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Influence of moderate electric fields on gelation of whey protein isolate

Rodrigues, Rui M.; Martins, Artur J.; Ramos, Óscar L.; Malcata, F. X.; Teixeira, J. A.; Vicente, A. A.; Pereira, Ricardo
Fonte: Elsevier BV Publicador: Elsevier BV
Tipo: Artigo de Revista Científica
Publicado em //2015 Português
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Proteins are one of the food constituents most affected by heating, and some of the changes involve their unfolding, denaturation and gelation. Ohmic heating has often been claimed to improve the quality of foodstuffs due to its uniform heating and (putative) presence of a moderate electric field (MEF). However, this is still subject to discussion, so it is important to determine the effect of ohmic heating and of its MEF upon food constituents. Hence, the aim of this work was to evaluate the effects of MEF on denaturation, aggregation and viscoelastic properties of whey protein isolate (WPI), and compare them with those obtained via conventional heating under identical treatment conditions (up to 30 min at 85 °C). Results have shown that MEF interferes with whey protein unfolding and aggregation pathways at relatively high temperatures. MEF treatments have resulted in WPI solutions possessing more 8 and 10% of native β-Lactoglobulin and α-Lactalbumin, respectively, after 30 s of heating at 85 °C, when compared with a conventional heating method. Protein aggregates from MEF-treated WPI solutions presented a maximum increase in size of 78 nm, whereas conventional heating produced an increase of 86 nm. Unlike in conventional heating...

Production of whey protein cold-set hydrogels through application of moderate electric fields

Rodrigues, Rui M.; Ramos, Óscar L.; Teixeira, J. A.; Vicente, A. A.; Pereira, Ricardo
Fonte: Sociedade Portuguesa de Química Publicador: Sociedade Portuguesa de Química
Tipo: Conferência ou Objeto de Conferência
Publicado em //2014 Português
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ISBN 978-989-98541-5-4; Whey, a liquid by-product from dairy industry is now recognized as a functional food due to the presence of bioactive and highly nutritional components, such as globular whey proteins. Denaturation and aggregation kinetic behavior of these proteins is of particular relevance when properly engineered and controlled, as it results in the production of novel micro and nano-systems with many potential uses in food compositions. Application of moderate electric fields (MEF) during heating process at relatively high temperatures has the potential to interfere with the secondary structure of globular whey proteins networks [1], eventually forming gels. However, the heat needed to produce these gels limits their application to formulations that do not contain heat-sensitive compounds [2]. The objective of this study is producing cold-set hydrogels from purified β-lactoglobulin and whey ingredients through combined application thermal and MEF treatment and salt induced gelation. Protein dispersions (3 % w/v and pH=3) were heated with and without presence of MEF treatments (0, 3 and 10 V/cm) at temperatures of 90 °C. Cold gelation was then induced through addition of different quantities of FeSO4 at 24 ºC. Nano-scale aggregation phenomena during the initial steps of whey protein aggregation as affected by the thermal treatment and applied electric field were assessed by dynamic light scattering (DLS). Rheological measurements were performed using a controlled stress rheometer with concentric cylinder geometry in order to assess the effects of MEF on macroscopic properties of the WPI hydrogels produced. Results shows that the extent of protein aggregation decreased both with the purity of the whey product used and the increase of the intensity of the applied MEF treatment. MEF treatments at 10 V/cm applied on whey protein isolate originated average mean particle sizes at nano-scale range...

Penetration of ceftibuten into middle ear fluid.

Lin, C; Kumari, P; Perrotta, R J; Reidenberg, B E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1996 Português
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The penetration of ceftibuten, an extended-spectrum oral cephalosporin, into middle ear fluid (MEF) was evaluated in pediatric patients during a course of daily oral doses of 9 mg/kg of body weight for 10 days. Plasma and MEF collected at 2, 4, 6, or 12 h after at least 3 days of dosing were analyzed for ceftibuten by a high-pressure liquid chromatography method, and the data were used to calculate pharmacokinetic parameters. Plasma and MEF had almost identical maximum concentrations (Cmax) of ceftibuten (14 micrograms/ml). These Cmax values in MEF during acute otitis media were well in excess of the MIC for 90% of the isolates of each of four major pathogens in this disease. The time to Cmax was longer in MEF (4 h) than in plasma (2 h). Excellent penetration (71%) of ceftibuten into MEF was observed on the basis of the area under the curve ratio (MEF/plasma). These data clearly indicate that ceftibuten penetrated well into the MEF to yield clinically effective concentrations.

Induction of a macrophage migration enhancement factor after desensitization of tuberculin-positive rabbits with purified protein derivative.

Gordon, M R; Takata, I; Myrvik, Q N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1986 Português
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The production of a macrophage migration enhancement factor (MEF) has been achieved as a consequence of administering a desensitizing dose of purified protein derivative (PPD) to Mycobacterium bovis BCG-sensitized rabbits. The migration-enhancing effect was first demonstrated when alveolar macrophages (AM) harvested from desensitized rabbits exhibited marked migration stimulation; whereas maximum migration enhancement was observed 8 days after the administration of PPD, migration enhancement of the AM from these rabbits persisted for up to 12 days. Sera from BCG-sensitized, PPD-desensitized animals exhibited a peak of MEF activity 4 days after desensitization. Maximal MEF activity was demonstrated in culture supernatants of nonadherent spleen cells harvested 8 days after the intravenous desensitizing dose of PPD was given. Control spleen cell culture supernatants did not produce detectable MEF. The route of desensitization with PPD was critical. When PPD was administrated intratracheally, MEF activity was not induced. The intravenous administration of BCG after PPD desensitization reversed migration enhancement to strong migration inhibition. Ammonium sulfate fractionation indicated that two fractions contained MEF activity. MEF activity was retained by dialysis membranes with a 15...

Endotoxin-induced tumor necrosis factor alpha synthesis in murine embryo fibroblasts.

Havell, E A; Rogerson, B J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1993 Português
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176.52293%
Murine embryo fibroblasts (MEF) were found to secrete tumor necrosis factor (TNF) in response to stimulation with endotoxin. Endotoxin-induced TNF production by MEF was inhibited by cycloheximide. However, reversal of the effect of this inhibitor on protein synthesis results in TNF being secreted in amounts equivalent to those produced by endotoxin-induced MEF not treated with cycloheximide. Actinomycin D treatment of MEF blocked the production of endotoxin-induced TNF. Maximal production of TNF required MEF gene transcription during the first 6 h of incubation with endotoxin. To determine whether endotoxin-induced TNF alpha (TNF-alpha) and/or TNF beta were produced by MEF, cDNA was synthesized from the total RNA isolated from endotoxin-induced MEF and amplified by the polymerase chain reaction in the presence of oligonucleotide primers specific for each cytokine. On the basis of the polymerase chain reaction analysis, it was determined that TNF-alpha mRNA levels were increased in endotoxin-induced MEF. Thus, production of TNF-alpha by fibroblasts in response to the endotoxin component of bacterial cell walls is likely to contribute to the expression of TNF-mediated effects occurring in fibroblast-rich tissues infected with gram-negative bacteria.

Differential expression of the myocyte enhancer factor 2 family of transcription factors in development: the cardiac factor BBF-1 is an early marker for cardiogenesis.

Goswami, S; Qasba, P; Ghatpande, S; Carleton, S; Deshpande, A K; Baig, M; Siddiqui, M A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1994 Português
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In the present study, we have used single chicken blastoderms of defined early developmental stages, beginning with the prestreak stage, stage 1 (V. Hamburger and H. L. Hamilton, J. Morphol. 88:49-92, 1951), to analyze the onset of cardiac myogenesis by monitoring the appearance of selected cardiac muscle tissue-specific gene transcripts and the functional expression of the myocyte enhancer factor 2 (MEF-2) proteins. Using gene-specific oligonucleotide primers in reverse transcriptase PCR assay, we have demonstrated that the cardiac myosin light-chain 2 (MLC2) and alpha-actin gene transcripts appear as early as stage 5, i.e., immediately after the cardiogenic fate assignment at stage 4. Consistent with this observation is the developmental expression pattern of DNA-binding activity of BBF-1, a cardiac muscle-specific member of the MEF-2 protein family, which also begins at stage 5 prior to MEF-2. Differential expression of DNA-binding complexes is also observed with another AT-rich DNA sequence (CArG box) as probe, but the binding pattern with the ubiquitous TATA-binding proteins remains unchanged during the same developmental period. Thus, the cardiogenic commitment and differentiation of the precardiac mesoderm, as exemplified by the appearance of cardiac MEF-2...

A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes.

Gossett, L A; Kelvin, D J; Sternberg, E A; Olson, E N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1989 Português
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Exposure of skeletal myoblasts to growth factor-deficient medium results in transcriptional activation of muscle-specific genes, including the muscle creatine kinase gene (mck). Tissue specificity, developmental regulation, and high-level expression of mck are conferred primarily by a muscle-specific enhancer located between base pairs (bp) -1350 and -1048 relative to the transcription initiation site (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909, 1988). To begin to define the regulatory mechanisms that mediate the selective activation of the mck enhancer in differentiating muscle cells, we have further delimited the boundaries of this enhancer and analyzed its interactions with nuclear factors from a variety of myogenic and nonmyogenic cell types. Deletion mutagenesis showed that the region between 1,204 and 1,095 bp upstream of mck functions as a weak muscle-specific enhancer that is dependent on an adjacent enhancer element for strong activity. This adjacent activating element does not exhibit enhancer activity in single copy but acts as a strong enhancer when multimerized. Gel retardation assays combined with DNase I footprinting and diethyl pyrocarbonate interference showed that a nuclear factor from differentiated C2 myotubes and BC3H1 myocytes recognized a conserved A + T-rich sequence within the peripheral activating region. This myocyte-specific enhancer-binding factor...

Immunoglobulins of the Middle Ear Fluid in Acute Otitis Media: Relationship to Serum Immunoglobulin Concentrations and Bacterial Cultures

Howie, Virgil M.; Ploussard, John H.; Sloyer, John L.; Johnston, Richard B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1973 Português
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Immunoglobulin concentrations were studied in 255 specimens of middle ear fluid (MEF) from 165 episodes of acute otitis media in children. There were significant amounts of all three major immunoglobulins (Ig) in MEF, the mean concentration of IgA being 39 mg/100 ml, of IgM 63 mg/100 ml, and of IgG 383 mg/100 ml. Secretory component was present in all 10 MEF specimens in which it was sought. In patients over 9 months of age, there was a decreased likelihood of isolating pathogenic bacteria from MEF if the patient had higher concentrations of IgA in MEF than in simultaneously obtained serum. IgA concentrations were greater in MEF than in serum in almost half the patients, and the mean MEF-serum ratio for IgA was 1.38. Thus, it would appear that in this disorder MEF represents primarily a secretory response to inflammation rather than a transudate.

Characterization and Prevalence of MefA, MefE, and the Associated msr(D) Gene in Streptococcus pneumoniae Clinical Isolates

Daly, Melissa M.; Doktor, Stella; Flamm, Robert; Shortridge, Dee
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2004 Português
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180.00125%
Recent work has shown that the efflux genes in Streptococcus pneumoniae that are responsible for acquired macrolide resistance can be distinguished as either mef(E) or mef(A). The genetic elements on which mef(A) and mef(E) are found also carry an open reading frame (ORF) that is 56% homologous to msr(A) in Staphylococcus. The prevalence of mef(A/E) and of the msr-like ORF [msr(D)] was evaluated in 153 mef+ S. pneumoniae clinical isolates collected in North America, Europe, Africa, and Asia from 1997 to 2002. Clinical isolates were screened with PCR primers specific for either mef(A) or mef(E) and for msr(D). mef(A), mef(E), and msr(D) were cloned from mef+ strains and transformed into a susceptible, competent strain of S. pneumoniae. The transformants were tested for antimicrobial susceptibilities and efflux pump induction. The results of this work demonstrated that mef(A) is more often isolated in parts of Europe, with some incidence in Canada, and that the msr-like gene alone can confer the efflux phenotype.

Erythromycin Resistance and Genetic Elements Carrying Macrolide Efflux Genes in Streptococcus agalactiae

Marimón, José María; Valiente, Adoración; Ercibengoa, María; García-Arenzana, José M.; Pérez-Trallero, Emilio
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2005 Português
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The macrolide resistance determinants and genetic elements carrying the mef(A) and mef(E) subclasses of the mef gene were studied with Streptococcus agalactiae isolated in 2003 and 2004 from 7,084 vaginorectal cultures performed to detect carrier pregnant women. The prevalence of carriage was 18% (1,276 isolates), and that of erythromycin resistance 11.0% (129 of the 1,171 isolates studied). erm(B), erm(A) subclass erm(TR), and the mef gene, either subclass mef(A) or mef(E), were found in 72 (55.8%), 41 (31.8%), and 12 (9.3%) erythromycin-resistant isolates, while 4 isolates had more than 1 erythromycin resistance gene. Of the 13 M-phenotype mef-containing erythromycin-resistant S. agalactiae isolates, 11 had the mef(E) subclass gene alone, one had both the mef(E) and the erm(TR) subclass genes, and one had the mef(A) subclass gene. mef(E) subclass genes were associated with the carrying element mega in 10 of the 12 mef(E)-containing strains, while the single mef(A) subclass gene found was associated with the genetic element Tn1207.3. The nonconjugative nature of the mega element and the clonal diversity of mef(E)-containing strains determined by pulsed-field gel electrophoresis suggest that transformation is the main mechanism through which this resistance gene is acquired.

Pref-1 (Preadipocyte Factor 1) Activates the MEK/Extracellular Signal-Regulated Kinase Pathway To Inhibit Adipocyte Differentiation▿

Kim, Kyung-Ah; Kim, Jung-Hyun; Wang, Yuhui; Sul, Hei Sook
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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177.64826%
Preadipocyte factor 1 (Pref-1) is found in preadipocytes but is absent in adipocytes. Pref-1 is made as a transmembrane protein but is cleaved to generate a biologically active soluble form. Although Pref-1 inhibition of adipogenesis has been well studied in vitro and in vivo, the signaling pathway for Pref-1 is not known. Here, by using purified soluble Pref-1 in Pref-1 null mouse embryo fibroblasts (MEF), we show that Pref-1 increases MEK/extracellular signal-regulated kinase (ERK) phosphorylation in a time- and dose-dependent manner. Compared to wild-type MEF, differentiation of Pref-1 null MEF into adipocytes is enhanced, as judged by lipid accumulation and adipocyte marker expression. Both wild-type and Pref-1 null MEF show a transient burst of ERK phosphorylation upon addition of adipogenic agents. Wild-type MEF show a significant, albeit lower, second increase in ERK phosphorylation peaking at day 2. This ERK phosphorylation, corresponding to Pref-1 abundance, is absent during differentiation of Pref-1 null MEF. Prevention of this second increase in ERK1/2 phosphorylation in wild-type MEF by the MEK inhibitor PD98059 or by transient depletion of ERK1/2 via small interfering RNA-enhanced adipocyte differentiation. Furthermore...

Tumor Necrosis Factor-like Weak Inducer of Apoptosis (TWEAK) activates proinflammatory signaling pathways and gene expression through the activation of TGF-beta activated kinase 1

Kumar, Mukesh; Makonchuk, Denys Y.; Li, Hong; Mittal, Ashwani; Kumar, Ashok
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/02/2009 Português
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TWEAK is a relatively recently identified proinflammatory cytokine which functions through binding to Fn14 receptor in target cells. Although TWEAK has been shown to modulate several biological responses, the TWEAK-induced signaling pathways remain poorly understood. In this study, we tested the hypothesis that TAK1 is involved in TWEAK-induced activation of NF-κB and MAPK, and expression of proinflammatory protein. TWEAK increased the phosphorylation and kinase activity of TAK1 in cultured myoblast and fibroblast cells. The activation of NF-κB was significantly inhibited in TAK1-deficinet (TAK1−/−) mouse embryonic fibroblasts (MEF) compared to wild-type MEF. Deficiency of TAK1 also inhibited the TWEAK-induced activation of IκB kinase and phosphorylation and degradation of IκBα protein. However, there was no difference in the levels p100 protein in TWEAK-treated wild-type and TAK1−/− MEF. Furthermore, TWEAK-induced transcriptional activation of NF-κB was significantly reduced in TAK1−/− MEF and in C2C12 myoblasts transfected with a dominant-negative TAK1 or TAK1 siRNA. TAK1 was also required for the activation of AP-1 in response to TWEAK. Activation of JNK1 and p38 MAPK but not ERK1/2 or Akt kinase was significantly inhibited in TAK1−/− MEF compared to wild-type MEF upon treatment with TWEAK. TWEAK-induced expression of proinflammatory genes such as MMP-9...

Induction of Efflux-Mediated Macrolide Resistance in Streptococcus pneumoniae ▿

Chancey, Scott T.; Zhou, Xiaoliu; Zähner, Dorothea; Stephens, David S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2011 Português
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The antimicrobial efflux system encoded by the operon mef(E)-mel on the mobile genetic element MEGA in Streptococcus pneumoniae and other Gram-positive bacteria is inducible by macrolide antibiotics and antimicrobial peptides. Induction may affect the clinical response to the use of macrolides. We developed mef(E) reporter constructs and a disk diffusion induction and resistance assay to determine the kinetics and basis of mef(E)-mel induction. Induction occurred rapidly, with a >15-fold increase in transcription within 1 h of exposure to subinhibitory concentrations of erythromycin. A spectrum of environmental conditions, including competence and nonmacrolide antibiotics with distinct cellular targets, did not induce mef(E). Using 16 different structurally defined macrolides, induction was correlated with the amino sugar attached to C-5 of the macrolide lactone ring, not with the size (e.g., 14-, 15- or 16-member) of the ring or with the presence of the neutral sugar cladinose at C-3. Macrolides with a monosaccharide attached to C-5, known to block exit of the nascent peptide from the ribosome after the incorporation of up to eight amino acids, induced mef(E) expression. Macrolides with a C-5 disaccharide, which extends the macrolide into the ribosomal exit tunnel...

Cellular Phenotype-Dependent and -Independent Effects of Vitamin C on the Renewal and Gene Expression of Mouse Embryonic Fibroblasts

Kuo, Shiu-Ming; Burl, Lana R.; Hu, Zihua
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 13/03/2012 Português
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178.57266%
Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF) and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10−5 M), but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells...

Antibody in Middle Ear Fluid of Children Originates Predominantly from Sera and Nasopharyngeal Secretions

Kaur, Ravinder; Kim, Thomas; Casey, Janet R.; Pichichero, Michael E.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2012 Português
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180.00125%
The human middle ear is devoid of any immunocompetent cells in normal mucosa. We sought to determine the source of antibody present in the middle ear of children. Total IgG, IgA, and secretory IgA antibodies were determined by enzyme-linked immunosorbent assay from the nasopharyngeal, middle ear, and serum samples of children with acute otitis media. The two-dimensional gel electrophoresis pattern of the entire array of IgA antibodies in the nasal wash (NW) and middle ear fluid (MEF) was compared from the MEF and NW samples using isoelectric focusing and Western blotting. The total IgG and IgA antibodies in the MEF and NW samples of 137 children were compared. The ratio of IgG to IgA in the MEF was significantly different (P < 0.008) compared to NW because IgA levels were higher and IgG levels lower in NW. The IgG/IgA ratio of MEF resembled serum consistent with transudation to the MEF. Small amounts of secretory IgA were detected in MEF but the electrophoresis patterns of the entire array of IgA antibodies in the MEF and NW were virtually identical in each child evaluated; thus, IgA in MEF derived predominantly from serum and the nasopharynx by reflux via the Eustachian tube. The IgG/IgA antibody levels in the MEF and the same composition of IgA antibody in the MEF and NW identifies the predominant source of antibody in the MEF as a transudate of serum combined with nasal secretions refluxed from the nasopharynx in children.

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

Kumagai, Tadahiro; Sato, Takeshi; Natsuka, Shunji; Kobayashi, Yukito; Zhou, Dapeng; Shinkai, Tadashi; Hayakawa, Satoru; Furukawa, Kiyoshi
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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178.57266%
Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5+/−- and B4galt5−/−-derived MEF cells are a half and null when compared to that of B4galt5+/+-derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5+/+-, B4galt5+/−- and B4galt5−/−-derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5+/+-, B4galt5+/−- and B4galt5−/−-derived MEF cells. However, when cell homogenates were incubated with glucosylcer-amide in the presence of UDP-[3H]Gal, Lac-Cer synthase activity in B4galt5+/−- and B4galt5−/−-derived MEF cells decreased to 41% and 11% of that of B4galt5+/+-derived MEF cells. Consistent with this...

Metal-Enhanced Fluorescence Lifetime Imaging and Spectroscopy on a Modified SERS Substrate

Ray, Krishanu; Lakowicz, Joseph R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/08/2013 Português
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In this paper, we developed a metal-enhanced fluorescence (MEF) substrate by modification of the commercially available surface enhanced Raman spectroscopy (SERS) substrate that may meet the reproducibility and sensitivity challenge of MEF. In spite of many studies and interest on MEF from a number of research groups, application to real-world situations and its commercial use remain challenging mainly due to the difficulties in fabricating reproducible MEF substrates. Specifically, one of the challenges is achieving a standardized MEF substrate for reproducible fluorescence intensity enhancement and/or changes in lifetime. The gold standard klarite substrates for SERS were coated with a thin layer of silver nanoparticles for MEF studies. To test the newly developed MEF substrates, a monolayer of streptavidin conjugated Alexa-647 was assembled on biotinylated-glass or MEF substrates. We observed over 50-fold increase in the fluorescence intensity from a monolayer of streptavidin conjugated Alexa-647 on the biotinylated MEF substrate compared to the same on glass substrate. A significant reduction in the lifetime and increased photostability of Alexa-647 on MEF substrate was observed. Fluorescence lifetime imaging was performed on the monolayer of dye assembled on the modified SERS substrates. We expect this study will serve as a platform to encourage the future use of a standardized MEF substrate for a plethora of sensing applications.

Dimethyl Fumarate and Monoethyl Fumarate Exhibit Differential Effects on KEAP1, NRF2 Activation, and Glutathione Depletion In Vitro

Brennan, Melanie S.; Matos, Maria F.; Li, Bing; Hronowski, Xiaoping; Gao, Benbo; Juhasz, Peter; Rhodes, Kenneth J.; Scannevin, Robert H.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 20/03/2015 Português
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Delayed-release dimethyl fumarate (also known as gastro-resistant dimethyl fumarate), an oral therapeutic containing dimethyl fumarate (DMF) as the active ingredient, is currently approved for the treatment of relapsing multiple sclerosis. DMF is also a component in a distinct mixture product with 3 different salts of monoethyl fumarate (MEF), which is marketed for the treatment of psoriasis. Previous studies have provided insight into the pharmacologic properties of DMF, including modulation of kelch-like ECH-associated protein 1 (KEAP1), activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway, and glutathione (GSH) modulation; however, those of MEF remain largely unexplored. Therefore, the aim of this study was to evaluate the in vitro effects of DMF and MEF on KEAP1 modification, activation of the NRF2 pathway, and GSH conjugation. Using mass spectrometry, DMF treatment resulted in a robust modification of specific cysteine residues on KEAP1. In comparison, the overall degree of KEAP1 modification following MEF treatment was significantly less or undetectable. Consistent with KEAP1 cysteine modification, DMF treatment resulted in nuclear translocation of NRF2 and a robust transcriptional response in treated cells...

Analysis of the Factors that Limit the Ability of Feeder Cells to Maintain the Undifferentiated State of Human Embryonic Stem Cells

Villa-Diaz, Luis G.; Pacut, Crystal; Slawny, Nicole A.; Ding, Jun; O'Shea, K. Sue; Smith, Gary D.
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
Português
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182.50129%
Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fibroblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC growth and differentiation, and to begin defining the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the influence of different FLs. Significant enhancement of hESC growth (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported significantly better hESC growth in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC growth were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identified 19 genes related to apoptosis with significantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifications associated with extracellular matrix (ECM) structural constituents and growth factors. Because members of Wnt signaling pathway were identified in the array analysis...

Un método de acople para mef - mec para análisis de interacción suelo - estructura

Rojas,Katherina; Lehmann,Lutz; Cerrolaza,Miguel
Fonte: Instituto de Materiales y Modelos Estructurales.Facultad de Ingenieria. Universidad Central de Venezuela. Publicador: Instituto de Materiales y Modelos Estructurales.Facultad de Ingenieria. Universidad Central de Venezuela.
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/08/2008 Português
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El Método de los Elementos Finitos y el Método de los Elementos de Contorno son las herramientas numéricas más utilizadas para análisis en mecánica de sólidos. Cada uno de estos métodos tiene sus ventajas y desventajas en diferentes casos. El MEF es adecuado para análisis de dominios finitos con materiales no homogeneos y comportamiento no lineal, mientras que MEC ofrece ventajas en el análisis de dominios infinitos con materiales homogéneos y comportamiento lineal. En este trabajo se presenta un acople iterativo del método de los elementos finitos y el método de los elementos de contorno. El dominio del problema es dividido en dos subdominios, donde cada subdominio es analizado por separado y sólo la información en la interfase es comunicada entre los dos subdominios. El ensamble y análisis de un sistema de ecuaciones general es evitado, obteniéndose ventaja de las características de las matrices, ya que el sistema de ecuaciones en el método de los elementos finitos es simétrico.Los resultados numéricos obtenidos concuerdan satisfactoriamente con resultados obtenidos por otros autores.