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Distortion of magnetic evoked fields and surface potentials by conductivity differences at boundaries in brain tissue.

Huang, J C; Nicholson, C; Okada, Y C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1990 Português
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We investigated the conditions under which inhomogeneity in electrical conductivity may significantly modify the magnetic evoked field (MEF) due to primary currents (i.e., neuronal currents) in the brain. In the case of an isolated turtle cerebellum immersed in a large bath of physiological saline, our theoretical analysis showed the cerebellar surface to significantly enhance the MEF due to a primary current, by a factor of as much as two, for experimentally determined values of the conductivities of the cerebellar tissue and saline. A further parametric investigation of the conductivity effect revealed that conductivity boundaries may significantly modify the MEF due to neuronal currents located within 1 mm of a conductivity boundary, as would be the case for active neurons near an edema, an anoxic fringe such as might occur during stroke, or a ventricle in the human head. For a stationary neural source, conductivity boundaries may modify the magnitude of its MEF without affecting its temporal waveform. However, this boundary effect was found to be small for a model geometry locally approximating cortical sources in a sulcus or a fissure, where the boundary effects from adjacent sulcal walls tend to cancel each other.

Complement activity in middle ear effusions.

Bernstein, J M; Schenkein, H A; Genco, R J; Bartholomew, W R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1978 Português
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Evidence for complement utilization in middle ear fluids (MEF) from patients with otitis media with effusion was sought. It was found that cleavage products of C3, C4 and Factor B could be demonstrated immunochemically in MEF, and that native C3 was present in much lower concentrations than other proteins, relative to their serum concentrations. Haemolytic assays for C1-C5 showed that early complement components are inactivated in MEF. Potential mechanisms for complement utilization in MEF are discussed.

A comparison of the migration patterns of normal and malignant cells in two assay systems.

Varani, J.; Orr, W.; Ward, P. A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1978 Português
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The migration patterns of normal mouse embryo fibroblast (MEF) cells and mouse fibrosarcoma (FS) cells were compared in two assay systems. The two assay systems used were themodified Boyden chamber (micropore membrane) assay and the agarose drop explant assay. In both assays the major population of MEF cells exhibited a greater rate of migration than the major population of FS cells. However, a small subpopulation of FS cells which had a much greater rate of migration than the major population of either MEF or FS cells was detected in the agarose drop assay. A number of drugs which are known to inhibit the migration of leukocytes were tested against the MEF and FS cells. Concentrations were found that inhibited the major population of both groups by greater than 90%. However, at concentrations which inhibited the migration of the major population of FS cells by greater than 90%, a small group of fast-moving cells was still detected. Although the fast-moving cells were relatively resistant to treatment with the various drugs, this group was sensitive to a factor in serum. When normal human serum was used in place of fetal calf serum, the migration of the major population of FS cells was inhibited very little but movement of the fast-moving population was completely eliminated. We speculate that the small subgroup of fast-moving cells may be responsible for the invasive nature of the FS cells.

The Proto-Oncogene LRF Is under Post-Transcriptional Control of MiR-20a: Implications for Senescence

Poliseno, Laura; Pitto, Letizia; Simili, Marcella; Mariani, Laura; Riccardi, Luisa; Ciucci, Alessia; Rizzo, Milena; Evangelista, Monica; Mercatanti, Alberto; Pandolfi, Pier Paolo; Rainaldi, Giuseppe
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 02/07/2008 Português
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MicroRNAs (miRNAs) are short 20–22 nucleotide RNA molecules that act as negative regulators of gene expression via translational repression: they have been shown to play a role in development, proliferation, stress response, and apoptosis. The transcriptional regulator LRF (Leukemia/lymphoma Related Factor) has been shown to prevent p19ARF transcription and consequently to inhibit senescence in mouse embryonic fibroblasts (MEF). Here we report, for the first time, that LRF is post-transcriptionally regulated by miR-20a. Using a gene reporter assay, direct interaction of miR-20a with the LRF 3′UTR is demonstrated. To validate the interaction miR-20a/3′UTR LRF miR-20a was over-expressed, either by transient transfection or retroviral infection, in wild type mouse embryo fibroblasts and in LRF-null MEF derived from LRF knock-out mice. We observed LRF decrease, p19ARF increase, inhibition of cell proliferation and induction of senescence. The comparison of miR-20a activity in wt and LRF-null MEF indicates that LRF is the main mediator of the miR-20a-induced senescence and that other targets are cooperating. As LRF down-regulation/p19ARF induction is always accompanied by E2F1 down-regulation and increase of p16, we propose that all these events act in synergy to accomplish miR-20a-induced senescence in MEF. Senescence has been recently revaluated as a tumor suppressor mechanism...

Application of Capillary Electrophoresis Mass Spectrometry and Liquid Chromatography Multiple-Step Tandem Electrospray Mass Spectrometry To Profile Glycoform Expression during Haemophilus influenzae Pathogenesis in the Chinchilla Model of Experimental Otitis Media ▿

Lundström, Susanna L.; Li, Jianjun; Månsson, Martin; Figueira, Marisol; Leroy, Magali; Goldstein, Richard; Hood, Derek W.; Moxon, E. Richard; Richards, James C.; Schweda, Elke K. H.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Otitis media caused by nontypeable Haemophilus influenzae (NTHi) is a common and recurrent bacterial infection of childhood. The structural variability and diversity of H. influenzae lipopolysaccharide (LPS) glycoforms are known to play a significant role in the commensal and disease-causing behavior of this pathogen. In this study, we determined LPS glycoform populations from NTHi strain 1003 during the course of experimental otitis media in the chinchilla model of infection by mass spectrometric techniques. Building on an established structural model of the major LPS glycoforms expressed by this NTHi strain in vitro (M. Månsson, W. Hood, J. Li, J. C. Richards, E. R. Moxon, and E. K. Schweda, Eur. J. Biochem. 269:808-818, 2002), minor isomeric glycoform populations were determined by liquid chromatography multiple-step tandem electrospray mass spectrometry (LC-ESI-MSn). Using capillary electrophoresis ESI-MS (CE-ESI-MS), we determined glycoform profiles for bacteria from direct middle ear fluid (MEF) samples. The LPS glycan profiles were essentially the same when the MEF samples of 7 of 10 animals were passaged on solid medium (chocolate agar). LC-ESI-MSn provided a sensitive method for determining the isomeric distribution of LPS glycoforms in MEF and passaged specimens. To investigate changes in LPS glycoform distribution during the course of infection...

Double-Stranded RNA-Dependent Protein Kinase-Dependent Apoptosis Induction by a Novel Small CompoundS⃞

Hu, Wenxian; Hofstetter, Wayne; Wei, Xiaoli; Guo, Wei; Zhou, Yanbin; Pataer, Abujiang; Li, Hong; Fang, Bingliang; Swisher, Stephen G.
Fonte: American Society for Pharmacology and Experimental Therapeutics Publicador: American Society for Pharmacology and Experimental Therapeutics
Tipo: Artigo de Revista Científica
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The interferon-induced, double-stranded RNA-dependent protein kinase (PKR) can play critical roles in inhibiting virus replication and inducing apoptosis. To develop new agents that may inhibit viral replication or induce apoptosis in cancer cells via the PKR signaling pathway, we screened a chemical library for compounds that have differential cytotoxic effects on wild-type [mouse embryonic fibroblast (MEF)/PKR(+/+)] and PKR-knockout [MEF/PKR(-/-)] mouse embryonic fibroblast cells. We identified a synthetic compound, BEPP [1H-benzimidazole1-ethanol,2,3-dihydro-2-imino-a-(phenoxymethyl)-3-(phenylmethyl)-,monohydrochloride], that induces a cytotoxic effect more effectively in MEF/PKR(+/+) cells than in MEF/PKR(-/-) cells. BEPP also relatively effectively inhibited the growth of a human lung cancer cell line overexpressing PKR, compared with other cancer cell lines. In sensitive cells, BEPP induced apoptosis with activation of caspase-3. Treatment with BEPP led to increased phosphorylation of PKR and eIF2α, increased expression of BAX, and decreased expression of Bcl-2. BEPP-induced apoptosis was PKR dependent and was blocked by the adenovector expressing the dominant-negative PKR. Furthermore, pretreatment of HeLa cells at a noncytotoxic dose of BEPP effectively inhibited Vaccinia virus replication. Together...

Lysophosphatidic acid induces cell migration through the selective activation of Akt1

Kim, Eun Kyoung; Yun, Sung Ji; Do, Kee Hun; Kim, Min Sung; Cho, Mong; Suh, Dong-Soo; Kim, Chi Dae; Kim, Jae Ho; Birnbaum, Morris J.; Bae, Sun Sik
Fonte: Korean Society of Medical Biochemistry and Molecular Biology Publicador: Korean Society of Medical Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration.

Polyelectrolyte Layer-by-Layer Assembly To Control the Distance between Fluorophores and Plasmonic Nanostructures

Ray, Krishanu; Badugu, Ramachandram; Lakowicz, Joseph R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 27/11/2007 Português
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In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles. MEF is largely dependent on several factors, such as chemical nature, size, shape of the nanostructure, and its distance from the interrogating fluorophore. Herein, we elucidate the potential of layer-by-layer (LbL) assembly to understand the distance dependence nature of MEF from sulforhodamine B (SRB) assembled on the plasmonic nanostructured surfaces [in the form of Silver Islands films (SIFs)]. The varied proximity of fluorophores from the SIF surfaces was controlled by constructing different numbers of alternate layers of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). An anionic laser dye SRB could be electrostatically attached to the positively charged PAH layer. Orientation of the SRB probe molecule adsorbed in PSS/PAH-layered assembly was determined by polarized absorption spectroscopy. The observed tilt angle of the probe transition dipole moment with respect to the surface normal was 40°. Our results show that MEF is indeed distance-dependent. Accordingly, we observed a maximum of a ~6-fold increase in the fluorescence intensity from a monolayer of the SRB at a distance of ~9 nm from the metal-nanostructured surface...

Genetic Inactivation of Poliovirus Infectivity by Increasing the Frequencies of CpG and UpA Dinucleotides within and across Synonymous Capsid Region Codons▿ †

Burns, Cara C.; Campagnoli, Ray; Shaw, Jing; Vincent, Annelet; Jorba, Jaume; Kew, Olen
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only ∼9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses...

Metal-Enhanced Fluoroimmunoassay on a Silver Film by Vapor Deposition

Zhang, Jian; Matveeva, Evgenia; Gryczynski, Ignacy; Leonenko, Zoya; Lakowicz, Joseph R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 28/04/2005 Português
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We studied a fluoroimmunoassay using metal-enhanced fluorescence (MEF) detection on silver film generated by vapor deposition method. The morphology of the silver film was controlled through the thickness of the film. A silica layer was coated on the silver film to protect the film and separate the fluorophore from the metal surface. Rabbit immunoglobulin G (IgG) was adsorbed on the silica by physiosorption and then dye-labeled anti-rabbit IgG was bound to the immobilized rabbit IgG. It was observed that the fluorophore was quenched on a thin silver film (2 nm), enhanced on a thick film (>5 nm), and reached saturation (ca. 10 times enhancement) at 20 nm. The MEF was also dependent on the thickness of the silica with a maximum at 10 nm. The lowest lifetime was observed on the 20 nm silver film, which was consistent with the saturation of MEF. These results showed the properties of a silver film needed for a maximum increase of fluorescence intensity in a fluoroimmunoassay. Dependence of the MEF on the emission wavelength was also studied using different dye-labeled anti-rabbit IgGs.

Neurofibromin physically interacts with the N-terminal domain of Focal Adhesion Kinase

Kweh, Frederick; Zheng, Min; Kurenova, Elena; Wallace, Margaret; Golubovskaya, Vita; Cance, William G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/2009 Português
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The NF1 gene that is altered in patients with type 1 neurofibromatosis encodes a neurofibromin (NF1) protein that functions as a tumor suppressor. In this report, we show for the first time physical interaction between neurofibromin and focal adhesion kinase (FAK), the protein that localizes at focal adhesions. We show that neurofibromin associates with the N-terminal domain of FAK, and that the C-terminal domain of neurofibromin directly interacts with FAK. Confocal microscopy demonstrates co-localization of NF1 and FAK in the cytoplasm, peri-nuclear and nuclear regions inside the cells. Nf1+/+ MEF cells expressed less cell growth during serum deprivation conditions, and adhered less on collagen and fibronectin-treated plates than Nf1−/− MEF cells, associated with changes in actin and FAK staining. In addition, Nf1+/+ MEF cells detached more significantly than Nf1−/− MEF cells by disruption of FAK signaling with the dominant-negative inhibitor of FAK, C-terminal domain of FAK, FAK-CD. Thus, the results demonstrate the novel interaction of neurofibromin and FAK and suggest their involvement in cell adhesion, cell growth, and other cellular events and pathways.

Calcineurin activates interleukin-6 transcription in mouse skeletal muscle in vivo and in C2C12 myotubes in vitro

Allen, David L.; Uyenishi, Jill J.; Cleary, Allison S.; Mehan, Ryan S.; Lindsay, Sarah F.; Reed, Jason M.
Fonte: American Physiological Society Publicador: American Physiological Society
Tipo: Artigo de Revista Científica
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Expression of the cytokine interleukin-6 (IL-6) by skeletal muscle is hugely increased in response to a single bout of endurance exercise, and this appears to be mediated by increases in intracellular calcium. We examined the effects of endurance exercise on IL-6 mRNA levels and promoter activity in skeletal muscle in vivo, and the role of the calcium-activated calcineurin signaling pathway on muscle IL-6 expression in vivo and in vitro. IL-6 mRNA levels in the mouse tibialis anterior (TA) were increased 2–10-fold by a single bout of treadmill exercise or by 3 days of voluntary wheel running. Moreover, an IL-6 promoter-driven luciferase transgene was activated in TA by both treadmill and wheel-running exercise and by injection with a calcineurin plasmid. Exercise also increased muscle mRNA expression of the calcineurin regulatory gene MCIP1, as did treatment of C2C12 myotubes with the calcium ionophore A23187. Cotransfection of C2C12 myotubes with a constitutively active calcineurin construct significantly increased while cotransfection with the calcineurin inhibitor CAIN inhibited activity of a mouse IL-6 promoter-reporter construct. Cotransfection with a myocyte enhancer-factor-2 (MEF-2) expression construct increased basal IL-6 promoter activity and augmented the effects of calcineurin cotransfection...

Choosing the Optimal Trigger Point for Analysis of Movements after Stroke Based on Magnetoencephalographic Recordings

Waldmann, Guido; Schauer, Michael; Woldag, Hartwig; Hummelsheim, Horst
Fonte: SAGE-Hindawi Access to Research Publicador: SAGE-Hindawi Access to Research
Tipo: Artigo de Revista Científica
Publicado em 13/01/2010 Português
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The aim of this study was to select the optimal procedure for analysing motor fields (MF) and motor evoked fields (MEF) measured from brain injured patients. Behavioural pretests with patients have shown that most of them cannot stand measurements longer than 30 minutes and they also prefer to move the hand with rather short breaks between movements. Therefore, we were unable to measure the motor field (MF) optimally. Furthermore, we planned to use MEF to monitor cortical plasticity in a motor rehabilitation procedure. Classically, the MF analysis refers to rather long epochs around the movement onset (M-onset). We shortened the analysis epoch down to a range from 1000 milliseconds before until 500 milliseconds after M-onset to fulfil the needs of the patients. Additionally, we recorded the muscular activity (EMG) by surface electrodes on the extensor carpi ulnaris and flexor carpi ulnaris muscles. Magnetoencephalographic (MEG) data were recorded from 9 healthy subjects, who executed horizontally brisk extension and flexion in the right wrist. Significantly higher MF dipole strength was found in data based on EMG-onset than in M-onset based data. There was no difference in MEF I dipole strength between the two trigger latencies. In conclusion...

Rapid and Sensitive Detection of Troponin I-T-C Complex from Human Serum using Microwave-Accelerated Metal-Enhanced Fluorescence

Aslan, Kadir
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2011 Português
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We present the rapid and sensitive detection of Troponin I-T-C (Tn I-T-C) complex from buffer and human serum samples using Microwave-Accelerated and Metal-Enhanced Fluorescence (MA-MEF) technique, which is based on the combined use of low power microwave heating, silver nanoparticle films (SNFs) and fluorescence spectroscopy. The detection of Tn I-T-C complex from buffer solutions and human serum samples on SNFs was carried out using fluorescence-based immunoassays at room temperature (control immunoassay, 2 hour total assay time) and using low-power microwave heating (MA-MEF-based immunoassay, 1 minute total assay time). A lower detection limit for Tn I-T-C complex from buffer solutions in the control immunoassay and MA-MEF-based immunoassay was 0.01 ng/ml and 0.005 ng/ml, respectively. However, the lower detection limit for Tn I-T-C complex from human serum in the control immunoassay was increased to 10 ng/ml. The use of MA-MEF technique afforded for the detection of Tn I-T-C complex from human serum samples in 1 min with a lower detection limit of 0.05 ng/ml.

Comparative Analysis of the Humoral Immune Response to Moraxella catarrhalis and Streptococcus pneumoniae Surface Antigens in Children Suffering from Recurrent Acute Otitis Media and Chronic Otitis Media with Effusion

Verhaegh, Suzanne J. C.; Stol, Kim; de Vogel, Corné P.; Riesbeck, Kristian; Lafontaine, Eric R.; Murphy, Timothy F.; van Belkum, Alex; Hermans, Peter W. M.; Hays, John P.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2012 Português
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A prospective clinical cohort study was established to investigate the humoral immune response in middle ear fluids (MEF) and serum against bacterial surface proteins in children suffering from recurrent acute otitis media (rAOM) and chronic otitis media with effusion (COME), using Luminex xMAP technology. The association between the humoral immune response and the presence of Moraxella catarrhalis and Streptococcus pneumoniae in the nasopharynx and middle ear was also studied. The levels of antigen-specific IgG, IgA, and IgM showed extensive interindividual variation. No significant differences in anti-M. catarrhalis and anti-S. pneumoniae serum and MEF median fluorescence intensity (MFI) values (anti-M. catarrhalis and antipneumococcal IgG levels) were observed between the rAOM or COME groups for all antigens tested. No significant differences were observed for M. catarrhalis and S. pneumoniae colonization and serum IgG levels against the Moraxella and pneumococcal antigens. Similar to the antibody response in serum, no significant differences in IgG, IgA, and IgM levels in MEF were observed for all M. catarrhalis and S. pneumoniae antigens between OM M. catarrhalis- or S. pneumoniae-positive and OM M. catarrhalis- or S. pneumonia-negative children suffering from either rAOM or COME. Finally...

Role of group A p21-activated kinases in the anti-apoptotic activity of the pseudorabies virus US3 protein kinase

Van den Broeke, C.; Radu, M.; Nauwynck, H.J.; Chernoff, J.; Favoreel, H.W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The alphaherpesvirus US3 kinase is a conserved multifunctional serine/threonine kinase that plays a role in several processes, including modulation of the actin cytoskeleton, egress of virus particles from the nucleus and inhibition of apoptosis. However, the mechanisms used by the US3 protein to exert its functions remain poorly understood. Recently, we identified the group A p21-activated kinases PAK1 and PAK2 as important effectors in the US3-mediated cytoskeletal rearrangements. Here, we investigated if group A PAKs are also involved in the anti-apoptotic properties of US3. Infection experiments using a group A PAK inhibitor pointed at a moderate role for group A PAKs in the anti-apoptotic properties of US3. Furthermore, infection assays using wild type and US3null PRV in wild type MEF, PAK1−/−MEF and PAK2−/− MEF indicated that PAK2 does not play a role in US3-mediated inhibition of apoptosis during infection, whereas PAK1 plays a significant, yet limited role. Experiments in US3-transfected MEF using staurosporine as apoptosis trigger confirmed these observations. These results show that PAK1 plays a significant, yet limited, role in the anti-apoptotic activity of US3.

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Turner, William S.; McCloskey, Kara E.
Fonte: MyJove Corporation Publicador: MyJove Corporation
Tipo: Artigo de Revista Científica
Publicado em 28/10/2012 Português
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Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture...

The role of microRNAs during the genesis of medulloblastomas induced by the hedgehog pathway

Luo, Xiaoju; Liu, Jun; Cheng, Steven Y
Fonte: Editorial Department of Journal of Biomedical Research Publicador: Editorial Department of Journal of Biomedical Research
Tipo: Artigo de Revista Científica
Publicado em /01/2011 Português
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Constitutive hedgehog (Hh) signaling is associated with the genesis of medulloblastomas (MB). The objective of this study is to identify special microRNAs (miRNAs) regulated by the Hh pathway, and to clarify the role of miRNAs during the genesis of MB induced by sustained Hh activation. In the primary screening, we used stem-loop RT-PCR to test the expression of 90 different miRNAs in the wildtype (WT) and Ptc-/- MEF cell lines. In the secondary screening, the miRNAs screened from the first screening were validated in the Sufu-/- MEF cell lines. We then verified the expression of miRNAs both in the normal cerebellar tissues and the MB induced by activated Hh pathway, and examined the expression of the other 21 miRNA members of the miR-154 cluster in the MB and normal cerebellum. In the first screening, 13 miRNAs showed significant differential expression in WT and Ptc-/- MEF cell lines, while 10 of them had significant difference in the Sufu-/- MEF cell line. Compared to the normal mouse cerebellum, only 2 miRNAs in 15 miRNAs were differentially expressed between the MB and normal cerebellar tissues. Among 21 members of the miR-154 cluster, 6 miRNAs were downregulated in the MB. Our study demonstrated that miR-154 may be regulated by the Hh pathway...

Molecular Resistance Mechanisms of Macrolide-Resistant Invasive Streptococcus pneumoniae Isolates from Alaska, 1986 to 2010

Rudolph, Karen; Bulkow, Lisa; Bruce, Michael; Zulz, Tammy; Reasonover, Alisa; Harker-Jones, Marcella; Hurlburt, Debby; Hennessy, Thomas
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2013 Português
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The rapid emergence of antibiotic-resistant pneumococcal strains has reduced treatment options. The aim of this study was to determine antimicrobial susceptibilities, serotype distributions, and molecular resistance mechanisms among macrolide-resistant invasive pneumococcal isolates in Alaska from 1986 to 2010. We identified cases of invasive pneumococcal disease in Alaska from 1986 to 2010 through statewide population-based laboratory surveillance. All invasive pneumococcal isolates submitted to the Arctic Investigations Program laboratory were confirmed by standard microbiological methods and serotyped by slide agglutination and the Quellung reaction. MICs were determined by the broth microdilution method, and macrolide-resistant genotypes were determined by multiplex PCR. Among 2,923 invasive pneumococcal isolates recovered from 1986 to 2010, 270 (9.2%) were nonsusceptible to erythromycin; 177 (66%) erythromycin-nonsusceptible isolates demonstrated coresistance to penicillin, and 167 (62%) were multidrug resistant. The most frequent serotypes among the macrolide-resistant isolates were serotypes 6B (23.3%), 14 (20.7%), 19A (16.7%), 9V (8.9%), 19F (6.3%), 6A (5.6%), and 23F (4.8%). mef and erm(B) genes were detected in 207 (77%) and 32 (12%) of the isolates...

Gsta4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity*

McElhanon, Kevin E.; Bose, Chhanda; Sharma, Rajendra; Wu, Liping; Awasthi, Yogesh C.; Singh, Sharda P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The alpha class glutathione s-transferase (GST) isozyme GSTA4–4 (EC2.5.1.18) exhibits high catalytic efficiency to-wards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated concentrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To examine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we disrupted the Gsta4 gene that encodes GSTA4–4 in mice. The effect of electrophile and oxidants on embryonic fibroblasts (MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of GSTA4–4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)3], a pro-drug form of 4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of caspase-3. Interestingly...