Yeast red pigment modifies cloned human α-synuclein pathogenesis in Parkinson disease models in Saccharomyces cerevisiae and Drosophila melanogaster.

Recently, we identified the yeast red pigment (RP), a polymer of 1-(5'-Phosphoribosyl)-5-aminoimidazole, as a novel potential anti-amyloid agent for the therapy of neurodegenerative diseases. The purpose of this study was to further validate RP for treatment of Parkinson's disease (PD) and to clarify molecular mechanisms involved in the reduction of amyloid cytotoxicity. We investigated RP effects in vivo using Saccharomyces cerevisiae and Drosophila melanogaster PD models. Western blot analysis revealed reduction in the levels of insoluble α-synuclein in both models, while soluble α-synuclein decreased only in Drosophila. In both models RP significantly reduced α-synuclein cytotoxicity, as was revealed by immunohistochemistry in Drosophila (p < 0.001, n = 27 flies per genotype/assay) and by flow cytometry in yeast (p < 0.05). Data obtained from the yeast PD model suggests that RP antitoxic effects are associated with a drop in ROS accumulation, and slower cellular transition from the early to late apoptotic stage. Using Drosophila brain tissue sections, we have demonstrated that RP helps to compensate for an α-synuclein-mediated reduction in the number of dopaminergic neurons and leads to better performance in animal climbing tests (p < 0.001, n = 120-150 flies per genotype/assay). Taken together, these results demonstrate the potential of RP for the treatment of PD, at least in model systems.

[Comparative structural and functional characteristics of different forms of Saccharomyces cerevisiae red pigment and its synthetic analogue].

Structural and functional characteristics of the yeast red pigment (product of polymerization of N1-(beta-D-ribofuranosyl)-5-aminoimadazole), isolated from adel 1 mutant cells of Saccharomyces cerevisiae, its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N1-methyl-5-aminoimadazole in vitro) has been obtained. Products of in vitro polymerization were identified using mass spectrometry. The ability of these pigments to inhibit amyloid formation using insulin fibrils was compared. The entire compounds studied were able to interact with amyloids and inhibit their growth. Electron and atomic force microscopy revealed a common feature inherent in the insulin fibrils formed in presence of these compounds--they were merged into conglomerates that were more stable and resistant to the effects of ultrasound in comparison with insulin aggregates grown without pigments. We speculate that all these compounds can cause coalescence of fibrils, partially block their loose ends and, thereby, inhibit the attachment of new monomers to growing fibrils.

[The effect of red pigment of Saccharomyces cerevisiae on insulin fibril formation in vitro].

The effect of the yeast red pigment, the result of polymerization of AIR, and of its low molecular weight derivative (presumably devoid of phosphoribosyl moiety) on the formation of amyloid fibrils in vitro was studied. Both the red pigment and its derivative, the result of acid hydrolysis of the original pigment, were shown to diminish the intensity of amyloid bound Thioflavine T fluorescence. Correlation between the decrease of the intensity of Thioflavine T fluorescence and the concentration of both forms of the red pigment was demonstrated. Both forms were also able to compete with Thioflavine T for amyloid fibrils. Electron microscopy permitted to visualize a drop of fibril size in the case of red pigments presence during their formation.

[The effect of red pigment on amyloidization of yeast proteins].

Amyloid bound thioflavine T fluorescence was studied in the lysates of yeast strains carrying mutations in genes ADE1 or ADE2 and accumulating red pigment, a result of polymerization of aminoimidazoleribotide (an intermediate of adenine biosynthesis). The fluorescence is drastically enhanced in the case of cells grown in media containing high concentration of adenine (100 mg/l) that blocks accumulation of red pigment. Blocks at first stages of purine biosynthesis de novo also impede red pigment and lead to the same effect on thioflavine fluorescence. At the same time induction of mutations in genes ADE1 or ADE2 in originally white prototrophic strains leads to considerable drop of fluorescence. A fraction of protein polymers was studied by agarose gel electrophoresis and this permitted to conclude that lowering of fluorescence intensity is indeed connected with the decrease of amyloid amount in cells accumulating red pigment. Model experiments with insulin fibers demonstrate that red pigment binds fibrils and blocks their interaction with Thioflavine T. 2D-electrophoretic comparison of pellet proteins of red and white isogenic strains, followed by MALDI, allowed identification of 23 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins, involved in glucose metabolism, closely corresponding to prion-dependent proteins characterized in our previous work. We suppose that, binding amyloid fibrils, red pigment hinders formation of prion aggregates and also, blocking fibril contact with chaperones, impedes prion propagation.

[Comparison of crude lysate pellets of isogenic strains of yeast with different prion composition: identification of a set of prion-associated proteins].

A new approach: comparative analysis of proteins of the pellets of crude cell lysates of isogenic strains of Saccharomyces cerevisiae differing by their prion composition permitted to identify a large group of prion-associated proteins in yeast cells. 2D-electrophoresis followed by MALDI-analysis of a recipient [psi-] strain and of [PSI+] cytoductant led to identification of 35 proteins whose aggregation state responded to a shift of prion(s) content. Approximately half of these proteins belonged to functional groups of chaperones and enzyme involved in glucose metabolism. Notable were also proteins involved in translation, in oxidative stress response and in protein degradation. The data obtained are compared with the results of other groups who used other approaches to detecting proteins involved in prion aggregates.

[Estimating of changes in the amyloid and prion content of yeast cells].

An attempt was made at estimating the overall amyloid content of yeast cells by treating crude cellular lysates with thioflavin T, the agent specifically staining amyloid fibrils. We demonstrated that overproduction of the yeast chaperone Hsp104p, as well as GuHCI treatment of the [PSI+] cells led both to elimination of the [PSI+] factor and to a stable decrease of the overall amyloid content estimated by intensity of fluorescence (IF) of the thioflavin T. At the same time, overexpression of gene SUP35, coding the protein prionizable to [PSI+], led to generation of [PSI+] clones with higher IF of thioflavin T. Cytoduction in the crosses involving PSI factor leads to considerable enhancement of IF; cytoductants with the nucleus of the recipient [psi-] strain not only got [PSI+] factor from the donor strain but also increased their amyloid content. In these model experiments all treatments modifying one of the yeast prions, [PSI+] factor, led to a predictable shift of IF of thioflavin T that behaved like a cytoplasmic hereditary determinant. The data obtained show that IF of thioflavin T staining gives reliable estimates of cellular amyloid content and that mitotically stable shift of IF after a battery of treatments modifying cellular prion set provides quantitative estimate of the input of prionizable protein molecules to the amyloid pool. The combination of thioflavin staining and prionotropic treatments applied here can be possibly used for future attempts of checking yeast strains for cryptic prions.

[Stereospecific DNA anomalies in the human chromosome 21]. / Stereospetsificheskie anomalii v strukture DNK 21-i khromosomy cheloveka.

Using our own original computer program, we analysed more than 10 millions b.p. of the complete nucleotide sequence in the human chromosome 21. A graphic catalogue of largest stereospecific anomalies of this sequence is presented. Clusters of different stereospecific anomalies, showing presumably areas of cooperative binding of different regulatory and structural proteins to DNA have been revealed. Most of the large stereospecific anomalies are situated in introns, being often accompanied by regions devoid of some specific dinucleotides.

[Detection of concealed "illegitimate" nuclei in tetrad analysis of the diploid progeny of heterokaryons in Saccharomyces cerevisiae]. / Vyiavlenie skrytykh "nezakonnykh" iaderpri tetradnom analize diploidnogo potomstva geterokarionov u Saccharomyces cerevisiae.

In this work, the studies on the previously detected phenomenon of concealed heterokaryosis in Saccharomyces cerevisiae were continued. In genetic and Southern blotting experiments, one of the nuclei in the heterokaryon was shown to be active (capable of division and ensuring the corresponding cell phenotype), whereas the other was not expressed until the heterokaryotic clone was transferred to the medium selective for this concealed nucleus. Moreover, the concealed nucleus was able to assume the active state after fusion with the second parental nucleus. It was analyzed whether the nuclei with new marker combinations occurring in meiosis can behave as exceptional nuclei. Tetrad analysis of hybrids carrying the kar1 mutation in their nuclei revealed the relatively high percentage of exceptional tetrads (more than 10%). One spore in these tetrads usually formed diploid cells capable of sporulation. The presented data of genetic and molecular biological studies testify in favor of the assumption that abnormal spores contain two nuclei, which form an "illegitimate" hybrid after fusion. An extraneous spore (termed x) has usually a genotype close to that of one of the spores in this tetrad. Thus, it was assumed that the additional DNA replication round occurs in the absence of cell division during one of meiotic divisions. Results of cytological analysis conducted by the method of specific DNA staining confirmed the existence of exceptional tetrads, one spore of which contains two nuclei.

[Cryptic heterokaryons in strains of Saccharomyces cerevisiae. Conditions for detecting a cryptic nucleus]. / Skrytye geterokariony v shtammakh Saccharomyces cerevisiae. Usloviia proiavleniia skrytogo iadra.

A phenomenon discovered earlier, cryptic heterokaryosis in Saccharomyces yeast, has been further investigated. A phenotypically silent nucleus in a yeast cell may resume its expression after fusion with another parental cell. The resulting hybrid is capable of sporulation. By the growth of a cytoductant with an expressing nucleus from one parent and a silent nucleus from the other parent on suitable selective media, the silent nucleus can be activated. The presence of deletion or insertion mutations in several genes in YPH strains allows nuclei of the YPH type to be traced not only genetically but also by blotting.

[Stereospecific anomalies in the structure of DNA from chromosomes V, XII, XIII, XIV, XV and XVI of saccharomycetes yeasts]. / Sterospetsificheskie anomalii v strukture DNK V, XII, XIII, XIV, XV i XVI kromosom drozhzhei-sakharomitsetov.

Using an original computer program we analysed complete nucleotide sequences of chromosomes V, XII, XIII, XIV, XV, and XVI in yeast. The full catalogue of 5 highest stereospecific anomalies and of stereospecific anomalies for 5 genes with highest CAI in each chromosome has been presented. Trains of different stereospecific anomalies, possibly showing areas of cooperative binding of different regulatory and structural proteins to DNA, are documented (see: Soidla, Lukina, 1998, 1999). Together with confirming the earlier noticed association between stereospecific anomalies and genes with high expression level (coding mostly proteins of translation machinery of the cell and glycolytic enzymes), here we can also notice an obviously high incidence of transcription apparatus genes being located at (or near) largest stereospecific anomalies. Intracellular transport genes and genes whose products function in the cell nucleus may also be often associated with largest stereospecific anomalies.

[Stereospecific anomalies in DNA structure of Saccharomyces chromosomes IV, VII, VIII, X and XI]. / Stereospetsificheskie anomalii v strukture DNK IV, VII, X i XI khromosom drozhzhei-sakharomitsetov.

Using an original computer program we analysed complete nucleotide sequences of chromosomes IV, VII, VIII, X and XI in yeast. Data about 5 largest stereospecific anomalies in each chromosome are presented together with those for 5 genes with highest CAI in each chromosome. Clusters of different stereospecific anomalies are demonstrated, including trains of not overlapping anomalies, possibly showing areas of cooperative binding of different regulatory and structural proteins to DNA (Soidla, Lukina, 1998). Together with confirming the earlier noticed connection between stereospecific anomalies and genes with high expression level (coding mostly proteins of translation machinery of cell and glycolytic enzymes), here we also noticed an obvious high incidence of transcription apparatus genes being located at (or near) largest stereospecific anomalies.

[Stereospecific anomalies in the structure of five completely sequenced yeast chromosomes]. / Stereospetsificheskie anomalii v strukture piati polnost'iu sekvenirovannykh khromosom drozhzhei.

Using an original computer program we analysed complete nucleotide sequences of chromosomes I, II, III, VI and IX in yeast cells. As a general rule, we found large stereospecific anomalies near genes with a presumed high expression level (a full catalogue of such anomalies for 5 genes with highest CAI in each chromosome is presented). As a rule, they are also present at mobile genetic elements. Many large stereospecific anomalies are situated next to the sites of specific anomalies of general nucleotide composition-regions devoid of specific dinucleotides. We have noticed many "trains" (lines) of different stereospecific anomalies, possibly showing areas of cooperative binding of different regulatory and structural proteins to DNA. In several, but not all, analysed chromosomes we found a new class of especially large stereospecific anomalies related to repetitive DNA of small length (less than or around 100 nucleotides).

[Cryptic heterokaryons in Saccharomyces cerevisiae strains: pseudopleiotropic suppression in the multiple marked YPH857 strain]. / Skrytye geterokariony v shtammakh Saccharomyces cerevisiae psevdopleiotropnaia supressiia v mnozhestvenno markirovannom shtamme YPH857.

The yeast strain YPH857 carrying multiple genetic markers was shown to segregate clones that had the pleiotropic suppression phenotype. The phenotype was designated Ppsu+. This suppression involves deletion alleles of the TRP1 and HIS3 genes and an insertion in the URA3 gene. Unlike the original YPH857 culture that carries an unidentified mutation of resistance to cycloheximide, the Ppsu+ clones exhibited a decreased level of resistance to this inhibitor of protein synthesis. In addition, they have a lower mating ability and can produce asci on a standard medium for sporulation. A comparative analysis of total DNA from the YPH857 strain and Ppsu+ segregants by Southern blotting provided evidence for the presence of an extraneous nucleus in these segregants. Ppsu+ strains were shown to contain wild-type alleles, apart from deletion and insertional alleles typical for the YPH857 strain. Moreover, they contain the 2 microns DNA of the Scp3 type with deletion of one of the two EcoRI and HpaI recognition sites (whereas the 2 microns DNA of YPH857 belongs to the Scp1 type) and exhibit heterogeneity with respect to the presence of one EcoR1 recognition site in the gene of 5S ribosomal RNA. It was supposed that the "cryptic" nucleus belongs to a strain of low viability and can survive as an unexpressed DNA in a small fraction of cells. Nuclei from the cryptic and YPH857 strains can be fused at a low rate to yield Ppsu+ cells capable of sporulation. In certain cases, a Ppsu+ clone may be heterogeneous: some of its cells contain nuclei in an unfused heterokaryotic state. This assumption has been confirmed by selecting Cyhr colonies with the original YPH857 genome among Ppsu+ clones on the cycloheximide-containing medium.

[Cryptic heterokaryons in Saccharomyces cerevisiae strains. Model experiments]. / Skrytye geterokariony v shtammakh Saccharomyces cerevisiae. Model'nye opyty.

Heterokaryons capable of segregating clones of different phenotypes were obtained in saccharomycetes yeast. Clones expressing phenotypic traits encoded by the nuclear genome of one parent and the mitochondrial genome of another were shown to contain cells that carried the second phenotypically silent nucleus. The cryptic nucleus can be uncovered by growing these "cytoductants" on the corresponding medium. The use of strains carrying an insertion in the URA3 gene and the endogenic plasmid, 2 microns DNA, made it possible to detect this nucleus by blot hybridization.

[Isolation of nucleotide sequences, possessing enhancer functions from the Saccharomyces cerevisiae genetic library]. / Vydelenie nukleotidnykh posledovatel'nostei, obladaiushchikh énkhansernoi funktsiei, iz genoteki Saccharomyces cerevisiae.

We propose a method for isolation of enhancer-like sequences from a yeast genomic library. The method was used to identify DNA inserts capable of increasing bacterial bla gene expression when located downstream from the transcription initiation site. Plasmids carrying different fragments of one such insert were used to localize the enhancer-like function on a 1.2 kb fragment.

[A 2-um plasmid of Saccharomyces]. / 2-mkm plazmida drozhzhei-sakharomitsetov.

The review of yeast endogenous 2 micron plasmid and of some 2 micron plasmid based vectors is devoted to structure--function relationships with a particular emphasis on the stable replication control mechanisms. Some original data on possible novel plasmid encoded proteins are provided.

[Induction and genetic analysis of mutations modifying phenotypic suppression at increased levels of carbon dioxide in yeasts]. / Poluchenie i geneticheskii analiz mutatsii, vliiaiushchikh na fenotipicheskuiu supressiiu pri povyshennykh kontsentratsiiakh uglekislogo gaza u drozhzhei.

A new class of yeast mutations modifying the phenotypic suppression by carbon dioxide is described. The mutations are designated ncm (for CO2 modification). Mutations ncm1 and ncm3 were shown not to block adenine biosynthesis. Ncm3 mutation inhibited CO2 suppression of the ade1 and ade2 mutations.

[Possible enzymatic role of imported tRNA1Lys during splicing of mitochondrial transcripts in yeasts]. / Vozmozhnaia fermentativnaia rol' importiruemoi tRNK1Lys pri splaisinge mitokhondrial'nykh transkriptov u drozhzhei.

A sequence of imported tRNA1Lys fitting the donor boundaries of mitochondrial first class introns is shown to exhibit a ribozyme consensus-like structure.