by WT PKCδ, were regulated by Rho GTPases. This study represents the first evidence that PKCα and δ are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch‐induced hypertrophic process. © 2004 Wiley‐Liss, Inc.
Steroids dilate nuclear pores imaged with atomic force microscopy
Authors: Shahin Victor , Albermann Lars , Schillers Hermann , Kastrup Lilian , Schäfer Claudia , Ludwig Yvonne , Stock Christian , Oberleithner Hans 
Published: August 2004

AbstractMacromolecules that act in the cell nucleus must overcome the nuclear envelope (NE). This barrier between cytosol and the nucleus is perforated by nuclear pore complexes (NPCs) that serve as translocation machineries. We visualized the translocation process at the NE surface, applying a nanotechnical approach using atomic force microscopy (AFM). In order to initiate protein targeting to NPCs, dexamethasone (dex) was injected into Xenopus laevis oocytes. Dex is a synthetic steroid of great therapeutic relevance that specifically binds to glucocorticoid receptors and thus triggers an intracellular signal cascade involving the cell nucleus. Ninety and 180 sec after dex injection cell nuclei were isolated, the NEs spread on glass and scanned with AFM. With single molecule resolution we observed that dex initiated proteins (DIPs) first bind to NPC‐free areas of the outer nuclear membrane. This causes NPCs to dilate. Then, in a second step, DIPs attach directly to NPCs and enter the dilated central channels. DIPs accumulation and NPC conformational changes were blocked by RU486, a specific glucocorticoid receptor antagonist. In conclusion, dex exposure induces NPC dilation. NPCs change conformation already prior to transport. The NPC dilation signal is most likely transmitted through NPC associated filaments or yet unknown structures in the NE outer membrane. NPC dilation could have significant impact on nuclear targeting of therapeutic macromolecules. © 2004 Wiley‐Liss, Inc.
Phosphoinositide 3‐kinase/Akt involvement in arsenic trioxide resistance of human leukemia cells
Authors: Tabellini Giovanna , Cappellini Alessandra , Tazzari Pier Luigi , Falà Federica , Billi Anna Maria , Manzoli Lucia , Cocco Lucio , Martelli Alberto M. 
Published: August 2004

AbstractThe purpose of this study was to evaluate the possible involvement of the phosphoinositide 3‐kinase (PI3K)/Akt survival pathway in determining resistance to arsenic trioxide (As2O3)‐induced apoptosis. We employed a HL60 cell clone (HL60AR) with a constitutively active PI3K/Akt survival pathway, as well as U937 and K562 cells. In addition, we used parental (PT) HL60 cells overexpressing a constitutively active Akt. Selective pharmacological inhibitors of the PI3K/Akt axis (LY294002, wortmannin) were employed to influence the sensitivity to As2O3. While HL60PT cells were sensitive to 2.5 μM As2O3 and died of apoptosis, HL60AR cells were resistant up to 5 μM As2O3. Treatment with either LY294002 or wortmannin lowered resistance of HL60AR cells to As2O3. Also in U937 and K562 cells, inhibitors of the PI3K/Akt axis caused a decrease in As2O3 resistance. Overexpression of constitutively active Akt in HL60PT cells caused the induction of resistance to 2.5 μM As2O3. Conversely, forced expression of a dominant negative Akt in HL60AR cells resulted in a decrease in As2O3 resistance. Moreover, HL60 cell resistance to 2.5 μM As2O3 could be significantly reduced by incubation with SN50, a peptide inhibitor selective for the NF‐κB transcription factor. Taken together our findings suggest that a constitutive activation of the PI3K/Akt pathway, which is increasingly detected in some types of acute myeloid leukemia, may contribute to As2O3 resistance, most likely through NF‐κB activation. Selective pharmacological inhibitors of this survival pathway, as well as of NF‐κB, might be usefully employed in the future to reverse resistance to this treatment. © 2004 Wiley‐Liss, Inc.
Intraocular gene transfer of pigment epithelium‐derived factor rescues photoreceptors from light‐induced cell death
Authors: Imai Daisuke , Yoneya Shin , Gehlbach Peter L. , Wei Lisa L. , Mori Keisuke 
Published: August 2004

AbstractIn this study, we investigated whether intraocular gene transfer of pigment epithelium‐derived factor (PEDF) ameliorates the extent of light‐induced photoreceptor cell death. Lewis rats received intravitreous injection of 3 × 109 particles of adenoviral vector expressing PEDF (AdPEDF.11) in one eye and 3 × 109 particles of empty adenoviral vector (AdNull.11) in the contralateral eye. The rats were then dark‐adapted for 3 days after which they were continuously exposed to fluorescent light (2,500 lux) for 0, 6, 24, 96, and 168 h. Both eyes were then enucleated and processed for morphometric analysis. Cell death in the retina was examined using TUNEL staining with a propidium iodide counterstain. The photoreceptor cell counts in each of the three groups were significantly different (P < 0.001). Eyes that received intravitreous injection of AdNull.11 or no injection showed a greater number of pyknotic photoreceptor cells and a reduced photoreceptor cell density as compared to eyes treated with intravitreous AdPEDF.11 injection. AdNull.11 treated eyes showed a lesser but still significant protection of photoreceptor cells when compared to untreated eyes. Fewer TUNEL‐positive photoreceptor cells were present in AdPEDF.11 treated eyes than in AdNull.11 treated or untreated eyes (P = 0.004). The amplitudes of the ERG a‐wave, b‐wave, and oscillatory potentials (OPs) were increased significantly by treatment (P < 0.05). These data suggest that adenovirus vector‐mediated intraocular expression of PEDF significantly increases photoreceptor cell survival following excessive light exposure. Neuroprotection may result from inhibition of light‐induced apoptotic processes. This study provides proof of concept for a gene transfer approach to modulating retinal cell death resulting from photo‐oxidative damage and supports the hypothesis that gene transfer of PEDF is broadly applicable to modulating apoptosis in the retina. © 2004 Wiley‐Liss, Inc.
Nuclear trafficking of metallothionein requires oxidation of a cytosolic partner
Authors: Takahashi Yukihisa , Ogra Yasumitsu , Suzuki Kazuo T. 
Published: August 2004

AbstractThe present study revealed the mechanism underlying the nuclear trafficking of metallothionein (MT). Nuclear localization of MT in digitonin‐permeabilized BALB 3T3 cells was enhanced in the presence of a cytosolic factor added as a rat red blood cell lysate by oxidation with H2O2 in a dose‐dependent manner, but inhibited with excess glutathione. A cytosolic partner was assumed to bind MT and retain it in the cytoplasm, and its oxidation can mobilize MT to the nuclei on cellular oxidation. Pre‐treatment of nuclei with H2O2 did not enhance the localization, and MT that had been localized in the nuclei was washed out, indicating that MT is in the nuclei as a result of a higher rate of uptake by the nuclei than the rate of diffusion from the nuclei. Nuclear localization of lysozyme and nuclear localization signal (NLS)‐bearing allophycocyanin were not enhanced by the oxidation in the presence of cytosolic factor, suggesting that the nuclear traffic occurring on oxidation is specific to MT. Moreover, when cells were arrested the cell cycle at the S phase, MT was localized in the nuclei in response to coincidental generation of a feeble reactive oxygen species (ROS). These observations suggest that MT comes localized in the nuclei on the sensing of intracellular oxidation, whereby a cytosolic partner specific to MT comes oxidized as a cargo system, MT being localized as a result of enhanced uptake in the nuclei and re‐localized in the cytoplasm diffusely. Nuclear MT was proposed to protect the nuclei from the oxidation occurring with progression of the cell cycle. © 2004 Wiley‐Liss, Inc.
Anti‐inflammatory effect of heme oxygenase 1: Glycosylation and nitric oxide inhibition in macrophages
Authors: Lin Hui‐Yi , Shen Shing‐Chuan , Chen Yen‐Chou 
Published: August 2004

AbstractFlavonoids including the aglycones, hesperetin (HT; 5,7,3′‐trihydroxy‐4′‐methoxy‐flavanone), and naringenin (NE; 5,7,4′‐trihydroxy flavanone) and glycones, hesperidin (HD; 5,7,3′‐trihydroxy‐4′‐methoxy‐flavanone 7‐rhamnoglucoside) and naringin (NI; 5,7,4′‐trihydroxy flavanone 7‐rhamno glucoside), were used to examine the importance of rutinose at C7 on the inhibitory effects of flavonoids on lipopolysaccharide (LPS)‐induced nitric oxide production in macrophages. Both HT and NE, but not their respective glycosides HD and NI, induced heme oxygenase 1 (HO‐1) protein expression in the presence or absence of LPS and showed time and dose‐dependent inhibition of LPS‐induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW264.7, J774A.1, and thioglycolate‐elicited peritoneal macrophages. Additive inhibitory effect of an HO‐1 inducer hemin and NE or NI on LPS‐induced NO production and iNOS expression was identified, and HO enzyme inhibitor tin protoporphyrin (SnPP) attenuated the inhibitory effects of HT, NE, and hemin on LPS‐induced NO production. Both NE and HT showed no effect on iNOS mRNA and protein stability in RAW264.7 cells. Removal of rutinose at C7 of HD and NI by enzymatic digestion using hesperidinase (HDase) and naringinase (NIase) produce inhibitory activity on LPS‐induced NO production, according to the production of the aglycones, HT and NE, by high‐performance liquid chromatography (HPLC) analysis. Furthermore, the amount of NO produced by LPS or lipoteichoic acid (LTA) was significantly reduced in HO‐1‐overexpressing cells (HO‐1/RAW264.7) compared to that in parental cells (RAW264.7). Results of the present study provide scientific evidence to suggest that rutinose at C7 is a negative moiety in flavonoid inhibition of LPS‐induced NO production, and that HO‐1 is involved in the inhibitory mechanism of flavonoids on LPS‐induced iNOS and NO production. © 2004 Wiley‐Liss, Inc.
Mitogen‐activated protein kinases and asthma
Authors: Pelaia Girolamo , Cuda Giovanni , Vatrella Alessandro , Gallelli Luca , Caraglia Michele , Marra Monica , Abbruzzese Alberto , Caputi Mario , Maselli Rosario , Costanzo Francesco S. , Marsico Serafino A. 
Published: August 2004

AbstractMitogen‐activated protein kinases (MAPKs) are evolutionary conserved enzymes which play a key role in signal transduction mediated by cytokines, growth factors, neurotransmitters and various types of environmental stresses. In the airways, these extracellular stimuli elicit complex inflammatory and structural changes leading to the typical features of asthma including T cell activation, eosinophil and mast cell infiltration, as well as bronchial hyperresponsiveness and airway remodelling. Because MAPKs represent an important point of convergence for several different signalling pathways, they affect multiple aspects of normal airway function and also significantly contribute to asthma pathophysiology. Therefore, this review focuses on the crucial involvement of MAPKs in asthma pathogenesis, thus also discussing their emerging role as molecular targets for anti‐asthma drugs. © 2004 Wiley‐Liss, Inc.
Developmental variability during early embryonic development of zebra fish, Danio rerio
Authors: Schmidt Kai , Starck J. Matthias 
Published: August 2004

AbstractEarly vertebrate embryos pass through a period of remarkable morphological similarity. Possible causes for such similarity of early embryos include modularity, developmental constraints, stabilizing selection, canalization, and exhausted genetic variability. Supposedly, each process creates different patterns of variation and covariation of embryonic traits. We study the patterns of variation of the embryonic phenotype to test ideas about possible evolutionary mechanisms shaping the early embryonic development. We use the zebra fish, Danio rerio, as a model organism and apply repeated measures of individual embryos to study temporal changes of phenotypic variability during development. In particular, we are looking at the embryonic development from 12 hours post fertilization until 27 hours post fertilization. During this time period, the development of individual embryos is documented at hourly intervals. We measured maximum diameter of the eye, length of embryo, number of somites, inclination of somites, and the yolk size (as a maternal effect). The coefficient of variation (CV) was used as a measure of variability that was independent of size. We used a principal component analysis for analysis of morphological integration. The experimental setup kept environment × genotype interactions constant. Nongenetic parental contributions had no significant effects on interindividual variability. Thus all observed phenotypic variation was based on additive genetic variance and error variance. The average CV declined from 14% to 7.7%. The decline of the CV was in particular expressed during 15–19 h post fertilization and occurred in association with multiple correlations among embryonic traits and a relatively high degree of morphological integration. We suggest that internal constraints determine the patterns of variability during early embryonic development of zebra fish. J. Exp. Zool. (Mol. Dev. Evol.) 302B:000000, 2004. © 2004 Wiley‐Liss, Inc.
Developmental fate of the mandibular mesoderm in the lamprey, Lethenteron japonicum: Comparative morphology and development of the gnathostome jaw with special reference to the nature of the trabecula cranii
Authors: Kuratani Shigeru , Murakami Yasunori , Nobusada Yoshiaki , Kusakabe Rie , Hirano Shigeki 
Published: August 2004

AbstractThe vertebrate jaw is a mandibular‐arch derivative, and is regarded as the synapomorphy that defines the gnathostomes. Previous studies (Kuratani et al., Phil. Trans. Roy. Soc. 356:15, 2001; Shigetani et al., Science 296:1319, 2002) have suggested that the oral apparatus of the lamprey is derived from both the mandibular and premandibular regions, and that the jaw has arisen as a secondary narrowing of the oral patterning mechanism into the mandibular‐arch domain. The heterotopy theory of jaw evolution states that the lamprey upper lip is a premandibular element, leaving further questions unanswered as to the homology of the trabecula in the lamprey and gnathostomes, and to the morphological nature of the muscles in the upper lip. Using focal injection of vital dyes into the cheek process core of lamprey embryos, we found that the upper lip muscle and trabecula are both derived from mandibular mesoderm. Secondary movement of the muscle primordium is also evident when the expression of the early muscle marker gene, LjMA2, is visualized. A nerve‐fiber labeling study revealed that the upper lip muscle‐innervating neurons are located in the rostral part of the brain stem, where the trigeminal motor nuclei are not found in gnathostomes. We conclude that the lamprey upper lip is composed of premandibular ectomesenchyme and a lamprey‐specific muscle component derived from the mandibular mesoderm innervated by lamprey‐specific motoneurons. Furthermore, the lamprey trabecula is most likely equivalent to a mesodermally derived neurocranial element, similar to the parachordal element in gnathostomes, rather than to the neural‐crest‐derived prechordal element. J. Exp. Zool. (Mol. Dev. Evol.) 302B:000–000, 2004. © 2004 Wiley‐Liss, Inc.
Internal energy and fragmentation of ions produced in electrospray sources
Authors: Gabelica Valérie , Pauw Edwin De 
Published: August 2004

AbstractThis review addresses the determination of the internal energy of ions produced by electrospray ionization (ESI) sources, and the influence of the internal energy on analyte fragmentation. A control of the analyte internal energy is crucial for several applications of electrospray mass spectrometry, like structural studies, construction of reproducible and exportable spectral libraries, analysis of non‐covalent complexes. Sections II and III summarize the Electrospray mechanisms and source design considerations which are relevant to the problem of internal energy, and Section IV gives an overview of the inter‐relationships between ion internal energy, reaction time scale, and analyte fragmentation. In these three sections we tried to make the most important theoretical elements understandable by all ESI users, and their understanding requires a minimal background in physical chemistry. We then present the different approaches used to experimentally determine the ion internal energy, as well as various attempts in modeling the internal energy uptake in electrospray sources. Finally, a tentative comparison between electrospray and other ionization sources is made. As the reader will see, although many reports appeared on the subject, the knowledge in the field of internal energy of ions produced by soft ionization sources is still scarce, because of the complexity of the system, and this is what makes this area of research so interesting. The last section presents some perspectives for future research. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:566–587, 2005