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1
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Plant ureases: Roles and regulation.

100%
Sirko A., Brodzik R.
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2000
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vol. 47
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issue 4
1189-1195
EN
Both urea and urease were subjects of early scientific investigations. Urea was the first organic molecule to be synthesized and jack bean urease was the first enzyme ever to be crystallized. About 50 years later it was shown to be the first nickel metalloenzyme. Since then, nickel-dependent ureases have been isolated from many bacteria, fungi and higher plants. They have similar structures and mechanisms of catalysis. A urease apoenzyme needs to be activated. This process requires participation of several accessory proteins that incorporate nickel into the urease forming catalytic site. In this review, ureases from various organisms are briefly described and the similarities of their structures discussed. Moreover, the significance of urea recycling in plants is explained and recent literature data about the function and activation of plant ureases are presented.
2
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Recent advances in understanding plant response to sulfur-deficiency stress

100%
Lewandowska M., Sirko A.
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2008
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vol. 55
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issue 3
457-471
EN
Sulfur is an essential macronutrient for all living organisms. Plants are able to assimilate inorganic sulfur and incorporate it into organic compounds, while animals rely entirely on organic sources of sulfur. In the last decades sulfate availability in soils has become the major limiting factor for plant production in many countries due to significant reduction of anthropogenic sulfur emission forced by introducing stringent environmental legislation. The sulfur flux after transferring plants from optimal conditions to sulfur deficiency is regulated on multiple levels including transcription, translation and activity of enzymes needed for sulfate assimilation and synthesis of sulfur-containing metabolites. Most of these regulatory steps are not yet fully characterized. Plant responses to sulfur limitation are complex and can be divided into phases depending on the degree of sulfur shortage. The initial responses are limited to adaptations within sulfur metabolic pathway, while multiple metabolic pathways and developmental process are affected when sulfur shortage becomes more severe. The major aim of this work is a comprehensive review of recent progress in understanding the regulation of plant adaptations to sulfur deficit.
3
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Plant expression systems for production of hemagglutinin as a vaccine against influenza virus

81%
Redkiewicz P., Sirko A., Kamel K., Góra-Sochacka A.
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2014
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vol. 61
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issue 3
551-560
EN
Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity.
4
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Influenza prevention and treatment by passive immunization

81%
Kalenik B., Sawicka R., Góra-Sochacka A., Sirko A.
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2014
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vol. 61
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issue 3
573-587
EN
Passive immunity is defined as a particular antigen resistance provided by external antibodies. It can be either naturally or artificially acquired. Natural passive immunization occurs during pregnancy and breast-feeding in mammals and during hatching in birds. Maternal antibodies are passed through the placenta and milk in mammals and through the egg yolk in birds. Artificial passive immunity is acquired by injection of either serum from immunized (or infected) individuals or antibody preparations. Many independent research groups worked on selection, verification and detailed characterization of polyclonal and monoclonal antibodies against the influenza virus. Numerous antibody preparations were tested in a variety of in vitro and in vivo experiments for their efficacy to neutralize the virus. Here, we describe types of antibodies tested in such experiments and their viral targets, review approaches resulting in identification of broadly neutralizing antibodies and discuss methods used to demonstrate their protective effects. Finally, we shortly discuss the phenomenon of maternal antibody transfer as a way of effective care for young individuals and as an interfering factor in early vaccination.
5
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DNA vaccines against influenza

81%
Stachyra A., Góra-Sochacka A., Sirko A.
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2014
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vol. 61
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issue 3
515-522
EN
Genetic vaccine technology has been considerably developed within the last two decades. This cost effective and promising strategy can be applied for therapy of cancers and for curing allergy, chronic and infectious diseases, such as a seasonal and pandemic influenza. Despite numerous advantages, several limitations of this technology reduce its performance and can retard its commercial exploitation in humans and its veterinary applications. Inefficient delivery of the DNA vaccine into cells of immunized individuals results in low intracellular supply of suitable expression cassettes encoding an antigen, in its low expression level and, in turn, in reduced immune responses against the antigen. Improvement of DNA delivery into the host cells might significantly increase effectiveness of the DNA vaccine. A vast array of innovative methods and various experimental strategies have been applied in order to enhance the effectiveness of DNA vaccines. They include various strategies improving DNA delivery as well as expression and immunogenic potential of the proteins encoded by the DNA vaccines. Researchers focusing on DNA vaccines against influenza have applied many of these strategies. Recent examples of the most successful modern approaches are discussed in this review.
6
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Modification of non-protein thiols contents in transgenic tobacco plants producing bacterial enzymes of cysteine biosynthesis pathway.

81%
Liszewska F., Błaszczyk A., Sirko A.
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2001
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vol. 48
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issue 3
647-656
EN
Conditions of achieving the maximal accumulation of sulfhydryl metabolites in the leaves of tobacco were explored. Simultaneous production of bacterial O-acetylserine (thiol)-lyase and serine acetyltransferase resulted in the increased thiols contents as compared to single transformants and controls. However, leaf discs feeding experiments differently affected thiols concentration in different plant groups and suggested that the most promising strategy to obtain plants with a high level of non-protein thiol-containing compounds might be sulfate feeding to plants overproducing serine acetyltransferase.Formation of cysteine from sulfide and O-acetyl-L-serine (OAS) is catalyzed by O-acetylserine (thiol)-lyase (OAS-TL) (EC 4.2.99.8) while OAS is synthesized by serine acetyltransferase (SAT) from acetyl-coenzyme A and serine. Molecular interactions between SAT and OAS-TL are involved in the regulation of the enzymatic activities of these proteins in bacteria (Mino et al., 2000) and in plants (Bogdanova & Hell, 1997; Droux et al., 1998). Experiments with Escherichia coli enzymes clearly indicated that OAS-TL activity was reduced to 30% in a complex (Mino et al., 2000). Similarly, a very dramatic decrease of the catalytic activity of OAS-TL bound in a complex has been reported for the plant enzymes (Droux et al., 1998). On the other hand, the bienzyme complex formation stabilizes bacterial SAT (Mino et al., 2000) and increases the apparent affinity for the substrates of plant SAT (Droux et al., 1998). The stability of the complex is negatively affected by OAS and positively by sulfide (Droux et al., 1998). Bacterial SAT is feedback regulated by cysteine, however, no relationship seems to exist between the complex formation and SAT sensitivity to this inhibition (Mino et al., 2000).Both enzymes, SAT and OAS-TL, are located in three compartments of the plant cell: the cytosol, chloroplasts and mitochondria. Different isoforms of these enzymes are in a different way regulated by sulfur nutrition (Nakamura et al., 1999; Takahashi et al., 1997; Warrilow & Hawkesford, 1998). The feedback regulation by L-cysteine of various isoforms of plant SAT has recently been studied (Inoue et al., 1999; Noji et al., 1998). According to the model proposed, the only role of the chloroplastic and mitochondrial isoforms (that are insensitive to the feedback inhibition) would be the production of OAS for cysteine biosynthesis. The cysteine-sensitive cystosolic isoform of SAT would, according to this model, have two roles: (i) OAS production for cysteine biosynthesis in the cystosol and (ii) control of OAS pool for regulatory purposes. The second postulated function is tightly connected with the fact that OAS acts as a positive regulator of genes whose expression is affected by sulfur status (Saito, 2000).Glutathione, the main low-molecular-mass thiol-containing compound in the plant cell, has multiple functions, including involvement in responses to various environmental stresses and maintenance of the redox homeostasis (Foyer & Rennenberg, 2000). Under non-stressing conditions the majority of glutathione is maintained in the reduced form (GSH) and its concentration is determined mainly by the rate of biosynthesis. GSH is produced from cysteine, glutamate and glycine in two steps catalyzed by γ-glutamylcysteinyl synthetase (γ-ECS) and glutathione synthetase (GS), respectively (Noctor et al., 1998). The biosynthesis and accumulation of GSH has been shown to depend on (i) the activity of γ-ECS, (ii) the availability of cysteine, and (iii) the light-dependent formation of glycine through the photorespiratory pathway (Foyer & Rennenberg, 2000).The main aim of this study was to identify the optimal conditions for the maximal accumulation of non-protein sulfhydryl metabolites in plant leaves. The transgenic tobacco plants with cytosolic production of bacterial enzymes of the cysteine biosynthesis pathway, SAT and OAS-TL, were obtained and analyzed for the transgenes expression. Additionally, leaf discs of either single or double transformants, as well as of control plants, were assayed for the thiol contents upon incubation in solutions of various compounds expected to have an influence on sulfur metabolism.
7
Content available remote

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

81%
Liszewska F., Gaganidze D., Sirko A.
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2005
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vol. 52
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issue 1
117-128
EN
We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol) lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.
8
Content available remote

Characterization of the Escherichia coli gene encoding a new member of the short-chain dehydrogenase/reductase (SDR) family

81%
Sirko A., Węgleńska A., Hryniewicz M., Hulanicka D. M.
Acta Biochimica Polonica
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1997
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vol. 44
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issue 1
153-157
9
Content available remote

Identification and mapping of the sulphate permease promoter region in Esherichia coli

80%
Hryniewicz M., Sirko A., Hulanicka D.
Acta Biochimica Polonica
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1989
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vol. 36
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issue 3-4
353-363
10
Content available remote

Polyadenylation and decay of 26S rRNA as part of Nicotiana tabacum response to cadmium

71%
Lewandowska M., Borcz B., Kamińska J., Wawrzyński A., Sirko A.
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2007
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vol. 54
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issue 4
747-755
EN
In contrast to mRNAs, ribosomal RNAs are generally not considered to be polyadenylated. Only a few recent reports describe non-abundant polyadenylated rRNA-related transcripts that have been detected and characterized in yeast and in human cells. Here we depict the phenomenon of 26S rRNA polyadenylation and degradation that was observed in shoots of Nicotiana tabaccum plants grown in the presence of cadmium. Fragments corresponding to 26S rRNA were identified using suppression subtractive hybridization during screening for genes induced in tobacco plants upon a three-week exposure to 15 µM cadmium chloride. Extracts prepared from the above-ground tissues of cadmium-treated tobacco plants were supposed to contain exclusively polyadenylated mRNAs. Surprisingly, numerous polyadenylated fragments matching parts of 26S rRNA were identified and their presence was confirmed by Northern blot and cDNA amplification techniques. To our knowledge this is the first report on rRNA polyadenylation in plants.
11
Content available remote

Antibody response to DNA vaccine against H5N1 avian influenza virus in broilers immunized according to three schedules

71%
Stachyra A., Góra-Sochacka A., Zagórski-Ostoja W., Król E., Sirko A.
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2014
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vol. 61
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issue 3
593-596
EN
Broiler type chickens were immunized intramuscularly with a DNA vaccine encoding hemagglutinin (HA) from H5N1 avian influenza virus. The chickens were divided into four groups: control group which was not immunized, a group which obtained only one dose, and two groups which were immunized twice, one group with a boost two weeks after the priming and the other four weeks. Blood samples were collected at several time points and the dynamics of the humoral response to the vaccine was studied. High level of anti-HA antibodies was detected only in the last two groups, that is in chickens immunized according to the prime-boost strategy, regardless of the schedule. An additional interesting observation of this study was detection of the cross-reactivity of an anti-H5 HA positive serum with H5N2 and H1N1 viruses, suggesting that the DNA vaccine tested can induce antibodies of a broad specificity.
12
Content available remote

Overexpression of genes involved in phytochelatin biosynthesis in Escherichia coli: effects on growth, cadmium accumulation and thiol level.

62%
Wawrzyńska A., Wawrzyński A., Gaganidze D., Kopera E., Piątek K., Bal W., Sirko A.
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2005
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vol. 52
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issue 1
109-116
EN
In Escherichia coli, heterologous production of Schizosaccharomyces pombe phytochelatin synthase (PCS) along with overproduction of E. coli serine acetyltransferase (SAT) and γ-glutamylcysteine synthase (γECS) was achieved and resulted in the accumulation of phytochelatins in bacterial cells. Overproduction of either γECS alone or simultaneous production of all three proteins in bacterial cells were accompanied by reduced growth rate in liquid cultures. Interestingly, bacteria overproducing either γECS or both SAT and γECS (with elevated level of γ-glutamylcysteine but not of phytochelatins) were able to accumulate more cadmium per dry weight than the control. However, the most efficient cadmium accumulation was observed in bacteria with elevated levels of all three proteins: SAT, γECS and PCS. Therefore, "pushing" the entire pathway might be the most promising approach in modification of bacteria for potential bioremediation purposes because the level of intermediates, cysteine and glutathione, can limit the rate of production of phytochelatins. However, in such bacteria other metabolic process might become limiting for efficient growth.
13
Content available remote

Genetic immunization of ducks for production of antibodies specific to Helicobacter pylori UreB in egg yolks.

62%
Kazimierczuk K., Cova L., Ndeboko B., Szczyrk U., Targosz A., Brzozowski T., Sirko A.
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2005
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vol. 52
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issue 1
261-266
EN
Following genetic immunization of laying ducks with a plasmid expressing Helicobacter pylori UreB (large subunit of urease), IgY against UreB were obtained from egg yolks. These polyclonal and monospecific IgY antibodies are of higher-titer and specifically recognize recombinant H. pylori urease purified from Escherichia coli. To our knowledge this is the first report describing generation of IgY antibodies directed against antigens of H. pylori by DNA-based immunization.
14
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Content available

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

52%
Sawicka R., Siedlecki P., Kalenik B., Radomski J., Sączyńska V., Porębska A., Szewczyk B., Sirko A., Góra-Sochacka A.
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2017
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vol. 64
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issue 1
85-92
EN
Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity- selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.
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