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  1. 7 Biotechnologia

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  2. 3 2002

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  1. 7 Salek A. T.

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1

Classic techniques for improvement of industrial yeast strains. Part II ? Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electrotransformation

100%
Salek A. T.
Biotechnologia
|
2002
|
issue 1
175-186
EN
Killer-sensitive strains of Saccharamocyces cerevisiae and Saccharomyces uvarum var. carlsbergensis were transformed by electroinjection using dsRNA isolated from a superkiller strain. Various recipient strains were used: both thermo-resistant and thermo-sensitive as well as mutants of industrial strains. Conversion of respiratory competent (rho+) into respiratory deficient (rho-) strains (mutants) resulted in a significant increase in the yield of electrotransformants and/or of long-term killer stability. Electrotransformation of rho- mutants of distillery and brewery strains resulted in more than 100 clones, which exhibited weak or strong killer activity over some or all of the experimental period of 10 months.
2

Classic techniques for improvement of industrial yeast strains: Part III ? A method for enucleation of Saccharomyces sp.

100%
Salek A. T.
Biotechnologia
|
2002
|
issue 1
187-194
EN
The first method for enucleation of yeast Saccharomyces cerevisiae is reported. Various strains, including some killer strain and respiratory-deficient mutants of Saccharomyces cerevisiae were enucleated after treatment with cytochalasin B. Removal of nuclei from protruding sphaeroplasts was induced by centrifugation in a Percoll density gradient. The enucleation yield (which averaged about 80%) and the quality of the cytoplasts were best when the yeast culture had been synchronized with nocodazole before the preparation. The presence of 1 mM CaCl2 and ATP (10 muM) in the enucleation medium prevented the formation of fragile products or aggregation. Cytoplasts could be stored for at least 1 day without visible deterioration.
3

Classic techniques for improvement of industrial yeast strains. Part I? Construction of ethanol-resistant and osmophilic industrial strains of Saccharomyces cerevisiae by electrofusion

100%
Salek A. T.
Biotechnologia
|
2002
|
issue 1
153-174
EN
The effective production of biomass or ethanol in industrial media of high osmolality requires new yeast strains. The present work focused on the development of such strains. Genetic engineering methods using cytoplasmatically-marked yeast (electrofusion of protoplasts of heterothallic haploids; electrotransformation of killer dsRNA or VLPs into haploids; generation of rho-) were used. The characteristics of the hybrids were evaluated by conventional analytical and instrumental methods, followed by statistical interpretation. After screening for a minimum 10% increase in industrially-relevant parameters, 3 osmophilic hybrids of baker's yeast, as well as 8 improved strains of distillery yeasts were selected. The baker?s yeasts showed optimum growth in a relatively concentrated molasses wort (1:5 ratio of molasses to final volume). The alcohol-resistant yeasts (including killer) produced up to 14.5% (w/w) ethanol in a medium containing 34% dissolved solids (a mixed mash of sucrose and potato). The characteristics of the alcohol-resistant and osmophilic yeasts were stable over several years of their industrial applications.The results show that electrical techniques (fusion to obtain hybrids, with interpretation by computer-aided image analysis, and transformation to give marked strains) can be used effectively enough for the construction of some industrially-productive yeasts.
4

The influence of yeast killer toxins on the cytotoxicity of shiga-like toxins Part I ? The effect of killer toxins on mammalian cells

100%
Salek A. T.
Biotechnologia
|
2003
|
issue 1
244-256
EN
It is evident that the results of preliminary experiments with 5 different yeast killer proteins did not show emphatic cytotoxicity or any adverse effect on any mammalian and embryo-cells. Moreover, they are likely to be harmless to animals? and humans? tissue cells. Therefore, they could be used to explain the pre-therapeutic effect on mammalian cells (mostly animals) in the case of infections by strains Escherichia coli, called EHEC. It was found that the yeast killer toxin from Williopsis mrakii can protect mammalian cells such as HeLa and Vero cells against the challenge by Shiga-Like-Toxins (derived from cultures of pathogenic strains of Escherichia coli). The final activities of tested mammalian cells are better when they are pre-treated by the killer protein, i.e. before the challenge with Shiga-Like-Toxins. It appears that this prophylactic effect could be very interesting for veterinary, which has been proved on a big population (about 2000) of healthy and ill (with diarrhoea, i.e. haemorrhagic colitis) pigs (manuscript ? confidential data). We can conclude that the yeast killer strains are probiotic, i.e. could reduce or eliminate fecal shedding of EHEC strains in pigs prior treated with the developed yeast toxins.
5

Yeast antimicrobial proteins

100%
Salek A. T.
Biotechnologia
|
2001
|
issue 4
135-162
EN
Many yeasts secrete proteins which are toxic for pathogenic and non-pathogenic microorganisms. These toxins, mostly glycoproteins, consist of membrane-binding subunits which interact with carbohydrates (e.g. 1,6--D-glucan or -mannan) on the cell wall of sensitive strains. The killing effect is presented by membrane permeation, cell lysis or inhibition of the cell cycle. It is also suggested that these killer glycoproteins, similar in structure to lectins, can mediate self-adhesion of the pathogenic microorganisms, thus stimulating their excretion from the intestines of infected mammals. It is supposed that the above interactions could be important for therapeutic applications, especially for enteric diseases. In order to fully understand the structural basis of the functions of killer glycoproteins, it is essential to characterize their glycosylation state and to determine the structure of all glycans attached to the proteins. In this paper, a strategic approach to the purification of yeast protein from complex biological mixtures is presented. The approach is structured into seven subassignments, each of which is essential for the successful isolation of a pure and biologically active yeast protein. The subassignments are: 1) decision on the use of the purified protein; 2) collecting information about the chemical, physical and biological properties of the protein; 3) establishing assays for the protein and its biological activity; 4) decision on the source of raw material; 5) development of an efficient extraction method; 6) development of a purification method; 7) establishment of optimum conditions for storage of the purified protein.
6

Development of an automated yeast identification system by the application of PCR technique: a comparison of inter-line PCR with reference methods

100%
Salek A. T.
|
EN
In the Inter-LINE (IL) PCR method, oligonucleotides GF, GR and 01, which originated from mammalian cells led to highly reproducible patterns of amplified template DNA, based on the consensus of LINE-sequences. These were used for the genomic fingerprinting of about 80 strains of yeast, consisting of 30 species from 13 genera). The IL-PCR technique using the above primers is described and compared to reference methods such as Arbitrarily Primed-PCR and Pulse Field Gel Electrophoresis (PFGE). A comparison of the two PCR variants was performed using suitable numbers of digitized PCR-fingerprints. A database for an automated yeast identification system is proposed.
7

The influence of yeast killer toxins on the cytotoxicity of shiga-like toxins Part II ? Binding to shiga-like toxin receptors

75%
Salek A. T.
Biotechnologia
|
2003
|
issue 1
257-266
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