Electrophoretic Forms Research Articles

Phosphorylation of the Herpes Simplex Virus Type 1 Tegument Protein VP22

The herpes simplex virus type 1 tegument protein VP22 is known to be highly phosphorylated during infection. Here we show that two electrophoretic forms of VP22 can be identified in infected cell extracts and that this heterogeneity is accounted for by phosphorylation. Furthermore, the nonphosphorylated form of VP22 appears to be specifically incorporated into virions. We also show that the phosphorylated form of VP22 is the only form detected during transient transfection and as such that VP22 can act as a substrate for a cellular kinase. Phospho-amino acid and phospho-peptide analyses ofin vivolabeled VP22 were utilized to demonstrate that the phosphorylation profiles of VP22 synthesized during transfection and infection are the same. In both cases VP22 was modified solely on serine residues located in the N-terminal 120 residues of the protein. Moreover,in vitrophosphorylation was utilized to show that the constitutive cellular kinase, casein kinase II, which has four serine consensus recognition sites at the N-terminus of VP22, phosphorylates VP22 in the same manner as observedin vivo.This kinase also phosphorylates VP22 at the N-terminus in intact capsid–tegument structures. Casein kinase II is therefore likely to be the major kinase of VP22 during infection.

Catalase activity in developing seedling of opium poppyPapaver somniferum L

Catalase is an enzyme unique to glyoxysomes in developing poppy seedlings. Catalase activity is very low in endosperm and in embryo of germinating poppy seeds. During postgerminative growth and development the enzyme activity increases rapidly with maximum in endosperm on day 2 and in developing seedling on day 3. A rapid decline of enzyme activity parallells the extension growth of poppy seedlings. Three electrophoretic forms of catalase have been detected in isolated glyoxysomes and partially purified catalase preparation. Electron microscopic observation indicates the presence of catalase in glyoxysomes of parenchyma cells of poppy seedling cotyledons. Numerous lipid bodies and electron-dense deposits in vacuoles are the most characteristic feature of these cells.

Structural characterization of variant forms of arylsulfatase A that associate with alcoholism.

Several electrophoretic forms of human platelet arylsulfatase A (ASA), including variant type IIIa and normal type IV(a), have been identified by nondenaturing polyacrylamide gel electrophoresis. An alcoholic population that we have analyzed is enriched in variant type IIIa compared with nonalcoholic psychiatric and normal controls. Individuals with the IIIa enzyme possess greatly reduced levels of ASA activity. To understand further the structural basis for the differences and their potential biological consequences, the nature of the ASA variant expressed by fibroblasts from different individuals was explored. The electrophoretic patterns of fibroblast ASA from the IIIa and IV(a) individuals differ in degree of phosphorylation. Furthermore, fibroblast ASA from IIIa individuals lacks an N-linked glycan found in ASA from IV(a) individuals. In addition, differences in peptide and/or posttranslational modification unrelated to the N-linked carbohydrate or phosphorylation exist between the fibroblast ASA from IIIa and IV(a) individuals. The finding that both fibroblasts and platelets exhibit related electrophoretic isoform patterns characteristic of the donor's ASA type allows for the use of fibroblasts to study the impact of ethanol on the metabolism of cells possessing different ASA types.

Analysis of phosphorylation sites of herpes simplex virus type 1 ICP4.

The herpes simplex virus ICP4 protein is required for induction of early and late viral gene transcription as well as for repression of expression of its own gene and several other viral genes. Several electrophoretic forms of ICP4 have been observed, and phosphorylation is thought to contribute to this heterogeneity and possibly to the multiple functions of ICP4. To define the complexity of the site(s) of phosphorylation of ICP4 and to initiate mapping of this site(s), we have performed two-dimensional phosphopeptide mapping of wild-type and mutant forms of ICP4 labeled in infected cells or in vitro. Wild-type ICP4 labeled in infected cells shows a complex pattern of phosphopeptides, and smaller mutant forms of ICP4 show progressively fewer phosphopeptides, arguing that multiple sites on ICP4 are phosphorylated. The serine-rich region of ICP4, residues 175 to 198, was shown to be a site for phosphorylation. Furthermore, the serine-rich region itself or the phosphorylation of this region increases phosphorylation of all phosphopeptides. A mutant ICP4 molecule lacking the serine-rich region showed low levels of phosphorylation by protein kinase A or protein kinase C in vitro. These results suggest that there may be a sequential phosphorylation of ICP4, with phosphorylation of the serine-rich region stimulating phosphorylation of the rest of the molecule. In addition, purified ICP4 showed an associated kinase activity or an autophosphorylation activity with properties different from those of protein kinase A or protein kinase C.

Expression and Purification of Recombinant Human Tryptase in a Baculovirus System

B2 is a mAb that recognizes a conformational determinant on the active form of native tryptase, but does not recognize native tryptase that spontaneously loses activity in physiologic buffer. Precursor forms of recombinant human (rh) α- and rhβ-tryptase have been expressed in a baculovirus system. In each case, multiple electrophoretic forms were detected in both culture media and cell lysates of infected insect cells by Western blots developed with the G3 mAb made against native human tryptase. Although only 4 of 30 amino acids in the leader sequences of α- and β-tryptase differ, rhα-tryptase appeared predominantly in the cell lysates, rhβ-tryptase predominantly in the culture media. B2 recognized rhα-tryptase and rhβ-tryptase found in the culture media of infected Sf-9 cells, but not that in cell lysates. Secreted forms of tryptase were purified to homogeneity by B2-immunoaffinity chromatography. From 1 liter of culture fluid 1.5 to 3 mg of rh-tryptase could be purified. Each rh-tryptase precursor was enzymatically inactive with synthetic substrates. Analysis of the N-terminal amino acid sequences of purified rhα- and rhβ-tryptase precursors (APAPVQA and APAPGQA, respectively) indicated that the initial 18 amino acids of the 30-amino-acid leader sequence had been removed. Differential N-glycosylation was found in both rhα-tryptase (one or two carbohydrate groups per molecule) and rhβ-tryptase (zero or one carbohydrate group per molecule). Thus, the baculovirus expression system is a useful tool for generation of rhα- and rhβ-tryptase precursors that exhibit a conformational epitope also present on natural tryptase and that are preferentially secreted into the culture media of infected cells.

A Streptomyces chitosanase is active in transgenic tobacco

Growth inhibition towards Rhizopus nigricans, Fusarium oxysporum f. sp. radicis-lycopersici, Verticillium albo-atrum and Pythium ultimum was observed in vitro using a purified chitosanase from an actinomycete, Streptomyces sp, strain N174. The corresponding gene, with its own signal peptide, was inserted into pBI121.7 shuttle vector to transform tobacco. Transgenic plants were analysed for chitosanase activity by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay. Two major and one minor active electrophoretic forms were detected in transgenic tobacco. Some chitosanases were recovered not only in leaf homogenates but also in leaf intercellular fluid extracts. One chitosanase electrophoretic form migrated very closely to the purified Streptomyces mature protein while the others corresponded to molecules of higher molecular mass. The N-terminus sequence was determined for one of the three chitosanase forms. It exhibited a different signal peptide cleavage site when compared to the mature chitosanase from Streptomyces. This is the first report on the expression of an active chitosanase gene with antimicrobial potential in plants.

Reply to “Comment on‘Electrophoretic Deposition Forming of SiC‐TZP Composites in a Nonaqueous Sol Media'”

Reply to “Comment on‘Electrophoretic Deposition Forming of SiC‐TZP Composites in a Nonaqueous Sol Media'”

Electrophoretic deposition of Al 2O 3-SiC composite

A ceramic composite with Al 2O 3 particles/SiC whiskers has been prepared by electrophoretic deposition in an isopropanol suspension. In the absence of a small amount of nitric acid (0.06–0.32 M), no deposition is observed. The amount of deposition increases with increasing applied voltage and solid loading, but decreases significantly with the amount of water present. The latter result is attributed to the fact that the colloidal stability of the suspension is degraded by water. Microstructural and compositional analyses show that the SiC whiskers are uniformly distributed in the green compact when the colloidally stable suspension is used during electrophoretic forming.

Secreted hen lysozyme in transgenic tobacco: recovery of bound enzyme and in vitro growth inhibition of plant pathogens

Less than 1% of secreted hen egg white lysozyme (HEWL) was isolated in intercellular fluid neutral phosphate buffer extracts of transgenic tobacco ( Nicotiana tabacum L.). When foliar tissue was infiltrated with 50 mM calcium chloride or 1% histamine, 10–20% of HEWL was isolated when compared to HEWL obtained in tissue homogenates boiled in the presence of sodium dodecyl sulfate. The presence of mature HEWL was shown by N-terminal amino acid microsequencing of the major electrophoretic form (14.4 kDa). A minor electrophoretic form (17 kDa) having the same N-terminus end (KVFGRC) as the major mature from was also found. Tissue imprinting of transgenic tobacco cut stem, petiole and leaf tissue for detecting lysis of Micrococcus luteus cells embedded as lysozyme substrate in polyacrylamide gels could be performed in the presence of histamine for release of HEWL. Despite tight binding of HEWL in transgenic tobacco, some HEWL activity could be detected around growing transgenic tobacco seedling rootlets. Moreover, HEWL recovered from transgenic tobacco extracellular extracts was shown to inhibit the growth of some bacterial and fungal plant pathogens.

The Vibrio cholerae O139 serogroup antigen includes an O-antigen capsule and lipopolysaccharide virulence determinants.

Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V. cholerae serogroup documented to cause epidemic cholera. Although related to V. cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS). Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS. All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody. A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms. The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule"). A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules. The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.

Synthesis and processing of mammalian protamines and transition proteins.

Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [3H]-arginine, [3H]-histidine, [35S]-cysteine, or [32P]-PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms.

Carboxy-terminal processing of the large subunit of [NiFe] hydrogenases

Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigas have been detected by Western analysis. The faster moving form co-migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C-terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C-terminal cleavage between His 536 and Val 537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His 582 and Val 583, in the E. coli hydrogenase-1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

Analysis of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus trans-activators IE-1 and IE-2 using monoclonal antibodies.

We have produced monoclonal antibodies against two Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) transcriptional trans-activators, IE-1 and IE-2. Temporal analysis of IE-1 and IE-2 proteins have shown that IE-1 continues to increase in steady-state levels from 0 to 120 h post-infection, whereas IE-2 declines by 36 h post-infection. At least five different electrophoretic forms of IE-1 are present in OpMNPV-infected LD652Y cells of which three appear to be from the non-spliced IE-1 gene. Cotransfection experiments also showed that IE-1 causes a reduction in the levels of IE-2 protein but concurrently IE-2 increases IE-1 expression. Western blot analysis indicated that a form of IE-1 copurified with budded virions but did not copurify with the polyhedron-derived virus.

電気泳動成形とその制御因子

There are two representative forming methods for ceramics such as pressure forming, which gives dynamic pressure to raw materials directly, and slip casting using capillalry attraction. In addition, there is an electrophoretic forming(electrophoretic and electroosmotic forming), which has been discussed as the third forming method, making use of surface charge of raw materials. Electrophoretic phenomena is explicated theoretically in an dilute system and there are many applications. However, it has a lot of unsolved problems in a concentrated system and its application is limited. And there are few industrial applications aiming at forming.In this paper, the fundamentals and the control factors of the electrophoretic (electroosmotic) forming, using natural inorganic materials as a starting material will be discussed. In addition, using these theory, continuous forming technology will also be discussed.

Plant chitinases

Plant chitinases

Characterization of different electrophoretic forms of cauliflower mosaic virus virions (strain Cabb-S)

The electrophoretic forms of purified cauliflower mosaic virus (CaMV), strain Cabb-S, were examined by electrophoresis on agarose gels. Three populations of viral particles were identified: a faster migrating component (the form F) and two slower migrating components (the forms S and S′). When the different forms of virions, after excision from gels, were subjected to analysis in SDS-polyacrylamide gel, the fast component consisted of the 37 and 42 kDa coat proteins whereas the slow components contained mainly the 39 kDa coat protein. However, there was no difference among the nucleic acids associated within the three forms. The biological significance of the different components is discussed.

Isolation and properties of a soluble sialidase from the culture fluid of Chinese hamster ovary cells.

A soluble sialidase that can degrade recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells has been isolated and purified to near homogeneity from the cell culture fluid of this host. Purification of approximately 34,000-fold was carried out using conventional purification techniques including sequential DEAE-Sepharose and S-Sepharose ion-exchange chromatography, followed by hydrophobic interaction chromatography with Phenyl-Toyopearl. Final purification was achieved by heparin-agarose and chromatofocusing chromatography. The minimum molecular weight of the sialidase on SDS-PAGE was approximately 43,000 Da. When the final preparation was examined under non-denaturing conditions, two major (pI = 6.8 and 7.0) and five minor electrophoretic forms with different isoelectric points were identified. The basis for the electrophoretic heterogeneity is not known, but it was not due to carbohydrate diversity since no carbohydrates were detected on the purified protein. The enzyme degraded a variety of sialyl-conjugate substrates, at a pH optimum of 5.9, including intact glycoproteins, oligosaccharides and gangliosides with a 4-fold preference for 2,3- versus 2,6-linked sialic acid residues. With ganglioside substrates, internally linked sialic acid residues were not cleaved by the enzyme. Delineation of this enzyme from the lysosomal and plasma membrane sialidases was made using inhibition studies with C-9 substituted 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2- enonic acid derivatives. The enzyme was identified in several CHO cell lines by immunoblotting using antiserum raised against a synthetic peptide based on amino acid sequence of a fragment derived by trypsin digestion of the purified sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)

ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae.

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae.

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

O-glycosylation of the coronavirus M protein. Differential localization of sialyltransferases in N- and O-linked glycosylation.

It has previously been shown that the M (E1) glycoprotein of mouse hepatitis virus strain A59 (MHV-A59) contains only O-linked oligosaccharides and localizes to the Golgi region when expressed independently. A detailed pulse-chase analysis was made of the addition of O-linked sugars to the M protein; upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three different electrophoretic forms could be distinguished that corresponded to the sequential acquisition of N-acetylgalactosamine (GalNAc), galactose (Gal), and sialic acid (SA). A fourth and fifth form could also be detected which we were unable to identify. Following Brefeldin A treatment, the M protein still acquired GalNAc, Gal, and SA, but the fourth and fifth forms were absent, suggesting that these modifications occur in the trans-Golgi network (TGN). In contrast, in the presence of BFA, the G protein of vesicular stomatitis virus (VSV), which contains N-linked oligosaccharides, acquired Gal and fucose but not SA. These results are consistent with earlier published data showing that Golgi compartments proximal to the TGN, but not the TGN itself, relocate to the endoplasmatic reticulum/intermediate compartment. More importantly, our data argue that, whereas addition of SA to N-linked sugars occurs in the TGN the acquisition of both SA on O-linked sugars and the addition of fucose to N-linked oligosaccharides must occur in Golgi compartments proximal to the TGN. The glycosylation of the M protein moreover indicates that it is transported to trans-Golgi and TGN. This was confirmed by electron microscopy immunocytochemistry, showing that the protein is targeted to cisternae on the trans side of the Golgi and co-localizes, at least in part, with TGN 38, a marker of the TGN, as well as with a lectin specific for sialic acid.