[Heat-shock protein HSP70 decreases activity of proteasomes in human neuroblastoma cells treated by amyloid-beta 1-42 with isomerized Asp7].

Experimental evidences indicate that heat-shock protein 70 (HSP70) can serve as a prospective therapeutic agent to treat Alzheimer's disease (AD). It has demonstrated a neuroprotective effect in vivo on mice models of AD. Moreover, HSP70 decreases oxidative stress in neurons induced by amyloid-ß (Aß42) and its more toxic form with isomerized Asp7 (isoAß42). The dysfunction of Ubiquitin-proteasome system (UPS) is observed in AD. UPS is responsible for the degradation of the majority of cellular proteins and plays an important role in protecting cells from oxidative stress. Here, we have shown that the incubation of human neuroblastoma cells SK-N-SH with isoAß42 increases the activity of intracellular proteasomes, which are the principal elements of the UPS. On the contrary, the proteasomal activity was decreased in isoAß42-treated cells in the presence of exogenous HSP70. These results highlight the existence of an interplay between Aß peptides, proteasomes, and HSP70.

[Rabies vaccines: Current status and prospects for development].

Rabies is an infectious disease among humans and animals that remains incurable, despite its longstanding research history. The only way to prevent the disease is prompt treatment, including vaccination as an obligatory component and administration of antirabies immunoglobulin as a supplement. Since the first antirabies vaccination performed in the 19th century, a large number of different rabies vaccines have been developed. Progress in molecular biology and biotechnology enabled the development of effective and safe technologies of vaccine production. Currently, new-generation vaccines are being developed based on recombinant rabies virus strains or on the production of an individual recombinant rabies antigen-glycoprotein (G protein), either as a component of nonpathogenic viruses, or in plants, or in the form of DNA vaccines. In this review, the main modern trends in the development of rabies vaccines have been discussed.

[Chromatin structure and transcription regulation in Saccharomyces cerevisiae].

Nucleosome organization of eukaryotic chromatin determines the DNA availability for regulatory factors during transcription. Nucleosome positioning at promoters is an important factor effecting gene expression. Recent studies showed that yeast promoters can be divided in two groups with the different chromatin organization. It has become clear that genes with the similar chromatin structure in promoters are regulated by the same mechanisms. In this review, using Saccharomyces cerevisiae as example, we discuss interplay between chromatin structure and transcription, dynamic changes in chromatin during transcription and a role of various factors (histone chaperones, remodeling complexes, histone variant H2A.Z) in these processes.

[Transcriptional regulation of proteasomal genes in eukaryotes].

The ubuquitin-proteasome system is involved in degradation of many intracellular proteins and is necessary for proper functioning of the cell under normal conditions and its survival under stress conditions. In this review the general principles of structure and functioning of the ubiquitin-proteasome proteolytic system is considered. The main attention was paid for regulation of proteasomal genes expression, and specifically for discovery and analysis of Rpn4p transcription factor, an activator for proteasomal genes in Saccharomyces cerevisiae. The data about regulation of proteasomal genes expression in higher eukaryotes is also discussed.

[Rpn4p is a positive and negative transcriptional regulator of the ubiquitin-proteasome system].

Ubiquitin-proteasome proteolytic system participates in metabolism of the majority of intracellular proteins and regulation of key cellular processes in eukaryotes. While the structure and functioning of this system is studied rather well, a little is known about regulation of its genes expression. At present time, the only regulatory system of transcription of proteasome genes is found in the yeast Saccharomyces cerevisiae. This system includes Rpn4p-proteasome-associated transcriptional regulator and its binding site called PACE (Proteasome Associated Control Element). To learn more about function of Rpn4p as a transcriptional regulator, there are following questions: 1) is the Rpn4p regulator for PACE-containing genes which encode for components of protein ubiquitinylation system 2) what is the contribution of Rpn4p in stress-activated level of mRNA of proteasome genes. In this work, using semiquantitative RT-PCR we have shown that deletion of RPN4 gene leads to decreasing in mRNA level of the genes of ubiqitination system RAD6, RAD23 and CDC48, while UBI4 mRNA level is increased in this strain. In the presence of alkylating agent methyl methanesulfonate or under heat shock we observed Rpn4 p-dependent elevation of mRNA level of the proteasomal genes RPT4 and RPNS. At the same time, CDC48 mRNA level is decreased in wild type yeast strain upon methyl methanesulfonate treatment. These data indicate that under normal or stress conditions Rpn4p may act as an activator or repressor for the genes of the ubiquitin-proteasome system.

[The legacy of Andrey Mirzabekov and his laboratory in the studies of chromatin structure using DNA-protein cross-linking].

The work of A.D. Mirzabekov and his collaborators on chromatin structure is reviewed. The methods of DNA-protein covalent binding with subsequent analysis of cross-linked products developed in the laboratory were used to localize the contacts in both DNA and individual proteins. This original approach played an important role in the deciphering of the molecular structure of nucleosome. In addition, it made possible the investigation of structural transitions in chromatin upon gene activation.

[Assays on comparing the local concentration of HU protein in the different regions of Escherichia coli genomic DNA].

HU, a nonspecific histone-like DNA binding protein is a major component of the bacterial nucleoid. HU is referred to as an accessory factor for complex protein-DNA assembly and as a protein involved in DNA compaction. In this study we investigated in vivo HU binding along the different regions of E. coli genome. For this purpose we used ChIP--in vivo formaldehyde crosslinking and immunoprecipitation of protein-DNA complexes with antiHU-antibodies. This technique allows to compare the local concentration of HU protein in the different regions of E. coli genomic DNA. In this study we analysed the HU-DNA crosslinking both in exponentially growing and stationary phase of bacteria in the following regions of E. coli genome: oriC region, promoter and structural regions of hupA and hupB genes coding two different subunits of HU, and structural parts of dps and glgS genes which are active only in stationary phase. Our results indicate that in exponentially growing E. coli cells the local concentration of HU protein is uniform for all analysed regions of genome and does not depend on their transcriptional status. The twofold increase of local concentration of HU protein was also shown for all analysed genome regions in the stationary phase cells.

[Isolation and identification of PACE-binding protein rpn4--a new transcription activator, participating in regulation of 26S proteosome and other genes]. / Vydelenie i identifikatsiia PACE-sviazyvaiushchego belka Rpn4--novogo transkriptsionnogo aktivatora, uchastvuiushchego v reguliatsii 26S proteasomykh i drugikh genov.

A new upstream activating sequence (UAS) 5'-GGTGGCAAA-3' was detected in the promoters of 27 out of the 33 proteasomal genes and of several genes related to the ubiquitin-proteasomal system of Saccharomices cerevisiae. The sequence was termed proteasome-associated control element (PACE). Gel retardation assay revealed specific binding of PACE with an extracted protein. The protein (64K) was purified by affinity chromatography and was homogeneous by SDS-PAGE. Microsequencing showed that the protein is Rpn4p. The ability of Rpn4p to activate transcription was demonstrated with constructs containing fragments of the RPN5 and CDC48 gene promoters and reporter cat. Binding to PACE, Rpn4p may act as a common transcription factor on the proteasomal and proteasome-related genes.

[Detection of nonhistone proteins, bound with regulatory elements of yeast rRNA genes, by UV-crosslinks in intact nuclei]. / Obnaruzhenie negistonovykh belkov, sviazannykh s reguliatornymi élementami genov rRNK drozhzhei metodom UF-sshivok na intaktnykh iadrakh.

We have studied the arrangement of DNA-binding proteins along yeast ribosomal non-transcribed spacer by UV-induced DNA-protein crosslinking on intact nuclei. We show binding of proteins with apparent Mw 120 and 30 kDa in promoter region, 120 kDa in enhancer region, 40 kDa in ARS. These proteins were identified preliminary as REB1 (promoter, enhancer), TFID (promoter), MCM3 (ARS). Good agreement between information supplied by this technics and literature data prove usefulness of our approach and make possibility for its wide appliance.

[Structural changes in chromatin of heat shock protein 70 gene of Drosophila during transcription]. / Strukturnye izmeneniia khromatina gena belka teplovogo shoka 70 Drosofily pri transkriptsii.

Using "protein-image" hybridization technique combined with various crosslinking methods, for formaldehyde-prefixed nuclei we have analysed changes induced by activation in the chromatin structure of HSP-70 genes. From the crosslinking data it follows that chromatin of actively transcribed genes undergoes some structural rearrangements resulting in certain weakening of the contacts between DNA and the globular parts of histones so that the histones remain bound to DNA through their N-terminal regions. In addition, there have been found two specific regions with a reduced content of histones: the 5'-promoter of HSP-70 gene and a region distanced by approximately 1 k.b. from the 3'-end of the HSP-70 gene.

[Chromatin structure of ribosomal genes of Drosophila melanogaster. Random location of nucleosomes in DNA and characteristics of organization of non-transcribed spacer]. / Struktura khromatina ribosomnykh genov Drosophila melanogaster. Sluchainoe raspolozhenie nukleosom na DNK i osobennosti v organizatsii netranskribiruemogo speisera.

Chromatin structure of ribosomal genes from nuclei of Drosophila melanogaster embryos was studied by using micrococcal nuclease cleavage. End-directed labelling with short cloned fragments of the ribosomal repeat was carried out. It shows, that the micrococcal nuclease prefers specific sites in naked DNA, and the pattern of DNA cleavage is essentially conserved when the nuclei are digested. Only minor differences are visible. Hence, there are no specific positions of nucleosomes on the ribosomal repeat. The DNA fragments from the nuclei treated with micrococcal nuclease were electrophoresed, transferred to DBM-paper and hybridized with different probes subcloned from the ribosomal repeat. The non-transcribed spacer and the region of the beginning of transcription are hydrolysed significantly faster than the coding region or inactive ribosomal insertion. The region of NTS and the beginning of transcription do not give normal nucleosomal fragment in the range of 145-185 bp; instead they produce a heterogeneous band 200-280 bp in length even after prolonged digestion. Dinucleosomal fragments are also slightly longer and more heterogeneous than in other parts of the ribosomal repeat. Higher oligomers are similar throughout the ribosomal repeat. We suggest that a hypothetical transcription factor interacts in a way with histones and protects unusual fragments of DNA from digestion.

[Structure of the transcription-active chromatin]. / Struktura khromatina, aktivnogo v transkriptsii.

A method of attaching proteins to specific sites of DNA has been developed. The proteins are fixed on DNA directly in the cell nucleus by covalent bonding. DNA's associated with different proteins are divided into several diagonals by the two-dimensional gel-electrophoresis. The nucleotide sequences linked to proteins are then identified by hybridizing them with cloned probes of DNA. This method was used for Drosophila cells to determine the presence of histones in the transcribed site of the hsp-22 gene, activated by heat shock, and also in the neighboring active gene and non-transcribed insertion in ribosomal RNA genes. It was found that moderate transcription removed H1 and part of core histones, whereas intensive transcription removed all histones from the structural region of the genes. The substitution of core histones by RNA-polymerase within the transcribed region seems to cause the removal of first H1 and then core histones (depending on the intensity of the process) from prolonged regions of chromatin, which leads to a 25 nm fibrilla being decondensed to a 10 nm thread and, finally, to a completely linearized histone-free DNA.