Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

A new clade of Dickeya spp. plays a major role in potato blackleg outbreaks in North Finland

Until recently Dickeya was regarded as a pathogen not established in Finland. As a result the blackleg symptom observed on potato was often associated with Pectobacterium atrosepticum. The occurrence of Dickeya spp. on potato in Finland was first reported in 2004. Since then the prevalence of Dickeya has been monitored through surveys and routine test of seed lots produced in the country. The results of monitoring of Dickeya spp. in seed lots produced in Finland between the years 2004 and 2008 indicated a steady increase in the incidence of Dickeya spp. The highest incidence was observed in samples from the 2006 growing season where about 37% were positive for Dickeya spp. The summer in 2006 was one of the warmest summers recorded in 100 years in Finland. The majority of infected lots were imported varieties. Since recently heavy blackleg outbreaks have occurred in production fields in the High Grade (HG) zone. A detailed study of these incidents of blackleg outbreaks in North Finland during the years 2008 and 2010 indicated that Dickeya spp. was the major component in the observed blackleg complex. It was detected and isolated from almost all symptomatic plants investigated. Repetitive sequences PCR (REP‐PCR) and Pulsed Field Gel Electrophoresis (PFGE) analysis of strains isolated in Finland showed identical pattern with those isolated recently in other European countries with a proposed name ‘Dickeya solani’. Moreover, the dnaX gene sequence of the representative strains isolated in Finland indicated 100% similarity to the dnaX sequences of ‘D. solani’. The study presents the first report of a detailed analysis of bacteria involved in potato blackleg complex from natural field outbreaks in North Finland HG zone and characterisation of the ‘D. solani’ strains playing the major role in the disease complex.     

Response of carrot protoplasts and protoplast-derived aggregates to selection using a fungal culture filtrate of Alternaria radicina

Protoplasts isolated from three accessions of cultivated carrot and 5-day-old protoplast-derived aggregates were subjected to selection to identify somaclonal variants with enhanced tolerance to the fungal disease black rot incited by Alternaria radicina. Different concentrations [1, 2, 3.5, 5, 10, 20, 35 and 50 % (v/v)] of a fungal culture filtrate (FCF) from 2-week-old liquid cultures of A. radicina were used. Protoplasts and aggregates were subjected to short-term selection for a period of 10 days. All FCF concentrations added to the cultures on the day of isolation decreased protoplast survival frequency and plating efficiency, while FCF applied 5 days later inhibited cell divisions in 5–50 % concentrations. The responses of protoplasts to the treatment were genotype dependent. Most R0 plants were regenerated in all accessions from cell lines grown with 1 % FCF, while only a few plants were produced from 2 to 3.5 % FCF-treated cultures of ‘Dolanka’ and the breeding line ‘9304B’, respectively. Nineteen-percent of putative stress-tolerant regenerants were tetraploids, while only 5 % tetraploids were observed in the control. The incidence of unique random amplified polymorphic DNA fragments indicating possible chromosomal rearrangements was low and did not differ among regenerants after selection and those derived from the control. Mobilization of miniature inverted repeat transposable elements was not observed. Some R0 individuals regenerated both from FCF-treated and untreated cultures showed lower susceptibility to A. radicina in a laboratory assay in comparison to control plants grown from seed. Regenerants from FCF-treated cultures showed lower frequency of flowering plants and a higher rate of male sterility. Pollen viability of the putative stress-tolerant regenerants varied over a wide range (6–98 %), independently of in vitro selection conditions. Our data suggest that A. radicina FCF may be feasible for the in vitro selection to generate plants with superior phenotypic performance against A. radicina.     

Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site

The aim of this study was to improve a useful molecular tool—TaqII restriction endonuclease-methyltransferase—by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue—TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.     

Characterization of heat-shock response of the marine bacterium Vibrio harveyi

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 C was the highest tempature at which V. harveyi was able to grow steadily. A shift from 30° C to 39° C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15 kDa protein. We showed that a protein of ～43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (σ³²) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.     

Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses

The IFNL4 gene is a recently discovered type III interferon, which in a significant fraction of the human population harbours a frameshift mutation abolishing the IFNλ4 ORF. The expression of IFNλ4 is correlated with both poor spontaneous clearance of hepatitis C virus (HCV) and poor response to treatment with type I interferon. Here, we show that the IFNL4 gene encodes an active type III interferon, named IFNλ4, which signals through the IFNλR1 and IL‐10R2 receptor chains. Recombinant IFNλ4 is antiviral against both HCV and coronaviruses at levels comparable to IFNλ3. However, the secretion of IFNλ4 is impaired compared to that of IFNλ3, and this impairment is not due to a weak signal peptide, which was previously believed. We found that IFNλ4 gets N‐linked glycosylated and that this glycosylation is required for secretion. Nevertheless, this glycosylation is not required for activity. Together, these findings result in the paradox that IFNλ4 is strongly antiviral but a disadvantage during HCV infection.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Fostering responsible research with genome editing technologies: a European perspective

In this consensus paper resulting from a meeting that involved representatives from more than 20 European partners, we recommend the foundation of an expert group (European Steering Committee) to assess the potential benefits and draw-backs of genome editing (off-targets, mosaicisms, etc.), and to design risk matrices and scenarios for a responsible use of this promising technology. In addition, this European steering committee will contribute in promoting an open debate on societal aspects prior to a translation into national and international legislation.