Bovine bone ash is the main raw material for fabrication of bone china, a special kind of porcelain that has visual and mechanical advantages when compared to usual porcelains. The properties of bone china are highly dependent on the characteristics of the bone ash. However, despite a relatively common product, the science behind formulations and accepted fabrication procedures for bone china is not completely understood and deserves attention for future processing optimizations. In this paper, the influence of the preparation steps (firing, milling, and washing of the bones) on the physicochemical properties of bone ash particles was investigated. Bone powders heat-treated at temperatures varying from 700 to 1000 degrees C were washed and milled. The obtained materials were analyzed in terms of particle size distribution, chemical composition, density, specific surface area, FTIR spectroscopy, dynamic electrophoretic mobility, crystalline phases and scanning electron microscopy. The results indicated that bone ash does not significantly change in terms of chemistry and physical features at calcination temperatures above 700 degrees C. After washing in special conditions, one could only observe hydroxyapatite in the diffraction pattern. By FTIR it was observed that carbonate seems to be mainly concentrated on the surface of the powders. Since this compound can influence in the dispersion stability...
A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-μl samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of Klebsiella strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains of Klebsiella pneumoniae and 24 strains of Klebsiella oxytoca demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains.
Esterase electrophoretic polymorphism in human and animal strains of Clostridium perfringens was studied by using polyacrylamide-agarose gel electrophoresis. Five types of esterases, designated E-I to E-V and defined by their hydrolytic specificities toward five synthetic substrates, were found in protein extracts of bacteria grown without glucose (glucose-containing media allowed only the expression of esterase E-I). Mobility variants of esterase E-I, which hydrolyzes alpha- and beta-naphthyl acetates and butyrates, were used as a basis for the distribution of strains into 11 zymogroups. When all five types of esterases and their electrophoretic variants were considered, 77 electrophoretic types (ETs) could be described for the 89 strains tested. Animal strains did not constitute a distinctive subpopulation, as revealed by their distribution in the zymogroups and by clustering analysis. Statistical analysis also emphasized the importance of esterase E-IV (which hydrolyzes only naphthyl acetates) and esterase E-V (which hydrolyzes only alpha-naphthyl acetate) in clustering by the relatedness of the ETs. ETs allowed the epidemiological characterization of stool isolates recovered from elderly inpatient residents and from adolescent chronic-care psychiatric patients. These results indicate that esterase electrophoretic typing may be a marker for epidemiological and ecological analyses.
Grossman, P D; Bloch, W; Brinson, E; Chang, C C; Eggerding, F A; Fung, S; Iovannisci, D M; Woo, S; Winn-Deen, E S; Iovannisci, D A
Fonte: PubMedPublicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/10/1994EN
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We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.
In vesicular stomatitis virus New Jersey serotype polyacrylamide gel electrophoresis was unable to distinguish the polypeptides of the temperature-sensitive (ts) mutants of complementation groups A, B, C, and F from those of the wild-type virus. However, the NS polypeptide of the representative mutant of group E, ts E1, had a significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide. The electrophoretic mobilities of the NS polypeptides of the three mutants of complementation group E varied, being greatest in the case of ts E1, slightly less for ts E2, and only a little greater than that of wild-type virus NS polypeptide in the case of ts E3. Since the NS polypeptides of the revertant clones ts E1/R1 and ts E3/R1 have mobilities identical to that of wild-type NS polypeptide, the observed altered mobilities of the group E mutants are almost certainly the direct result of the ts mutations in the E locus. The electrophoretic mobilities of the intracellular NS polypeptides of the group E mutants were indistinguishable from those of their virion NS polypeptides. The electrophoretic mobilities of the NS polypeptides of the group E mutants synthesized in vitro using mRNA synthesized in vitro by TNP were identical to those of the NS polypeptides of their purified virions. The NS polypeptides of all three mutants were labeled with 32Pi to approximately the same extent as wild-type virus NS polypeptide...
The envelope glycoproteins of several avian tumor virus recombinants selected for the host range of a leukosis virus and the transforming function of a sarcoma virus were compared with each other and with those of their parents. It was found that the glycoproteins of different recombinant viruses, derived from the same parents, differed in their electrophoretic mobilities measured in polyacrylmide gels. The glycoproteins that had lower electrophoretic mobilities had higher precentages of carbohydrate. The carbohydrate of viral glycoproteins was estimated to range between 8 and 18% from their buoyant densities in CsCl, using known glycoproteins as standards. After exhaustive Pronase digestion, the carbohydrate was recovered from viral glycoproteins as a mixture of glycopeptides with molecular weights ranging from 2,500 to 5,000. It was estimated that distinct viral glycoproteins contained between two and five such oligosaccharide chains and that the glycoproteins of different recombinants expressing the same host range marker may differ in the number of oligosaccharide chains and consequently also in their polypeptide structure. Those with lower electrophoretic mobility contain more oligosaccharide chains per molecule than those with higher electrophoretic mobilities. It is suggested that not oligosaccharide chains define the viral host range.
When subjected to electrophoresis in polyacrylamide gels, the virions of wild-type Qβ bacteriophage are found in a single, major, anomalously wide band. With Qβ mutant 27-2, this wide band is replaced by a set of narrow, well-defined bands. The most rapidly migrating band of the mutant, comprising less than 10% of the total, contains defective virions. These virions have sedimentation coefficients ranging from 70 to 100% of the bulk of the unfractionated mutant, they contain no read-through protein (protein IIb), and they are deficient in maturation protein and contain fragmented RNA. The second band, comprising less than 3% of the total virus, has not been well characterized. The virions in the remaining electrophoretic bands are infective. Their distribution into bands is believed due to differences in their effective volume resulting from differences in their content of protein IIb. The most rapidly migrating band of this series contains virions with a few molecules of IIb protein, whereas the more slowly migrating bands contain virions with a larger number of IIb molecules. The adjacent bands in the series contain virions which differ by approximately three IIb molecules. Wild-type Qβ virus is similar to the mutant in that the more slowly migrating virions contain more protein IIb than the more rapidly migrating virions. Their failure to resolve into distinct bands upon electrophoresis is believed due to a less restricted packing of protein IIb into their virions. Both wild-type Qβ and mutant 27-2 also have 1 to 5% of the virions in the form of dimers which migrate with approximately one-half the mobility of the respective monomer forms. When the average amount of IIb per virion is increased by growth of the virus in a UGA suppressor strain...
Plasma fibronectin was depleted within 15 min following sublethal burn, followed by partial recovery at 8 h and complete restoration by 24 h in anesthetized rats. Radiolabeled 75Se-plasma fibronectin, injected intravenously before burn, was rapidly sequestered in burn skin as well as the liver. Fibronectin levels at 2 h postburn as detected by immunoassay vs. 75Se-plasma fibronectin indicated that more fibronectin was in the plasma than detected by electroimmunoassay. Crossed immunoelectrophoretic analysis of fibronectin in early postburn plasma demonstrated a reduced electrophoretic mobility of the fibronectin antigen. Addition of heparin or fibrin, both of which have affinity for fibronectin, to normal plasma was unable to reproduce this altered fibronectin electrophoretic pattern. In contrast, addition of gelatin or native collagen to normal plasma reproduced the abnormal electrophoretic pattern of fibronectin seen in burn plasma. Extracts of burned skin, but not extracts of normal skin, when added to normal plasma, elicited a similar altered electrophoretic pattern for fibronectin. By gel filtration, fibronectin in burn plasma had an apparent molecular weight approximately 40% greater than that observed in normal plasma. These data suggest the release into the blood of a gelatinlike ligand from burned skin...
The electrophoretic mobilities of mononuclear white cells from the peripheral blood of normal subjects and leukemic patients were measured. Analysis of both fresh and cryopreserved samples of each type showed that cryopreservation had no distinguishable effect on the electrophoretic distribution of either normal or leukemic cells. Mode mobilities (mobilities at which maximum intensities are observed) of cells from nine leukemic patients were 7-28% below the average mode mobility of normal cells. The standard deviation of the electrophoretic mobilities of normal cells was 2%. Normal cells had an asymmetric, often bimodal, electrophoretic distribution; leukemic cells consistently had a single symmetric distribution.
Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of −3.55μm·s−1·V−1·cm in saline–sorbitol, pH7.2, at 25°C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pKa in the range 3.0–3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn2+ or Ca2+ was more effective in decreasing vesicle mobility than was Mg2+, Sr2+ or Ba2+. The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.
Electrophoretic properties of eight lysosomal hydrolases and 36 nonlysosomal enzymes were investigated in cultured fibroblasts from children with the inherited storage disease mucolipidosis II (ML II); fibroblasts from a child with a related disorder, mucolipidosis III (ML III); and two obligate heterozygous cell lines from parents of a ML II child. Cell homogenates of ML II fibroblast lines showed altered mobilities for lysosomal beta-hexosaminidase, acid phosphatase2, and alpha-mannosidase and deficient activity for the esterase-A4 and lysosomal alpha-mannosidase-B electrophoretic phenotypes. Altered mobility was also detected for the nonlysosomal enzyme adenosine deaminase-d. Deficient activities of other lysosomal enzymes were observed as previously reported. In a single ML III fibroblast line, only beta-hexosaminidase showed an abnormal electrophoretic pattern suggesting a difference between these cells and ML II fibroblasts. Thirty-five nonlysosomal enzymes associated with other cellular organelles and metabolic pathways were electrophoretically normal in all mucolipidosis cell lines. Heterozygous ML II cells showed normal expression for all enzymes. Two major patterns of altered lysosomal enzymes and adenosine deaminase were demonstrated in ML II cell lines...
To detect new genetic variation in human plasma proteins, a panel of 63 radioactive substances were screened as potential radioligands using polyacrylamide gel electrophoresis (PAGE) and autoradiography. Vitamins, hormones, drugs, amino acids, purines, pyrimidines, sugars and lipids labeled with 14C or other radionuclides were among those substances tested. A majority bound to albumin and a smaller fraction to prealbumins and lipoproteins. Several vitamins and hormones bound to specific alpha and beta globulins. (1) Electrophoretic polymorphisms of vitamin D-binding protein (group-specific component), a vitamin B12-binding protein (transcobalamin II), and thyroxine-binding alpha-globulin are described elsewhere. (2) Testosterone-binding beta-globulin (TeBG) showed an electrophoretic polymorphism in Caucasians and a possible deficiency allele. (3) Transcortin showed an electrophoretic doublet in all persons tested but no electrophoretic variation. (4) A protein binding derivative of norepinephrine or epinephrine was identified as transferrin. (5) A nonpolymorphic protein running cathodal to albumin and binding a derivative of riboflavin was tentatively identified as a fraction of albumin with mobility altered as a result of interaction with the ligand.
Electrophoretic patterns of normal dog plasma in veronal buffer at pH 8.5 are shown to be essentially similar to patterns of human plasma. Dog albumin has a higher mobility than human albumin and in a mixture of dog and human plasmas migrates as a partially separated peak. Normal dog plasma frequently shows four alpha globulin peaks. Rates of restoration of plasma protein components in dogs subjected to acute plasmapheresis have been studied by electrophoresis. During the first 24 hours following such acute depletion, appreciable quantities of all electrophoretic components of the plasma proteins enter the circulating blood stream even when food is not given and has not been given for 12 hours before plasmapheresis. In such fasting periods albumin and total globulin appear in approximately the proportions present in normal plasma. Alpha and beta globulins continue relatively elevated during subsequent days in which caloric and protein intakes are adequate for weight and nitrogen gains. Initial albumin levels, however, are regained more slowly than those of total globulin. The relative proportions of the electrophoretic components of plasma proteins may be disturbed from normal following a single acute depletion for as long as 2 to 3 weeks after the total protein level has returned to normal. Abnormally high beta globulin and fibrinogen...
Agarose-gel electrophoresis has been used for more than thirty years to characterize the linking-number (Lk) distribution of closed-circular DNA molecules. Although the physical basis of this technique remains poorly understood, the gel-electrophoretic behavior of covalently closed DNAs has been used to determine the local unwinding of DNA by proteins and small-molecule ligands, characterize supercoiling-dependent conformational transitions in duplex DNA, and to measure helical-repeat changes due to shifts in temperature and ionic strength. Those results have been analyzed by assuming that the absolute mobility of a particular topoisomer is mainly a function of the integral number of superhelical turns, and thus a slowly varying function of plasmid molecular weight. In examining the mobilities of Lk topoisomers for a series of plasmids that differ incrementally in size over more than one helical turn, we found that the size-dependent agarose-gel mobility of individual topoisomers with identical values of Lk (but different values of the excess linking number, ΔLk) vary dramatically over a duplex turn. Our results suggest that a simple semi-empirical relationship holds between the electrophoretic mobility of linking-number topoisomers and their average writhe in solution.
A pH controlled flow cell device was constructed to allow electrophoretic movement of charged lipids and membrane associated proteins in supported phospholipid bilayers. The device isolated electrolysis products near the electrodes from the electrophoresis process within the bilayer. This allowed the pH over the bilayer region to remain within ±0.2 pH units or better over many hours at salt concentrations up to 10 mM. Using this setup, it was found that the electrophoretic mobility of a dye conjugated lipid (Texas Red-DHPE) was essentially constant between pH 3.3 and 9.3. By contrast, streptavidin, which was bound to biotinylated lipids, shifted from migrating cathodically at acidic pH values to migrating anodically under basic conditions. This shift was due to the modulation of the net charge on the protein, which changed the electrophoretic forces experienced by the macromolecule. The addition of a PEG cushion beneath the bilayer or the increase in the ionic strength of the buffer solution resulted in a decrease of the electroosmotic force experienced by the streptavidin with little effect on the Texas Red-DHPE. As such, it was possible in part to control the electrophoretic and electroosmotic contributions to streptavidin independently of one another.
Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite’s life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by “click” chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment...
Fonte: Centro Comum de Pesquisa da Comissão EuropéiaPublicador: Centro Comum de Pesquisa da Comissão Européia
Tipo: Articles in JournalsFormato: Printed
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Four mammalian metallothioneins (MTs), rabbit liver MT (RL MT), rabbit liver MT-1 (RL MT-1) and rabbit liver MT-2 (RL MT-2) and horse kidney MT (HK MT), weresubjected to capillary zone electrophoresis (CZE) with on-line diode array detection (DAD) by using tris-borate buffer at neutral or nearly neutral conditions thus giving information about the putative isoforms in conditions also found in tissues. The MTs were found to exhibit a different polymorphism. Rabbit liver MT exhibited 3 main peaks and horse kidney 5 main peaks. Isoform abundance between batches was found to vary noticeably. Differences in electrophoretic behaviour were observed. As expected, the 2-charged main peak of RL MT-2 showed highest electrophoretic mobility. Main peak of RL MT-1 and two of rabbitliver MT had lower electrophoretic mobilities corresponding to single charge. Two later migrating horse kidney MT peaks had nearly similar electrophoretic behaviour as the single charged rabbit liver MT peaks. interestingly, horse kidney MT exhibited also peaks with lower electrophoretic mobilities and different behaviour than the other peaks observed. The ultra violet (UV) spectra obtained by using diode array detection (DAD) showed clear difference in the structure of these peaks compared to other peaks observed. These peaks showed no or nearly no absorbance ate wavelenghts 225 and 250 nm that corresponds to cation-thiol bondage...
alpha 2-Macroglobulin (alpha 2M) undergoes a major conformational change when reacting with proteinases or primary amines. This conformational change has been referred to as the 'slow' to 'fast' transformation based on the increase in alpha 2M mobility shown by non-denaturing PAGE. Previous studies demonstrated that many cytokines, including transforming growth factor beta 1 (TGF-beta 1) and interleukin-1 beta, bind preferentially or exclusively to alpha 2M which has undergone conformational change. In this study, we demonstrate that platelet-derived growth factor-BB (PDGF-BB) also binds preferentially to conformationally transformed alpha 2M (alpha 2M-methylamine, alpha 2M-trypsin) in vitro. Purified 125I-PDGF-BB-alpha 2M-methylamine complex cleared rapidly from the circulation of mice via the alpha 2M receptor/low-density-lipoprotein-receptor-related protein (alpha 2M-R/LRP). In order to determine whether PDGF-BB or TGF-beta 1 binds to native alpha 2M, we defined the native conformation by lack of interaction with alpha 2M-R/LRP instead of electrophoretic mobility. 125I-PDGF-BB was incubated with 4.3 microM native alpha 2M and 0.47 microM alpha 2M-methylamine. The 125I-PDGF-BB distributed evenly between slow-form and fast-form alpha 2M without shifting the electrophoretic mobility of either species. When the mixed preparation was injected intravenously in mice...
We recently found, using cultured mouse cell systems, that newly synthesized catalytic (C) subunits of cyclic AMP-dependent protein kinase undergo a posttranslational modification that reduces their electrophoretic mobilities in sodium dodecyl sulfate (SDS)-polyacrylamide gels and activates them for binding to a Sepharose-conjugated inhibitor peptide. Using an Escherichia coli expression system, we now show that recombinant murine C alpha subunit undergoes a similar modification and that the modification results in a large increase in protein kinase activity. Threonine phosphorylation appears to be responsible for both the enzymatic activation and the electrophoretic mobility shift. The phosphothreonine-deficient form of C subunit had reduced affinities for the ATP analogs p-fluorosulfonyl-[14C]benzoyl 5'-adenosine and adenosine 5'-O-(3-thiotriphosphate) as well as for the Sepharose-conjugated inhibitor peptide; it also had markedly elevated Kms for both ATP and peptide substrates. Autophosphorylation of C-subunit preparations enriched for this phosphothreonine-deficient form reproduced the changes in enzyme activity and SDS-gel mobility that occur in intact cells. A mutant form of the recombinant C subunit with Ala substituted for Thr-197 (the only C-subunit threonine residue known to be phosphorylated in mammalian cells) was similar in SDS-polyacrylamide gel electrophoresis mobility and activity to the phosphothreonine-deficient form of wild-type C subunit. In contrast to the wild-type subunit...
The charge inversion phenomenon is studied by molecular dynamics simulations,
focusing on size and valence asymmetric salts, and a threshold of surface
charge density for charge inversion. The charge inversion criteria by the
electrophoretic mobility and the radial distribution functions of ions coincide
except around the charge inversion threshold. The reversed electrophoretic
mobility increases with the ratio of coion to counterion radii, while it
decreases with the ratio of coion to counterion valences. The monovalent salt
enhances charge inversion of a strongly charged macroion at small ionic
strength, but it reduces reversed mobility otherwise. A cylindrical macroion is
more persistent to monovalent salt than a spherical macroion of the same radius
and surface charge density.; Comment: 9 pages, 9 figures (two in color). Physical Review E, in press