Edmund Ziomek

Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease

Abstract The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) carries out N-terminal processing of the viral replicase polyprotein, and also exhibits Lys48-linked polyubiquitin chain debranching and ISG15 precursor processing activities in vitro. Here, we used SDS–PAGE and fluorescence-based assays to demonstrate that ISG15 derivatives are the preferred substrates for the deubiquitinating activity of the PLpro. With k cat/K M of 602,000M−1 s−1, PLpro hydrolyzes ISG15-AMC 30- and 60-fold more efficiently than Ub-AMC and Nedd8-AMC, respectively. Data obtained with truncated ISG15 and hybrid Ub/ISG15 substrates indicate that both the N- and C-terminal Ub-like domains of ISG15 contribute to this preference. The enzyme also displays a preference for debranching Lys48- over Lys63-linked polyubiquitin chains. Our results demonstrate that SARS-CoV PLpro can differentiate between ubiquitin-like modifiers sharing a common C-terminal sequence, and that the debranching activity of the PLpro is linkage type selective. The potential structural basis for the demonstrated specificity of SARS-CoV PLpro is discussed.

Effect of polydimethylsiloxane oxygen carriers on the biological bleaching of hardwood kraft pulp by Trametes versicolor

SummaryImproving the availability of oxygen by adding polydimethylsiloxanes (PDMS) oxygen carriers to Trametes versicolor cultures increased pulp brightening. The presence of the oxygen carriers in cultures of T. versicolor with hardwood kraft pulp increased the growth rate of the fungus, but not the ultimate biomass yield. The PDMS also stimulated brightening of hardwood kraft pulp by it T. versicolor immobilized in polyurethane foam. A threefold increase in the oxygen uptake rate in T. versicolor cultures with PDMS was observed. This increase can be explained by elevated oxygen transfer rate and attributed to the surfactant properties of PDMS.

Influence of group A streptococcal acid glycoprotein on expression of major virulence factors and internalization by epithelial cells.

A single transposon insertion upstream to the open-reading-frame identified as the streptococcal acid glycoprotein (sagp) gene rendered a Tn916 isolate of Streptococcus pyogenes with elevated susceptibility to internalization by the epithelial cells. The role of SAGP in S. pyogenes internalization was further studied using isogenic mutant containing an in-frame deletion within the sagp gene. The sagp mutant displayed slower growth-rate and showed 5-fold higher internalization efficiency than the parent strain. Transcription of sagp at the logarithmic phase, but not at the stationary phase of the growth was repressed by csrR, the global regulator gene. At the same time, mutation of the sagp gene partially decreased the transcription of hasA, a gene that is required for capsule synthesis. The mutation had no effect on transcription of the emm3 gene, encoding for the M protein. The most striking effect of the sagp mutation was a down-regulation of the streptococcal pyrogenic exotoxin B (SpeB) at both translational and transcriptional level. Treatment of the SAGP mutant cells with the exogenous mSpeB (mature protease) only partially reduced their susceptibility to internalization. The exogenous mSpeB was more effective in reducing the internalization efficiency of a speB mutant and brought it to the level observed for the parent strain. In overall, results show that CsrR, directly or indirectly, affects the expression of SAGP, and that the SAGP modulates expression of not only SpeB, but also other genes that facilitate S. pyogenes internalization.

Interaction of a poly(dimethylsiloxane) with triglycerides in monomolecular films and application to lipase kinetics

Abstract It has been known for some time that poly(dimethylsiloxanes) (PDMS) can form monolayers at the air-water interface. In this paper we show that formation of mixed monolayers of PDMS with fatty acids, di- and triglycerides is also possible. At the air-water interface silicone can interact and increase the retention of the fatty acids, which otherwise easily desorb into the aqueous subphase. Unlike fatty acids, long acyl chain tri- and diglycerides did not desorb into the aqueous subphase and their interaction with silicone was manifested by a condensing effect and a change in the collapse pressure of the mixed monolayers. Geotrichum candidum lipase (GCL) showed a similar level of binding to silicone monolayer as to an air-water interface. At low surface pressure (7.5 mN/m) the lipase hydrolyzed dicaprin in mixed 1,2-sn-dicaprin-PDMS monolayers at a rate 1.5-fold lower than the desorption rate of the capric acid from mixed monolayers of capric acid and PDMS. Compression of the dicaprin-PDMS monolayer to 20 mN/m led to a 15-fold increase in lipase activity (70% of the activity observed for monolayer of pure dicaprin compressed to the same surface pressure), indicating an increase in dicaprins surface concentration and its accessibility to the enzyme. The lipase activity profile determined titrimetrically against varying amounts of glycerides in mixed glyceride-PDMS emulsions (at constant 5% oil-phase concentration) resembles the lipase activity-surface pressure profile obtained with the monolayer method when using pure glycerides. This suggests that the location of glycerides in condensed mixed monolayers with PDMS should be similar to that in mixed glyceride-PDMS emulsions. By changing the molar fraction of tristearin and tripalmitin in the mixture with PDMS, it was possible to regulate the quality of the interface in emulsions and to optimize the enzymatic hydrolysis of these solid triglycerides.

Application of Hydrophobic Chromatography to the Large Scale Purification of Pesulfovibri0 Desulfur Icans Cytochrome C3

Hydrophobic chromatography on Phenyl-Sepharose was employed for large scale purification of cytochrome c3 from Desulfovibrio desulfuricans. This chromatographic procedure minimizes operational volumes and eliminates the lengthy dialysis ordinarily used to remove large amounts of salts. Pure cytochrome c3 was obtained after one additional purification by ion-exchange chromatography.

Immobilization of isolated and cellular hydrogenase of D. desulfuricans in radiation-polymerized polyacrylamides

Purified hydrogenase fromDesulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6–7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65‡C) and long term storage (4‡C) of the hydrogenase activity of both the purified enzyme and whole cells.

Use of δ-(α-aminoadipoyl) chromogenic amides in screening for aminoadipoyl amidohydrolases

Abstract The synthesis of δ-(α-aminoadipoyl) aromatic amides and their use in screening for enzymes able to cleave δ-(α-aminoadipoyl) residues off the synthetic amides and cephalosporin C are described. A number of commercially available proteases and peptidases were not active with δ-(α-aminoadipoyl) chromogenic amides. Also, most tested microbial strains known to produce acylases did not hydrolyze these compounds. Only one microbial strain, Xanthomonas maltophila , had an appreciable activity toward the racemic form of chromogenic substrates. Activity measured in crude extracts from Xanthomonas cells indicated that this bacterium produces predominantly l -specific aminoadipoyl amidohydrolase and γ-glutamyl hydrolase. A low level of cephalosporin C and glutaryl-cephalosporin acylase activities was also found.

Immobilization of Desulphovibrio desulphuricans: cell-associated hydrogenase in beaded matrices

Abstract The immobilization of the hydrogenase (cytochrome c 3 hydrogenase, hydrogen: ferricytochrome c 3 oxidoreductase, EC 1.12.2.1) activity associated with Desulphovibrio desulphuricans whole cells is described. The periplasmic hydrogenase was prevented from leaking from the cells by glutaraldehyde treatment. This modification left the hydrogenase activity of the cells unchanged. The resulting whole-cell preparation was then immobilized in beaded gels formed by either calcium alginate or low temperature radiation-polymerized polyacrylamide(s). The alginate matrix was used to study the effect of bead diameter and retention of hydrogenase activity after immobilization. Polyacrylamide matrices were used to study the effects of immobilization on hydrogenase activity vs. pH profiles and also matrix charge effects on enzymatic activity and long-term storage.