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Scalable Culture and Cryopreservation of Human Embryonic Stem Cells on Microcarriers

Nie, Ying; Bergendahl, Veit; Hei, Derek J.; Jones, Jeffrey M.; Palecek, Sean P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2009 Português
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As a result of their pluripotency and potential for unlimited self-renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large-scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor-intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel-coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF-microcarriers was less than that on MEF-plates, the doubling time of hESCs on Matrigel-microcarriers was indistinguishable from that of hESCs expanded on Matrigel-coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here...

In Vitro Activity of CEM-101 against Streptococcus pneumoniae and Streptococcus pyogenes with Defined Macrolide Resistance Mechanisms ▿

McGhee, Pamela; Clark, Catherine; Kosowska-Shick, Klaudia M.; Nagai, Kensuke; Dewasse, Bonifacio; Beachel, Linda; Appelbaum, Peter C.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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CEM-101 had MIC ranges of 0.002 to 0.016 μg/ml against macrolide-susceptible pneumococci and 0.004 to 1 μg/ml against macrolide-resistant phenotypes. Only 3 strains with erm(B), with or without mef(A), had CEM-101 MICs of 1 μg/ml, and 218/221 strains had CEM-101 MICs of ≤0.5 μg/ml. CEM-101 MICs were as much as 4-fold lower than telithromycin MICs against all strains. For Streptococcus pyogenes, CEM-101 MICs ranged from 0.008 to 0.03 μg/ml against macrolide-susceptible strains and from 0.015 to 1 μg/ml against macrolide-resistant strains. Against erm(B) strains, erythromycin, azithromycin, and clarithromycin MICs were 32 to >64 μg/ml, while 17/19 strains had telithromycin MICs of 4 to 16 μg/ml; CEM-101 MICs were 0.015 to 1 μg/ml. By comparison, erm(A) and mef(A) strains had CEM-101 MICs of 0.015 to 0.5 μg/ml, clindamycin and telithromycin MICs of ≤1 μg/ml, and erythromycin, azithromycin, and clarithromycin MICs of 0.5 to >64 μg/ml. Pneumococcal multistep resistance studies showed that although CEM-101 yielded clones with higher MICs for all eight strains tested, seven of eight strains had clones with CEM-101 MICs that rose from 0.004 to 0.03 μg/ml (parental strains) to 0.06 to 0.5 μg/ml (resistant clones); for only one erm(B) mef(A) strain with a parental MIC of 1 μg/ml was there a resistant clone with a MIC of 32 μg/ml...

Rapid Whole Blood Bioassays using Microwave-Accelerated Metal-Enhanced Fluorescence

Aslan, Kadir
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
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The proof-of-principle demonstration of rapid whole blood bioassays based on microwave-accelerated metal-enhanced fluorescence (MAMEF) method using silver nanoparticle-deposited surfaces is presented. In this regard, spherical silver nanoparticles were deposited onto glass slides (silver nanoparticle films, SNFs) in a highly reproducible manner, which was assessed by optical absorption spectroscopy. Atomic force microscopy was employed to determine the size of the deposited silver nanoparticles. A model bioassay, based on the well-known interactions of biotinylated bovine serum albumin (b-BSA) and streptavidin was constructed on SNFs. The model bioassay was run at room temperature (metal-enhanced fluorescence (MEF)-based bioassay without microwave heating) for 60 minutes and with microwave heating (MAMEF-based bioassay) for 1 minute. In contrast to MEF-based bioassays that only allowed the use of samples in buffer solution, MAMEF-based bioassays afforded the use of whole blood samples. A lower detection limit of 1 nM and 0.01 nM for b-BSA was determined in MEF-based and MAMEF-based bioassays, respectively.

Further Activation of FLT3 Mutants by FLT3 Ligand

Zheng, Rui; Bailey, Emily; Nguyen, Bao; Yang, Xiaochuan; Piloto, Obdulio; Levis, Mark; Small, Donald
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Somatic mutations of FLT3 involving internal tandem duplication (ITD) of the juxtamembrane domainor point mutations in the kinase domain (TKD) appear to activate FLT3 in a FLT3 ligand (FL) - independent manner. To determine whether or not FLT3 mutants respond to FL for their activation, a FL-deficient (FL−/−) murine embryo fibroblast cell line (MEF) was established. Expression of FLT3/ITD and FLT3/TKD mutations in FL−/− MEF cells resulted in low levels of constitutive phosphorylation of FLT3.However, a more than 4-fold increase of FLT3 autophosphorylation was induced by exogenous FL. Rescue of endogenous FL expression in FL−/− MEF cells expressing FLT3 mutants led to more than a 3-fold increase of FLT3 phosphorylation. FL addition led to further activation of the FLT3 receptors and enhanced survival and/or decreased apoptosis in leukemia-derived cell lines and primary leukemic cells expressing FLT3 mutations. Functional studies revealed that exogenous FL promoted the colony-forming and recloning abilities of FLT3 mutant transduced primary bone marrow cells derived from FL−/− mice. Endogenous FL contributes in vivo to functional signaling through FLT3 as noted by the decreased survival of FL+/+ITD+/+ mice compared with FL−/−ITD+/+ mice. These data suggest that FL leads to further activation of FLT3 mutants and is especially important in light of recent findings of elevated FL levels in AML patients in response to chemotherapy.

TRPC1 expression and distribution in rat hearts

Huang, H.; Wang, W.; Liu, P.; Jiang, Y.; Zhao, Y.; Wei, H.; Niu, W.
Fonte: PAGEPress Publications Publicador: PAGEPress Publications
Tipo: Artigo de Revista Científica
Publicado em 29/12/2009 Português
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Transient receptor potential canonical (TRPC) proteins have been identified as a family of plasma membrane calcium-permeable channels. TRPC proteins can be activated by various stimuli and act as cellular sensors in mammals. Stretch-activated ion channels (SACs) have been proposed to underlie cardiac mechano-electric feedback (MEF), although the molecular entity of SAC remains unknown. There is evidence suggesting that transient receptor potential canonical 1 (TRPC1) is a stretch-activated ion channel. As a non-selective cation channel, TRPC1 may cause stretch-induced depolarization and arrhythmia and thus may contribute to the MEF of the heart. In this study, we examined the expression patterns of TRPC1 in detail at both the mRNA and protein levels in rat hearts. We isolated total RNA from the left and right atria, and the left and right ventricles, and detected TRPC1 mRNA in these tissues using reverse-transcriptase polymerase chain reaction (RT-PCR). To study the protein localization and targeting, we performed immunohistochemistry and immunofluorescence labeling with the antibody against TRPC1. TRPC1 was detected in the cardiomyocytes of the ventricle and atrium at both the mRNA and protein levels. The cell membrane and T-tubule showed strong fluorescence labeling in the ventricular myocytes. Purkinje cells...

Quantitative Comparison of Protein Surface Coverage on Glass Slides and Silver Island Films in Metal-Enhanced Fluorescence-based Biosensing Applications

Grell, Tsehai A.J.; Paredes, Eduardo; Das, Subha R.; Aslan, Kadir
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
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The use of Metal-Enhanced Fluorescence (MEF) phenomenon in fluorescence-based bioassays affords for increased sensitivity to be realized by incorporating metal nanoparticles onto planar surfaces. The close-range interactions of metal-fluorophores result in increased fluorescence emission from the bioassays, which in turn affords for the detection of target biomolecules at lower concentrations. Moreover, the use of silver nanoparticles increases the photostability of fluorophores improving the detectability of fluorescence emission under prolonged use of excitation light. Although numerous reports on MEF-based biosensing applications exist, the contribution of protein coverage on Silver Island Films (SIFs) on the increased fluorescence emission was never investigated. This work presents our findings on the quantitative comparison of protein surface coverage on SIFs and blank glass slides. In this regard, identical protein bioassay for a model protein (biotinylated bovine serum albumin, b-BSA) on these surfaces is constructed and the relative extent of protein surface coverage on SIFs and blank glass slides was determined using radio-labeled biomolecules. It was found that the total scintillation counts on SIFs and blank glass slides were similar for BSA concentrations ranging from 1 μM to 1 pM...

Adhesive forces in embryonic stem cell cultures

Blancas, Alicia A; Chen, Chi-Shuo; Stolberg, Sarah; McCloskey, Kara E
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
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Most cell culture systems grow and spread as contact-inhibited monolayers on flat culture dishes, but the embryonic stem cell (ESC) is one of the cell phenotypes that prefer to self-organize as tightly packed three-dimensional (3D) colonies. ESC also readily form 3D cell aggregates, called embryoid bodies (EB) that partially mimic the spatial and temporal processes of the developing embryo. Here, the rationale for ESC aggregation, rather than “spreading” on gelatin-coated or mouse embryonic fibroblast (MEF)-coated dishes, is examined through the quantification of the expression levels of adhesion molecules on ESC and the calculation of the adhesive forces on ESC. Modeling each ESC as a dodecahedron, the adhesive force for each ESC-ESC binding was found to be 9.1 × 105 pN, whereas, the adhesive force for ESC-MEF binding was found to be an order of magnitude smaller at 7.9 × 104 pN. We also show that E-cadherin is the dominating molecule in the ESC-ESC adhesion and blocking E-cadherin leads to a significant reduction in colony formation. Here, we mathematically describe the preference for ESC to self-assemble into ESC-ESC aggregates and 3D colonies, rather than to bind and spread on gelatin or MEF-coated dishes, and have shown that these interactions are predominantly due to E-cadherin expression on ESC.

Feeder Cells Support the Culture of Induced Pluripotent Stem Cells Even after Chemical Fixation

Yue, Xiao-Shan; Fujishiro, Masako; Nishioka, Chieko; Arai, Takashi; Takahashi, Eiki; Gong, Jian-Sheng; Akaike, Toshihiro; Ito, Yoshihiro
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 02/03/2012 Português
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Chemically fixed mouse embryonic fibroblasts (MEFs), instead of live feeder cells, were applied to the maintenance of mouse induced pluripotent stem (miPS) cells. Formaldehyde and glutaraldehyde were used for chemical fixation. The chemically fixed MEF feeders maintained the pluripotency of miPS cells, as well as their undifferentiated state. Furthermore, the chemically fixed MEF feeders were reused several times without affecting their functions. These results indicate that chemical fixation can be applied to modify biological feeders chemically, without losing their original functions. Chemically fixed MEF feeders will be applicable to other stem cell cultures as a reusable extracellular matrix candidate that can be preserved on a long-term basis.

Distance-Dependent Metal-Enhanced Intrinsic Fluorescence of Proteins Using Polyelectrolyte Layer-by-Layer Assembly and Aluminum Nanoparticles

Akbay, Nuriye; Lakowicz, Joseph R.; Ray, Krishanu
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Previously reported studies indicate that aluminum nanostructured substrates can potentially find widespread use in metal-enhanced fluorescence (MEF) applications particularly in the UV or near-UV spectral region toward label-free detection of biomolecules. MEF largely depends on several factors, such as chemical nature, size, shape of the nanostructure and its distance from the fluorophore. A detailed understanding of the MEF and its distance-dependence are important for its potential application in biomedical sensing. Our goal is to utilize intrinsic protein fluorescence for label-free binding assays. This is made possible by the use of metallic nanostructures which provide localized excitation and enhanced fluorescence of UV fluorophores and will also provide a way to separate the surface-bound proteins from the bulk samples. We evaluated varied probe distances from plasmonic nanostructures by the well-established layer-by-layer (LbL) technique. The investigated proteins were adsorbed on different numbers of alternate layers of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). Bovine serum albumin (BSA) was electrostatically attached to the positively charged PAH layer, and goat and rabbit IgG were attached to negatively charged PSS layer. We obtained a maximum of a ~ 9 fold increase in fluorescence intensity from BSA at a distance of ~9 nm from the Al nanostructured surface. Approximately 6- and 7- fold increases were observed from goat and rabbit IgG at a distance of ~8 nm...

Early Maternal Psychosocial Factors Are Predictors for Adolescent Caries

Nelson, S.; Lee, W.; Albert, J.M.; Singer, L.T.
Fonte: SAGE Publications Publicador: SAGE Publications
Tipo: Artigo de Revista Científica
Publicado em /09/2012 Português
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Few studies have investigated the role of early maternal enabling and psychosocial factors on subsequent adolescent caries experience. In this retrospective cohort study of 224 adolescents, we hypothesized that the causal pathway between early maternal enabling factors (education, cognitive abilities, psychological distress) and adolescent caries experience (DMFT) at age 14 yrs is mediated by maternal psychosocial factors (stress, coping, social support) and adolescent dental behavior/access. Maternal data on socio-demographic, medical, and psychosocial variables were measured when the child was 3, 8, and 14 yrs old. A structural equations model (SEM) evaluated the causal pathway, with latent variables for maternal enabling factors (MEF), stress, coping, and social support. Poor MEF was associated with increased stress and poorer coping when the child was 3 yrs old, which in turn affected adolescent dental visits and behavior. Greater social support at child’s age 3 was directly associated with lower mean DMFT in adolescence. Maternal psychosocial factors measured when children are young are important mediators for adolescent mean DMFT, but these factors measured when children are adolescents are not. Better early and concurrent MEF...

RNase L Triggers Autophagy in Response to Viral Infections

Chakrabarti, Arindam; Ghosh, Prabar Kumar; Banerjee, Shuvojit; Gaughan, Christina; Silverman, Robert H.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2012 Português
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Autophagy is a programmed homeostatic response to diverse types of cellular stress that disposes of long-lived proteins, organelles, and invading microbes within double-membraned structures called autophagosomes. The 2′,5′-oligoadenylate/RNase L system is a virus-activated host RNase pathway that disposes of or processes viral and cellular single-stranded RNAs. Here we report that activation of RNase L during viral infections induces autophagy. Accordingly, infections with encephalomyocarditis virus or vesicular stomatitis virus led to higher levels of autophagy in wild-type mouse embryonic fibroblasts (MEF) than in RNase L-null MEF. Similarly, direct activation of RNase L with a 2′,5′-oligoadenylate resulted in p62(SQSTM1) degradation, LC3BI/LC3BII conversion, and appearance of autophagosomes. To determine the effect of RNase L-mediated autophagy on viral replication, we compared viral yields in wild-type and RNase L-null MEF in the absence or presence of either chemical inhibitors of autophagy (bafilomycin A1 or 3-methyladenine) or small interfering RNA (siRNA) against ATG5 or beclin-1. At a low multiplicity of infection, induction of autophagy by RNase L during the initial cycle of virus growth contributed to the suppression of virus replication. However...

Culture Conditions Affect Cardiac Differentiation Potential of Human Pluripotent Stem Cells

Ojala, Marisa; Rajala, Kristiina; Pekkanen-Mattila, Mari; Miettinen, Marinka; Huhtala, Heini; Aalto-Setälä, Katriina
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 31/10/2012 Português
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Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are capable of differentiating into any cell type in the human body and thus can be used in studies of early human development, as cell models for different diseases and eventually also in regenerative medicine applications. Since the first derivation of hESCs in 1998, a variety of culture conditions have been described for the undifferentiated growth of hPSCs. In this study, we cultured both hESCs and hiPSCs in three different culture conditions: on mouse embryonic fibroblast (MEF) and SNL feeder cell layers together with conventional stem cell culture medium containing knockout serum replacement and basic fibroblast growth factor (bFGF), as well as on a Matrigel matrix in mTeSR1 medium. hPSC lines were subjected to cardiac differentiation in mouse visceral endodermal-like (END-2) co-cultures and the cardiac differentiation efficiency was determined by counting both the beating areas and Troponin T positive cells, as well as studying the expression of OCT-3/4, mesodermal Brachyury T and NKX2.5 and endodermal SOX-17 at various time points during END-2 differentiation by q-RT-PCR analysis. The most efficient cardiac differentiation was observed with hPSCs cultured on MEF or SNL feeder cell layers in stem cell culture medium and the least efficient cardiac differentiation was observed on a Matrigel matrix in mTeSR1 medium. Further...

Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells

Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A.; Chock, P. Boon
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signalling via a Gαq/11/PLCβ/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gαq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analysis. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in Gαq/11 double knockout MEF cells. Although CTGF expression is regulated by TGFβ, rPMT did not activate TGFβ pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly...

Adaptation of Human Pluripotent Stem Cells to Feeder-Free Conditions in Chemically Defined Medium with Enzymatic Single-Cell Passaging

Stover, Alexander E.; Schwartz, Philip H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2011 Português
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This protocol describes the culture of human pluripotent stem cells (PSCs) under feeder-free conditions in a commercially available, chemically defined, growth medium, using Matrigel as a substrate and the enzyme solution Accutase for single-cell passaging. This system is strikingly different from traditional PSC culture, where the cells are co-cultured with feeder cells and in medium containing serum replacement. PSCs cultured in this new system have a different morphology than those cultured on feeder cells but retain their characteristic pluripotency. This feeder-free PSC culture system is conceptually similar to feeder-free systems that use mouse embryonic fibroblast (MEF)-conditioned medium (MEF-CM) and Matrigel substratum. Instead of MEF-CM, a very complex and undefined medium, this new system uses StemPro SFM, a chemically defined medium that permits enzymatic passaging with Accutase to disaggregate the colonies into single cells. Accutase passaging has been used in conjunction with Stempro in our hands for 20+ passages without detectable karyotypic abnormalities. We will also review techniques for adapting cultures previously grown on MEFs, routine passaging of the cells, and cryopreservation.

INK4a/ARF limits the expansion of cells suffering from replication stress

Monasor, Angela; Murga, Matilde; Lopez-Contreras, Andres J.; Navas, Carolina; Gomez, Gonzalo; Pisano, David G.; Fernandez-Capetillo, Oscar
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
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Replication stress (RS) is a source of DNA damage that has been linked to cancer and aging, which is suppressed by the ATR kinase. In mice, reduced ATR levels in a model of the ATR-Seckel syndrome lead to RS and accelerated aging. Similarly, ATR-Seckel embryonic fibroblasts (MEF) accumulate RS and undergo cellular senescence. We previously showed that senescence of ATR-Seckel MEF cannot be rescued by p53-deletion. Here, we show that the genetic ablation of the INK4a/Arf locus fully rescues senescence on ATR mutant MEF, but also that induced by other conditions that generate RS such as low doses of hydroxyurea or ATR inhibitors. In addition, we show that a persistent exposure to RS leads to increased levels of INK4a/Arf products, revealing that INK4a/ARF behaves as a bona fide RS checkpoint. Our data reveal an unknown role for INK4a/ARF in limiting the expansion of cells suffering from persistent replication stress, linking this well-known tumor suppressor to the maintenance of genomic integrity.

GSK-3 and lysosomes meet in Alzheimer’s disease

Avrahami, Limor; Eldar-Finkelman, Hagit
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
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Aberrant regulation of glycogen synthase kinase-3 (GSK-3) is implicated in Alzheimer’s disease (AD), but the mechanisms involved remain elusive. Our recent study shows that GSK-3 impairs lysosomal acidification and that inhibition of GSK-3 re-acidified lysosomes in brains of AD mice. This effect was accompanied by reductions in β-amyloid pathology and amelioration of cognitive deficits. Presenilin-1 (PS1) is an essential factor in lysosomal acidification. To determine whether the inhibition of GSK-3 restores lysosomal malfunction caused by dysfunctional PS1, we treated MEF cells deficient in presenilin proteins (MEF-PS1/2−/−) with a selective substrate competitive GSK-3 inhibitor, L803-mts. L803-mts enhanced the acidic lysosomal pool in MEF-PS1/2−/− cells and increased levels of activated cathepsin D in the lysosomes. We conclude that GSK-3 and PS1 operate via similar mechanisms to disrupt lysosomal acidification. Importantly, these data indicate that GSK-3 inhibitors have potential in treatment of conditions associated with defective PS1.

Connective Tissue Growth Factor (CTGF) Expression Modulates Response to High Glucose

James, Leighton R.; Le, Catherine; Doherty, Heather; Kim, Hyung-Suk; Maeda, Nobuyo
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 12/08/2013 Português
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Connective tissue growth factor (CTGF) is an important mediator of fibrosis; emerging evidence link changes in plasma and urinary CTGF levels to diabetic kidney disease. To further ascertain the role of CTGF in responses to high glucose, we assessed the consequence of 4 months of streptozotocin-induced diabetes in wild type (+/+) and CTGF heterozygous (+/−) mice. Subsequently, we studied the influence of glucose on gene expression and protein in mice embryonic fibroblasts (MEF) cells derived from wildtype and heterozygous mice. At study initiation, plasma glucose, creatinine, triglyceride and cholesterol levels were similar between non-diabetic CTGF+/+ and CTGF+/− mice. In the diabetic state, plasma glucose levels were increased in CTGF+/+ and CTGF+/− mice (28.2 3.3 mmol/L vs 27.0 3.1 mmol/L), plasma triglyceride levels were lower in CTGF+/− mice than in CTGF+/+ (0.7 0.2 mmol/L vs 0.5 0.1 mmol/L, p<0.05), but cholesterol was essentially unchanged in both groups. Plasma creatinine was higher in diabetic CTGF+/+ group (11.7±1.2 vs 7.9±0.6 µmol/L p<0.01), while urinary albumin excretion and mesangial expansion were reduced in diabetic CTGF+/− animals. Cortices from diabetic mice (both CTGF +/+ and CTGF +/−) manifested higher expression of CTGF and thrombospondin 1 (TSP1). Expression of nephrin was reduced in CTGF +/+ animals; this reduction was attenuated in CTGF+/− group. In cultured MEF from CTGF+/+ mice...

Fluorescence enhancement using silver-gold nanocomposite substrates

Choudhury, Sharmistha Dutta; Badugu, Ramachandram; Ray, Krishanu; Vanam, Prasanna Sai; Lakowicz, Joseph R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 09/02/2012 Português
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Metal-enhanced fluorescence (MEF) is a newly emerging phenomenon in which the near-field interactions of fluorophores with the plasmons in metallic nanostructures can lead to substantial fluorescence enhancements. In the present study, we have investigated the use of silver-gold nanocomposite (Ag-Au-NC) structures, prepared by the galvanic replacement reaction of silver with gold, as plasmonic substrates for MEF. We have observed significant enhancement in the fluorescence intensities and decrease in the fluorescence lifetimes of two commonly used dyes, ATTO655 and Cy5, using the fabricated Ag-Au-NC substrates. Interestingly, the fluorescence enhancement depends on the amount of residual silver present in the substrates after the galvanic replacement reaction. Our results show that the galvanic replacement reaction is a very facile and powerful route to prepare Ag-Au-NC substrates that can be suitable for various MEF based applications.

T-cell Intracellular Antigen (TIA)-Proteins Deficiency in Murine Embryonic Fibroblasts Alters Cell Cycle Progression and Induces Autophagy

Sánchez-Jiménez, Carmen; Izquierdo, José M.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 24/09/2013 Português
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Mice lacking either T-cell intracellular antigen 1 (TIA1) or TIA1 related/like protein (TIAR/TIAL1) show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF). Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO) MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together...

Transgenic expression of the human LEDGF/p75 gene relieves the species barrier against HIV-1 infection in mouse cells

Tada, Takuya; Kadoki, Motohiko; Liu, Yang; Tokunaga, Kenzo; Iwakura, Yoichiro
Fonte: Frontiers Media S.A. Publicador: Frontiers Media S.A.
Tipo: Artigo de Revista Científica
Publicado em 17/12/2013 Português
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Attempts to create mouse models for AIDS have been hampered by species barriers in HIV-1 infection. We previously showed that the nuclear accumulation of HIV-1 preintegration complex (PIC) was suppressed in mouse cells. Lens epithelium-derived growth factor (LEDGF/p75) is a host factor identified as a binding partner of integrase (IN), and has been suggested to be involved in promoting viral integration by tethering PIC to the chromatin, which are observed as nuclear accumulation of IN by LEDGF/p75. Therefore, we here hypothesized that this host factor might act as one of the species-specific barriers in mouse cells. We generated transgenic (Tg) mice that constitutively express human (h) LEDGF/p75. The GFP-fused IN was efficiently accumulated into the nucleus of hLEDGF/p75 expressing Tg mouse embryonic fibroblast (MEF) cells in contrast to the control MEF cells. Importantly, hLEDGF/p75 Tg MEF cells were significantly more susceptible to HIV-1 infection. These results suggest that LEDGF/p75 is one of the host factors that constitute species barrier against HIV-1 in mouse cells.