Este trabalho apresenta o estudo de um sistema polimérico bi-componente formado por Poli(Ácido carboxílico)-Poli(Eter-poliol), constituído primariamente de um polímero acrílico polimerizado pelo processo de polimerização em solução aquosa por mecanismo de radicais livres ao qual foi adicionado seqüencialmente, um Poli(Eter-poliol), ambos de baixo peso molecular médio. Tal sistema tem por finalidade atuar como dispersante-ligante em sistema cerâmico à base de caulim CADAM, uma vez que este sistema apresenta propriedades termoplásticas e termorrígidas a diferentes temperaturas. Foram sintetizados 03 protótipos de um sistema polimérico e o critério para escolha teve como base o pH do sistema em sentido generalizado, isto é, compreendendo as diversas fases de preparação dos polímeros e sua aplicação final. É do conhecimento comum que a reação de esterificação entre grupos COOHOH requer catálise ácida para ocorrer, o qual usualmente é realizada em pH abaixo de 4 e, neste ensaio, este pH ácido é devido à presença de ácido para-toluenosulfônico, que atua como catalisador de esterificação. Em tal intervalo de pH, em virtude do ponto isoelétrico do caulim ser comumentemente abaixo de 4...
The rifampin-resistance mutation of LS3,an asporogenous mutant of Bacillus subtilis 3610, leads to altered mobility of the beta subunit of ribonucleic acid polymerase in sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. This finding argues that the rifampin-resistance mutation is located in the structural gene coding for the beta polypeptide.
Crude cholera toxin detoxified by treatment with Formalin showed an increased mobility and a decrease in the number of detectable protein constituents when compared with the parent toxin by disc electrophoresis. The significance of these observations in relation to the enhanced antigenic activity of cholera toxoid as compared to parent toxin is discussed.
Homogenates of the mid gut and digestive glands of Rhynchosciara angelae were prepared in 40% sucrose, with and without 0·5% Triton-X-100. These were subjected to disc electrophoresis and zymograms for esterases and alkaline phosphatase were prepared. In both groups the effect of Triton-X-100 was to increase the electrophoretic mobility of the enzymes concerned.
1. Rabbit anti-(rat foetal liver) serum, absorbed with adult rat liver cells, decreased the electrophoretic mobility of foetal liver cells by 51% and rat hepatoma cells by 45%, indicating the presence of a foetal-type antigen on the hepatoma cell membrane. 2. The chemical nature of the surface antigen was investigated. Incubation with neuraminidase had no effect on adult liver cells but decreased the electrophoretic mobility of foetal liver cells by 51% and of hepatoma cells by 34%; the effect of antiserum was decreased to one-fifth. 3. Sialic acid, or the supernatant from neuraminidase-treated cells, partially blocked the decrease in electrophoretic mobility induced by antiserum. 4. The pH–electrophoretic mobility curves of hepatoma cells treated with antisera were consistent with a sialic acidcontaining antigen on the surface of the tumour cells. 5. Treatment with ribonuclease did not decrease the electrophoretic mobility of adult-liver cells, but decreased that of the foetal liver cells by 17% and hepatoma cells by 29%. 6. In parallel studies made with mouse BP8 ascites-tumour cells ribonuclease decreased the electrophoretic mobility by 39%, that of normal mouse lymph-node cells by 4.8% and allergized mouse lymph-node cells by 13.3%. 7. Trypsin treatment also decreased the electrophoretic mobility of hepatoma cells by 22%.
1. The pH–mobility relationships for saline-washed cells from a mouse strain of acute lymphoblastic leukaemia were examined before and after treatment with lower aldehydes, diazomethane and neuraminidase (EC 126.96.36.199). 2. The content of sialic acid released into the supernatant fluid of neuraminidase-treated cells was measured. 3. The stability of the charge-determining structures to temporary changes in environment (pH and ionic strength) was established. 4. Similar measurements were made on lymph-node cells obtained from non-leukaemic mice (a resistant and a leukaemia-susceptible strain were examined). 5. It is deduced that both the malignant and the non-malignant cell possess two dissociable acid functions at the cell surface, a carboxyl group of sialic acid and another acidic group(s), probably carboxyl, of pK 3·0–4·5. The malignant cells, however, have a basic dissociable function not present in the non-malignant types. 6. Suggestions are made as to how the difference in surface chemistry may be related to the problem of malignancy.
The stepwise mutation model of Ohta and Kimura (1973) was proposed to explain patterns of genetic variability revealed by means of electrophoresis. The assumption that electrophoretic mobility was principally determined by unit changes in net molecular charge has been criticized by Johnson (1974, 1977). This assumption has been tested directly using hemoglobin. Twenty-seven human hemoglobin variants with known amino acid substitutions, and 26 nonhuman hemoglobins with known sequences were studied by starch gel electrophoresis. Of these hemoglobins, 60 to 70% had electrophoretic mobilities that could be predicted solely on the basis of net charge calculated from the amino acid composition alone, ignoring tertiary structure. Only four hemoglobins showed a mobility that was clearly different from an expected mobility calculated using only the net charge of the molecule. For the remaining 30% of hemoglobins studied, mobility was determined by a combination of net charge and other unidentified components, probably reflecting changes in ionization of some amino acid residues as a result of small alterations in tertiary structure due to the amino acid substitution in the variant. For the nonhuman hemoglobins, the deviation of a sample from its expected mobility increased with increasing amino acid divergence from human hemoglobin A.—It is concluded that the net electrostatic charge of a molecule is the principal determinant of electrophoretic mobility under the conditions studied. However...
Free-flow electrophoretic separation of mouse spleen cells provides three distinct progenitor cells of direct PFC, showing high, medium and low electrophoretic mobility. All progenitor cells possess surface immunoglobulin and mouse B-lymphocyte specific antigen. The progenitor cells of high electrophoretic mobility show high cycling turnover, a spleen seeking capacity of 16%, provide PFC with a maximum 8 days after transfer and reveal an isometrical increase of the PFC dose response line as a function of the graft size. The progenitor cells of medium electrophoretic mobility are low cycling, 16% home to the spleen, a maximum of PFC is developed eight days after transfer and the PFC dose response line increases allometrically. The progenitor cells of low EPM show low cycling activity, 20% home to the spleen, a maximum of PFC is attained six days after transfer and the PFC dose response line rises isometrically. These results suggest that the electrokinetically different PFC progenitors represent biologically distinct subsets. In double transfer experiments, some evidence was obtained that progenitor cells of low electrophoretic mobility are derived from progenitors of higher electrophoretic mobility. The same observation accounts also for the formation of B lymphocytes of low EPM. Since it seemed likely that the PFC progenitor cells represent virgin cells of a single lineage...
N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO3, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1...
Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO3, pH 7.2 at 25°C the tritosomes had an electrophoretic mobility of -1.77 ± 0.02 µm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm2, and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10-7 m. Treatment of the tritosomes with 50 µg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 ± 0.02 µm/s/V/cm under the same conditions and caused the release of 2.01 µg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven...
Calcitriol (1α, 25-dihydroxyvitamin D3) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA...
The electrophoretic mobility of microscopically visible particles is independent of size, shape and conductivity of the particle within the limits of the experimental error. This is valid for extreme variations in size, shape and conductivity.
The separation of organelles by capillary electrophoresis (CE) produces large numbers of narrow peaks, which commonly are assumed to originate from single particles. In this paper, we show this is not always true. Here, we use established methods to partition simulated and real organelle CEs into regions of constant peak density and then use statistical-overlap theory to calculate the number of peaks (single particles) in each region. The only required measurements are the number of observed peaks (maxima) and peak standard deviation in the regions, and the durations of the regions. Theory is developed for the precision of the estimated peak number and the threshold saturation above which the calculation is not advisable due to fluctuation of peak numbers. Theory shows that the relative precision is good, when the saturation lies between 0.2 and 1.0, and is optimal when the saturation is slightly greater than 0.5. It also shows the threshold saturation depends on the peak standard deviation, divided by the region’s duration. The accuracy and precision of peak numbers estimated in different regions of organelle CEs are verified by computer simulations having both constant and non-uniform peak densities. The estimates are accurate to 6%. The estimated peak numbers in different regions are used to calculate migration-time and electrophoretic-mobility distributions. These distributions are less biased by peak overlap than ones determined by counting maxima and provide more correct measures of the organelle properties. The procedure is applied to a mitochondrial CE...
Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC-nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC-nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract...
Study of the post-translational modification of methionine to its sulfoxide has been receiving increasing attention because of its implication in regulation of activity, but techniques for the detection of this modification remain limited. In particular, there has been no method to detect the oxidation of methionine on polyacrylamide gels. We demonstrate that alkylation of methionine introduces a charge change that shifts the mobility of the protein on an acidic gel relative to the alkylation resistant sulfoxide form.
Lipoprotein particles (LPPs) are biological nanoparticles whose physiological roles are greatly influenced by their sizes. The four major classes of LP are: very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein (LDL) and high density lipoprotein. Since the predominance of small, dense LDLs is associated with increased risk of coronary artery disease (CAD) and diabetes mellitus, LPP profiling can be used to predict metabolic risk factors. Highly tunable LPP mimics can be synthesized using nanoparticles to carefully control for size, lipid composition and surface charge to facilitate the study LPPs in CAD. Here, we engineered LPP mimics using gold nanoparticles between 10–50 nm in diameters. We measured the mobility and zeta potential of these LPP mimics and showed that each mimics have distinct electrokinetic properties and are electrostatically stable.
Tumour supernatants were tested on normal spleen cells electrophoretic mobility. In the case of two tumours, characterized by a fast growth rate (RV2 and VFM1), supernatants produced an increase in the mean electrophoretic mobility. For the two other tumours, characterized by a slow growth rate (VMM2 and VMM1), supernatants produced a decrease in the mean electrophoretic mobility. Electrophoretic mobility analysis of mouse spleen cells showed that after treatment with RV2 tumour supernatant, the percentage of 'slow' cells decreased by about 20%. VMM2 supernatant produced an increase in 'slow' cells of about 20%. The effect of dialysed supernatants (with mol. wt cutoff 12,000) was different. VMM2 and VMM1 dialysed supernatants modify spleen cells mobility as VMM2 and VMM1 undialysed supernatants. RV2 and VFM1 dialysed supernatants induced a significant slowing in the mobility in regard to RV2 and VFM1 undialysed supernatants. RV2 and VMM2 tumour supernatants were fractionated into four fractions on Sephacryl S-300. Fraction II (mol. wt of about 400,000) from VMM2 supernatant was found to reduce significantly the spleen cells' mobility. In contrast, fraction IV (mol. wt less than 12,000) from RV2 supernatant increases significantly the mobility...
In embryonic nuclei of Drosophila virilis, 45% of the DNA is satellite, and congruent to 50% of the H1 histone is phosphorylated. In polytene salivary gland nuclei, less than 1% of the DNA is satellite, and less than 10tion. The phosphorylated H1's migrate 4% slower than the unphosphorylated H1's on SDS-acrylamide gels. The mobility difference may arise because the phosphorylated and unphosphorylated H1's have different conformations in SDS. This putative conformational difference could be essential to the compaction of satellite DNA into heterochromatin.
Numerical solutions of the standard electrokinetic model provide a basis for
interpreting a variety of electrokinetic phenomena involving `bare' colloids.
However, the model rests on the classical notion of a shear or slipping plane,
whose location is unknown when surfaces are coated with permeable polymer.
Consequently, an electrokinetic model for `soft', `hairy' or `fuzzy' colloids
has been developed, but until recently solutions were available only for
several restricted cases, most notably for particles with thin, uniform layers,
and without polarization and relaxation. Here we present numerically exact
solutions of the full model for a variety of soft colloids, including
PEG-coated liposomes, PEO-coated latices, human erythrocytes, and
polyelectrolyte micelles. Particular attention is given to linking the
thickness, density and permeability of the coatings, which are key parameters
in the model, to ``physical'' quantities, such as the polymer molecular weight,
adsorbed amount, and hydrodynamic layer thickness. This paper also identifies
limits--on the ionic strength, particle size, layer thickness and
permeability--beyond which earlier theories breakdown. In short, polarization
and relaxation are as influential on the mobility of soft colloids as they are
for `bare' particles.
Cugia, Francesca; Monduzzi, Maura; Ninham, Barry; Salis, Andrea
Fonte: Royal Society of ChemistryPublicador: Royal Society of Chemistry
Tipo: Artigo de Revista Científica
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Ion specificity is a long standing unsolved puzzle of chemistry and biology. Myriad experiments underline its universality. We have studied ion specific effects on different buffers at the same nominal bulk pH. We used both electrophoretic light scatterin