PubMed Health⌕ Search

Biomedical subjects

N P Sharova

Publications and source records attributed to N P Sharova.

15 recordsLinked to original sources
PubMed2006

Peculiarities of proteasome pool formation in rat spleen and liver during postnatal development.

Changes in the specific activity and amounts of 26S and 20S proteasome pools in rat spleen and liver during postnatal development and appearance in them of immune subunits were studied. Two decreases in chymotrypsin-like activity of the proteasome pools were recorded during the first three weeks after birth. The activity minimum fell on the 11th and 19th days, and the first decrease was more prolonged and pronounced than the second. The decrease in the specific activity of the 26S proteasome pools was associated with a reduction of their quantity. The 20S proteasome pools displayed no such decreases. Noticeable quantities of immune subunits LMP7 and LMP2 were revealed by Western blotting in the spleen on the 7th day and on the 19th day in the liver, concurrently with the beginning of the decrease in the proteasome activity. It was concluded that during the first three weeks of postnatal development the proteasome pools in rat spleen and liver were replaced twice, and in the spleen (a lymphoid organ) a qualitatively new pool containing immune subunits appeared nearly two weeks earlier than in the liver (a non-lymphoid organ). The appearance of immune proteasomes in different organs and tissues during some weeks after birth seems to explain the immune system inefficiency during embryogenesis and early postnatal development.

Animals↗
PubMed2005

How does a cell repair damaged DNA?

DNA in living cells is constantly subjected to different chemical and physical factors of the environment and to cell metabolites. Some changes altering DNA structure occur spontaneously. This raises the potential danger of harmful mutations that could be transmitted to offspring. To avoid the danger of mutations and changing genetic information, a cell is capable to switch on multiple mechanisms of DNA repair that remove damage and restore native structure. In many cases, removal of the same damage may involve several alternative pathways; this is very important for DNA repair under the most unfavorable conditions. This review summarizes data about all known mechanisms of eukaryotic DNA repair including excision repair (base excision repair and nucleotide excision repair), mismatch repair, repair of double-strand breaks, and cross-link repair. Special attention is given to the regulation of excision repair by different proteins--proliferating cell nuclear antigen (PCNA), p53, and proteasome. The review also highlights problem of bypassing irremovable lesions in DNA.

DNA Damage↗
PubMed2002

Initiation of DNA replication in eukaryotes is an intriguing cascade of protein interactions.

Initiation of eukaryotic DNA replication is a complex process including the recognition of initiation sites on DNA, multi-step DNA preparation for duplication, and assembly of multi-protein complexes capable of beginning DNA synthesis at initiation sites. The process starts at the late M phase and lasts till the appropriate time of the S phase for each initiation site. A chain of interesting interactions between Orc1p-6p, Cdc6p, Mcm2p-7p, Mcm10p, Cdt1, Cdc45p, Dbf4/Cdc7p, RPA, and DNA polymerase alpha takes place during this period. The sequence of these interactions is controlled by cyclin-dependent kinases, as well as by ubiquitin-dependent proteolysis in the proteasome. This review summarizes the data on proteins initiating DNA replication and factors controlling their activities.

Binding Sites↗
PubMed2001

Identification of DNA polymerase delta in eggs of a teleost fish (loach).

DNA polymerase found in an extract from eggs of the teleost fish Misgurnus fossilis (loach) has been identified as an enzyme of the delta type. The enzyme was purified 4000- to 5000-fold from the extract by liquid chromatography. The DNA polymerase activity was sensitive to the inhibiting action of aphidicolin but resistant to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP). The enzyme activity correlates with the presence of a polypeptide with molecular mass of 120-130 kD that interacts specifically with polyclonal antibodies against calf thymus DNA polymerase delta as revealed by Western blotting and is presumably the catalytic subunit of the enzyme. The loach DNA polymerase possesses the 3'-->5'-exonuclease activity specific to single-stranded DNA and catalyzes distributive elongation of primers in primer-template complexes.

Animals↗
PubMed2001

Interaction of loach DNA polymerase delta with DNA duplexes with single-strand gaps.

The interaction of DNA polymerase delta purified from eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes with single-strand gaps of 1-13 nucleotides was studied. In the absence of template-restricting DNA, the enzyme elongated primers on single-stranded DNA templates in a distributive manner. However, in the presence of the proximal 5;-terminus restricting the template, the enzyme activity significantly increased. In this case, the enzyme was capable of processive synthesis by filling gaps of 5-9 nucleotides in DNA duplexes. These data indicate that DNA polymerase delta can interact with both the 3;- and 5;-termini located upstream and downstream from the gap. Analysis of the complexes formed by DNA polymerase delta and different DNA substrates by electrophoretic mobility shift assay confirmed the assumption that this enzyme can interact with the proximal 5;-terminus restricting the gap. DNA polymerase delta displayed much higher affinity in duplexes with gaps of approximately 10 nucleotides compared to the standard template-primer complexes. Maximal affinity was observed in experiments with DNA substrates containing unpaired 3;-tails in primers. The results of this study suggest that DNA polymerase delta exerts high activity in the cell nuclei during repair of DNA intermediates with single-strand gaps and unpaired 3;-termini.

Animals↗
PubMed2000

Preferential interaction of loach DNA polymerase delta with DNA duplexes containing single-stranded gaps.

We studied the interaction of DNA polymerase delta (pol delta) purified from the eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes containing single-stranded gaps of 1-13 nucleotides (nt). In the absence of processivity factors (PCNA, RF-C, and ATP), pol delta elongated primers on single-stranded DNA templates in a distributive manner. However, the enzyme was capable of processive synthesis by filling gaps of 5-9 nt in DNA duplexes. These data suggest that, upon filling a small gap, pol delta interacts with the 5'-terminus downstream of the gap as well as with the 3'-terminus of the primer. Interaction of pol delta with the proximal 5'-terminus restricting the gap was confirmed by electrophoretic mobility shift assay. Analysis of the enzyme binding to DNA duplexes containing gaps of various sizes showed a much higher affinity of pol delta for duplexes with gaps of about 10 nt than for DNA substrates with primers annealed to single-stranded templates. The most efficient pol delta binding was observed in experiments with DNA substrates containing unpaired 3'-tails in primers. The data obtained suggest that DNA molecules with small gaps and single-stranded tails may serve as substrates for direct action of pol delta in the course of DNA repair.

Adenosine Triphosphate↗
PubMed1989

Cyclic nucleotide phosphodiesterase and 5'-nucleotidase: a coupled system.

Evidence is presented that multiple forms of cyclic nucleotide phophodiesterase (PDE) activity chromatographically separated from the soluble fraction of bovine hypothalamus are co-eluted with multiple forms of 5'-nucleotidase (5'N) activity. The enzymes could not be resolved from each other by anion-exchange chromatography on DEAE-TSK; by affinity chromatography on phenyl-, blue-, concanavalin A-, 5' AMP-sepharose, cAMP-silica gel; or by gel filtration on sephacryl S-200. The catalytic activities were found to be associated with the tetrameric, dimeric, and monomeric forms of the enzymes. The molecular weights determined by gel filtration or by SDS-gel electrophoresis were 220, 114, and 57 kDa, respectively. Kinetic analysis revealed that the first-order rate constant for 5'AMP hydrolysis measured in the reactions: cAMP----5'AMP----adenosine was 100 times higher than that in the reaction: 5'AMP----adenosine. Thus, functional interrelation between PDE and 5'N was expressed in drastic acceleration of the consecutive reactions: cAMP----5'AMP----adenosine. The results confirm the conclusion about the existence of a multienzyme system involving PDE and 5'N or of a single bifunctional enzyme in brain tissue.

5'-Nucleotidase↗
PubMedDate not supplied

[The proteasome: destroy to live].

The importance of protein degradation for controlling individual protein levels is long beyond doubt. Yet only recent experimental evidence confirmed the key role of the ubiquitin-proteasome degradation pathway in the cell cycle, carcinogenesis, transcription, cell differentiation, tissue growth and atrophy, metabolism of various substrates, selective elimination of abnormal proteins, and antigen processing. The review considers the structures of the 26S proteasome and its 20S proteolytic core, the system of ubiquitination of proteins bearing a degradation signal, the role of the 26S proteasome in various metabolic processes, and possible mechanisms regulating the proteasomal activity in the cell.

Animals↗
PubMedDate not supplied

[The peculiarities of repair DNA-polymerases in the loach embryogenesis].

The current concept of eukaryotic DNA polymerases is considered, which are involved in nuclear DNA repair. The data are given on a new group of DNA polymerases that maintain the integrity of DNA structures without preliminary excision of damaged regions. A special attention is paid to specific features of the functioning of repair DNA polymerases in embryogenesis of the loach. A possible existence is discussed of the previously unknown pathway of DNA repair with participation of DNA polymerase delta as independent from the nuclear antigen of proliferating cells.

Animals↗
PubMedDate not supplied

[Multiple forms of the proteasomes and some approaches to their separation].

A technique for proteasome isolation from rat liver was developed; it allows isolation of 26S proteasome fraction free from 20S proteasome admixture. The technique includes consecutive stages of protein fractionation by different concentrations of ammonium sulfate, gel filtration on Sepharose 2B, and high-speed ultracentrifugation.

Ammonium Sulfate↗
PubMedDate not supplied

[Changes in proteasome activity and subunit composition during postnatal development of rat].

The dynamics of the activities of 26S and 20S proteasomes in the rat liver and spleen have been studied during postnatal development from 1 to 90 days. The activities of proteasome forms both in spleen and in liver increased in adult animals as compared to one day rats. The activities of both proteasome forms in the liver did not differ significantly from those in the spleen at all stages of postnatal development. Using Western blot with monoclonal antibodies to Rpt6 subunit, we confirmed the presence of 26S proteasome in both organs at all stages of postnatal development. Studies with polyclonal antibodies to beta1i (LMP2) subunit showed the appearance of the immune subunit in the spleen by day 9 and in the liver only by day 23 of postnatal development. This result suggests the earlier formation of the spleen as an organ with immune functions.

Animals↗
PubMedDate not supplied

[Exclusion of immune proteasomes from mouse ascitic carcinoma Krebs-II cells].

Pools of 26S and 20S proteasomes were studied in the spleen, liver, lung, and ascitic carcinoma Krebs-II of mouse. Western blotting demonstrated that the pool of 26S proteasomes in ascitic carcinoma Krebs-II was twice that in control lung cells and did not significantly differ by total 26S proteasome quantities from the spleen and liver. At the same time, the level of immune subunit LMP7 was 12 times lower in it compared to lung proteasomes and 4-5 times lower compared to spleen and liver proteasomes. Immune subunit LMP2 was undetectable by this technique in the ascitic carcinoma in contrast to the lung, spleen, and liver. All immune subunits in the studied organs and ascitic carcinoma Krebs-II are components of 26S but not 20S proteasomes.

Animals↗
PubMedDate not supplied

[Immune proteasomes and immunity].

A lot of facts that require understanding have been accumulated since immune proteasomes were discovered and their relationship with the immune response was established. For example, why are immune proteasomes present in all studied mammalian organs and tissues, including nonlymphoid tissues? What is responsible for differences in the ratio of immune to constitutive proteasomes in different organs? Are the functions of immune proteasomes related to the T-cell immune response alone, as was shown initially, or not? Are immune proteasomes formed simultaneously in different organs during ontogenesis? An attempt is made in this review to answer these and other related questions.

Animals↗