Electrophoretic Forms Research Articles

The level of matrix metalloproteinases in the blood in a model of adjuvant-induced arthritis and its correction with antisense oligonucleotides

Rheumatoid arthritis is characterized by significant rearrangements in bone and cartilage tissues mediated by matrix metalloproteinases (MMPs). One of the directions in the development of new drugs for rheumatoid arthritis is the search for new targets, including MMPs or their inhibitors. The work aimed to determine the levels of MMP-1, 3, and 9 in the peripheral blood of laboratory rats with a model of adjuvant-induced arthritis during the correction of this condition with antisense oligonucleotides that block the synthesis of tumor necrosis factor and interleukin 6. In laboratory Wistar rats, arthritis was modeled by administering Freund's adjuvant and then correcting the disease by introducing antisense oligonucleotides subcutaneously and using electrophoresis. The level of MMP in peripheral blood was determined at the beginning of treatment, after 1, 2, and 3 months. Chronic inflammation in the experiment was accompanied by an increase in the level of MMP-1, 3, and 9 in the peripheral blood by 2, 4.5, and 0.5 times, respectively. Blocking the proinflammatory cytokines TNF-α and IL-6 with antisense oligonucleotides was effective in reducing MMP levels. In most cases, it decreased significantly faster than in the untreated arthritis model. These changes were most pronounced in groups using tocilizumab, as well as in group with an electrophoretic form of administration of a combination of two oligonucleotides.

Novel α-1,3/α-1,4-Glucosidase from Aspergillus niger Exhibits Unique Transglucosylation to Generate High Levels of Nigerose and Kojibiose.

α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of N-glycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)- and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4-glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short- and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (<15 min) synthesized 3II- O-α-glucosyl-maltose and maltotriose, indicating α-1,3-/α-1,4-transferring specificity. A long-term reaction (<24 h) accumulated kojibiose and nigerose using formed glucose as an acceptor substrate. AgdA and AgdB are distinct α-glucosidases. At a high concentration of glucose added exogenously, AgdB largely generated the rare sugars kojibiose and nigerose (exhibiting beneficial physiological functions) with 19% and 24% yields from maltose, respectively.

Geographic and climatic differentiation of electrophoretic forms of esterase, glutamate dehydrogenase and peroxidase in Scots pine tissues

The enzymatic systems have been investigated in individual trees derived from 17 distant localities in Europe and Asia. Esterase and glutamate dehydrogenase polymorphism was determined in extracts from mixture of 50 macrogametophytes, iperoxidase polymorphism was investigated in needle extracts from 3-month-old seedlings. While testing by disc electrophoresis needle extracts of Scots pine deriving from specimens of different geographic ranges it was found that those from the South displayed mare isoperoxidases than those from the North. Most pronounced differences in number and relative activity of esterase and glutamate dehydrogenase isozymes were found in the specimens originating from isolated stands. It is suggested that hight temperatures and dry summer periods could influence the number and activity of the isozymes.

Phosphorylation of Yeast Phosphatidylserine Synthase by Protein Kinase A: IDENTIFICATION OF SER46 AND SER47 AS MAJOR SITES OF PHOSPHORYLATION

The CHO1-encoded phosphatidylserine synthase from Saccharomyces cerevisiae is phosphorylated and inhibited by protein kinase A in vitro. CHO1 alleles bearing Ser(46) --> Ala and/or Ser(47) --> Ala mutations were constructed and expressed in a cho1Delta mutant lacking phosphatidylserine synthase. In vitro, the S46A/S47A mutation reduced the total amount of phosphorylation by 90% and abolished the inhibitory effect protein kinase A had on phosphatidylserine synthase activity. The enzyme phosphorylation by protein kinase A, which was time- and dose-dependent and dependent on the concentration of ATP, caused a electrophoretic mobility shift from a 27-kDa form to a 30-kDa form. The two electrophoretic forms of phosphatidylserine synthase were present in exponential phase cells, whereas only the 27-kDa form was present in stationary phase cells. In vivo labeling with (32)P(i) and immune complex analysis showed that the 30-kDa form was predominantly phosphorylated when compared with the 27-kDa form. However, the S46A/S47A mutations abolished the protein kinase A-mediated electrophoretic mobility shift. The S46A/S47A mutations also caused a 55% reduction in the total amount of phosphatidylserine synthase in exponential phase cells and a 66% reduction in the amount of enzyme in stationary phase cells. In phospholipid composition analysis, cells expressing the S46A/S47A mutant enzyme exhibited a 57% decrease in phosphatidylserine and a 40% increase in phosphatidylinositol. These results indicate that phosphatidylserine synthase is phosphorylated on Ser(46) and Ser(47) by protein kinase A, which results in a higher amount of enzyme for the net effect of stimulating the synthesis of phosphatidylserine.

Capillary zone electrophoresis of Cowpea mosaic virus and peak identification

Cowpea mosaic virus (CPMV) was analyzed by CZE and the two separated subcomponents recorded at approximately 2.2 and approximately 2.7 min were respectively designated to be the slow electrophoretic form of CPMV (CPMV(s)) and the fast electrophoretic form of CPMV (CPMV(f)), the two electrophoretic forms of CPMV, by the following methods: first, CZE analysis of the biospecific complexes of CPMV and anti-CPMV polyclonal antibody proved the properties of CPMV; second, isolation and CZE analysis of CPMV(f) and CPMV(s). Because CPMV(s) was precipitated and CPMV(f) was left in the supernatant at pH 5.6, the only one peak at approximately 2.7 min from the supernatant was assigned for CPMV(f), while the peak at approximately 2.2 min dominating for the precipite was deemed to be CPMV(s). Third, viral characterization by directly analyzing the capsid proteins using MALDI-TOF-MS. Based on the theoretical molecular weights calculated from the sequence of the capsid proteins, the three peaks at m/z 21 516.75, 23 745.8 and 42 509.22 for natural CPMV were destined for the small (S) protein of CPMV(f), the S protein of CPMV(s) and the large (L) protein of both of them, respectively. As expected, the non-appearance of the peak at m/z 23 745.8 for the isolated CPMV(f) sample indicated the absence of CPMV(s), and the peak at m/z 23 745.8 was predominant in the spectrum for the precipitated CPMV(s) sample. After the confirmation of CPMV(s) and CPMV(f), the CZE separation of them was optimized. The developed analysis method has proven useful in investigating the stability of CPMV and the effect of 2,4-dinitrophenyl-decoration on the modification of the electrophoretical behavior of CPMV.

Esterases of the snails Helix pomatia and Cepaea hortensis. Variation and characterization of different molecular forms.

The esterase isoenzyme variation within two populations of Cepaea hortensis and one population of Helix pomatia was investigated. Different molecular forms of esterases were characterized by electrophoretic mobility, substrate preference, inhibitor/activator characteristics, molecular weight determined by gel filtration and ultracentrifugation, behaviour in response to different enzymes affecting enzyme structure and isoenzyme variation. In C. hortensis, 23 different molecular forms were identified and in H. pomatia, 14. The use of several different compounds to characterize the enzymatic specificity and the determination of the molecular weights render possible the individualization of the molecular forms. Ultracentrifugation and gelfiltration gave different molecular weight values for the same electrophoretic molecular form, which might be explained by the properties of the enzyme molecule and attachment to membrane structures. Several of the molecular forms appear to be lipoproteins, and data point to the possibility of non-specific esterases being aggregates of lipoproteins (subunits or different enzymes) with a possible dissociation into low molecular weight units.

Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus).

Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was undertaken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.

Regulation of the Yeast Ace2 Transcription Factor during the Cell Cycle*

Previous studies have revealed many parallels in the cell cycle regulation of the Ace2 and Swi5 transcription factors. Although both proteins begin entry into the nucleus near the start of mitosis, here we show that Ace2 accumulates in the nucleus and binds DNA about 10 min later in the cell cycle than Swi5. We used chimeric fusions to identify the N-terminal region of Ace2 as responsible for the delay, and this same region of Ace2 was required for interaction with Cbk1, a kinase necessary for both transcriptional activation by Ace2 and asymmetric distribution of Ace2. Ace2 and Swi5 also showed differences in prevalence during the cell cycle. Swi5 is apparently degraded soon after nuclear entry, whereas constant Ace2 levels throughout the cell cycle suggest Ace2 is exported from the nucleus. Our work suggests that the precise timing of Ace2 accumulation in the nucleus involves both a nuclear export sequence and a nuclear localization signal, whose activities are regulated by phosphorylation.

Bridging the gap between molecular epidemiologists and evolutionists

Molecular epidemiology designates the various molecular methods that aim to identify the relevant units of analysis of pathogens involved in transmissible diseases: species, subspecies, strains, clones and genes of interest. It is frequently based on an empirical approach. I advocate that evolutionary concepts enrich this discipline considerably and should be considered as an integral part. In turn, the experience and questioning of field experts are crucial to evolutionists who use transmissible diseases as models. A molecular epidemiology aim gives evolutionary studies a practical goal, putting a stop to approaches that are overly speculative.

Changes in the proteomic profile during differentiation and maturation of human monocyte-derived dendritic cells stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 and lipopolysaccharide

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation.

Novel electrophoresis mechanism based on synchronous alternating drag perturbation

We present a novel means of transporting molecules in solution by applying a zero-time-average alternating motive force to the molecules, and perturbing the molecular drag coefficient synchronously with the applied force, thus causing a net drift in a direction determined by the phase of the alternating drag perturbation relative to the alternating force. We apply an electrophoretic form of the method to transport and concentrate DNA in a gel, such that all molecules migrate on average away from the nearest electrode and toward a central region. Since an electrode does not occupy this central region, this method presents the possibility of transporting and focusing DNA and other charged molecules in regions free from electrodes and the associated electrochemistry.

Characterization of porcine plasma ficolins that bind Actinobacillus pleuropneumoniae serotype 5B

Ficolins are collagenous lectins implicated in resistance to infection by some micro-organisms with surfaces containing N-acetylglucosamine residues. Pigs have two known ficolin genes, alpha and beta, but ficolins in pig plasma appear in various electrophoretic forms, the origin and function of which are unclear. In this investigation the forms of ficolin in pig plasma that bind to the pneumonic pathogen Actinobacillus pleuropneumoniae serotype 5b (APP5) were compared with affinity-purified porcine plasma ficolins. APP5-bound ficolins consisted of two distinct multimeric (non-denatured) forms composed of subunits that migrated as multiple 38,40 and 42 kDa forms (apparent MW) with pI range 5.2-6.0. The APPS-binding forms of ficolin were consistent with ficolin alpha and were indistinguishable from affinity-purified plasma ficolins by peptide mass fingerprint analysis. Subunit bands separated by 2D-PAGE were consistent with porcine ficolin alpha. Affinity-purified plasma ficolins had a major subunit mass of 35081 Da by MALDI-TOF MS. Affinity-purified ficolins digested with N-glycosidase F retained APP5-binding activity and migrated faster as a single band of 38 kDa. These studies indicate that ficolin alpha is the major APPS-binding form in porcine plasma and suggest that N-glycosylation contributes to the apparent electrophoretic heterogeneity of porcine ficolins but is not required for APP5-binding activity.

Mutation of the protein tyrosine kinase consensus site in the herpes simplex virus 1 alpha22 gene alters ICP22 posttranslational modification.

We previously reported that at least eight HSV-1 and five HSV-2 proteins were tyrosine phosphorylated in infected human and mouse cells and the first phosphotyrosine-modified gene product identified was the ICP22 regulatory protein (Blaho, J. A., Zong, C. S., and Mortimer, K. A., 1997, J. Virol. 71, 9828-9832). All electrophoretic forms of ICP22 are tyrosine phosphorylated with the exception of the fastest migrating (unmodified) isoform. We now report the following. (i) ICP22 that reacted with a specific anti-phosphotyrosine antibody contained a significant amount of phosphotyrosine based on phospho-amino acid analysis. These results validate the discovery of ICP22 tyrosine phosphorylation. (ii) Wild-type ICP22 extracted from infected HEp-2 cells migrated as at least seven isoforms, termed ICP22a-g, in denaturing gels. (iii) The primary structure of ICP22 possesses a sequence that is homologous to protein tyrosine kinase recognition sites. A virus, termed RF141, was generated in which ICP22 tyrosine(193) in the kinase target site was mutated to an alanine. (iv) Biochemical analyses of infected HEp-2 and primary HFF cells indicated that the distributions of ICP22 isoforms differed between RF141 and control HSV-1(F). (v) The accumulations of representative viral polypeptides in RF141-infected HEp-2 cells appeared similar to wild-type virus. (vi) RF141 had reduced efficiencies of plating in HFF cells compared to control Vero cells. These differences increased as the multiplicity of infection decreased. Based on these results, we conclude (vii) that ICP22 tyrosine(193) is required for optimal posttranslational modification of the protein in HSV-1 infected human epithelial HEp-2 and primary human fibroblast cells.

Herpes Simplex Virus Infection Blocks Events in the G1 Phase of the Cell Cycle

Infection of cells in G1 phase with herpes simplex virus (HSV) prevents their progression into S phase (de Bruyn Kops, A., and Knipe, D. M., 1988, Cell 55, 857–868). We have examined G1-phase events in infected cells to determine whether this effect was the result of inhibition of G1 phase progression or of entry into S phase. We observed that HSV infection decreased pRb phosphorylation and induced a new phosphorylated form of pRb. Furthermore, HSV infection prevented the normal G1 increases in cyclin D1 and D3 protein levels, and blocked the normal G1 appearance of new electrophoretic forms of cdk2 and cdk4. Thus, HSV infection inhibits several events that normally occur in the cell cycle during G1 phase, arguing that the HSV-induced block in the cell cycle occurs in early to mid-G1 phase.

Isozyme variation within and among populations of Microsporum species.

Isozyme variation among 54 isolates of Microsporum canis, 18 Microsporum cookei isolates and two Diheterospora isolates were studied using starch gel electrophoresis. Of eight enzymes examined, four were polymorphic (EST, G6P, MDH and PEP), having from two to four electrophoretic forms. Within each species, consistent and reproducible isozyme patterns of the eight enzyme systems were obtained. Phenotypic diversity (H) in M. canis was higher than in M. cookei (H = 0.459 and H = 0.408 respectively), but phenotypic differentiation of M. canis isolates from different geographical regions (Auckland, Wellington and Palmerston North, New Zealand) was low, with a proportion of total diversity (Gst) of 0.151 found among the localities. The results suggest that the isolates of M. canis from different geographical regions are closely related, supporting the theory of a common lineage.

Electrophoretic forming of ceramics in aqueous media

La deposici.n electrofor.tica es un proceso de conformado particularmente sencillo en el que, al hacer pasar una corriente a trav.s de una suspensi.n coloidal o una disoluci.n, se obtienen recubrimientos sobre materiales conductores. La mayor parte de los materiales org.nicos y minerales pueden ser procesados por electrodeposici.n, siendo la mayor dificultad la selecci.n del veh.culo l.quido y el ajuste de las condiciones experimentales al sistema seleccionado. En el conformado por v.a l.quida el .xito del proceso radica en la preparaci.n de suspensiones estables de determinadas caracter.sticas el.ctricas. En este sentido el uso de veh.culos org.nicos presenta grandes ventajas, ya que evitan en gran medida los procesos electrol.ticos que tienen lugar en el entorno del electrodo, y que por tanto afectan a la homogeneidad y posible contaminaci.n del recubrimiento. No obstante, el uso de org.nicos supone el encarecimiento de un proceso que se caracteriza por sus ventajas econ.micas y ecol.- gicas. Por ello, los procesos de electroforesis a partir de medios acuosos, est.n recibiendo una creciente atenci.n dadas las enormes posibilidades para su implantaci.n en ciclos de producci.n de una amplia gama de materiales, como de hecho ocurre en la obtenci.n de recubrimientos de arcillas y esmaltes. En este trabajo se lleva a cabo una revisi.n de la fisicoqu.mica del proceso as. como de sus posibilidades de aplicaci.n en la fabricaci.n de productos cer.micos y v.treos en medio acuoso

Electrophoretic Forming of Al 2O 3/Y-TZP Layered Ceramics from Aqueous Suspensions

Electrophoretic forming is emerging as an economical and reliable method for the fabrication of coatings and laminate ceramics. The method is very simple, but a major problem is the toxicity and environmental problems associated with the organic solvents usually employed as vehicle media. This work aims to obtain Al 2O 3/Y-TZP laminates by electrophoretic deposition using water as the liquid vehicle. The slip characteristics have been studied in terms of viscosity, zeta potential, and conductivity for both starting powders. Electrophoretic deposition was performed at constant values of current density to obtain the monolithics and layered materials. Very small variations in the deflocculating conditions allow control over the thickness of each layer in the laminate, providing a powerful method for obtaining tailored complex microstructures.

Effects of Mg 2+, K +, and H + on an equilibrium between alternative conformations of an RNA pseudoknot

A complex pseudoknot structure surrounds the first ribosome initiation site in the Eschericlia coli α mRNA and mediates its regulation by ribosomal protein S4. A 112 nt RNA fragment containing this pseudoknot exists in two conformations that are resolvable by gel electrophoresis below room temperature. Between 30°C and 45°C the conformers reach thermodynamic equilibrium on a time scale ranging from one hour to one minute, and the interconversion between conformers is linked to H +, K + and Mg 2+ concentrations. Mg 2+ favors formation of the “fast” electrophoretic form: a single Mg 2+ is bound in the rate-limiting step, followed by cooperative binding of ∼1.7 additional ions. Binding of the latter ions provides most of the favorable free energy for the reaction. However, the “slow” form binds about the same number of Mg ions, albeit more weakly, so that saturating Mg 2+ concentrations drive the equilibrium to only ∼70% fast form. A single H + is taken up in the switch to the “slow” conformer, which has apparent pK≈5.9; low pH also stabilizes part of the pseudoknot structure melting at ∼62°C. Mg 2+ and H + appear to direct α mRNA folding by relatively small (10 to 100-fold) differences in their affinities for alternative conformers. K + has very little effect on the conformational equilibrium, but at high concentrations accelerates interconversion between the conformers. The α mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg 2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA tertiary structures, though it differs from these in the association of a single Mg 2+ with the rate-limiting step.

Electrophoretic Forming of Laminated Ceramic Composite Tubes

Electrophoretic Forming of Laminated Ceramic Composite Tubes

Electrophoretic forming of silicon carbide ceramics

Electrophoresis is the movement of particles suspended in a liquid under influence of an electric field. Electrophoretic forming of silicon carbide (SiC) requires the simultaneous deposition of SiC and sintering aids such as carbon and boron. The incorporation of the sintering additives was studied in two ways: co-deposition of a boron-containing SiC powder and carbon black, as well as the co-deposition of SiC, boron carbide and carbon black. A high final density after sintering (95%) was obtained with the two approaches.