Reduction of the number of mutant copies of mitochondrial DNA in tissues of irradiated mice in the postradiation period

Changes in the number of mutant copies of mitochondrial DNA (mtDNA) were studied in the brain and spleen tissues of mice after their X-irradiation at a dose of 5 Gy. For this purpose, heteroduplexes obtained via hybridization of the products of PCR amplification of mtDNA (ND3 gene and two D-loop regions) from irradiated and control mice were digested with the CelI nuclease capable of specific mismatch cleavage. Heteroduplexes obtained via hybridization of the products of PCR amplification of mtDNA from irrradiated and control mice were digested by the CelI nuclease to a greater degree than heteroduplexes of the PCR products of mtDNA of mice from the control group. This suggests the presence of mutations in mtDNA regions in irradiated mice. Digestion by the CelI nuclease of heteroduplexes obtained via hybridization of the PCR products of mtDNA (ND3 gene and D-loop regions) on day 8 after irradiation is essentially more efficient than digestion of heteroduplexes obtained via hybridization of the PCR products of mtDNA isolated from mouse tissues on days 14 and 28 of the postradiation period. These results indicate a reduction in the number of mtDNA copies with mutations in tissues of irradiated mice by day 28 of the postradiation period. The reduction in the level of mutant mtDNA copies by this term is especially significant in the spleen. The total number of mtDNA copies in the mouse brain and spleen tissues estimated by real-time PCR, relative to the nuclear β-actin gene, is also decreased by 30–50% as compared to the control on days 8 to 28 after irradiation. The results of the study suggest that mutant mtDNA copies are eliminated from tissues of irradiated animals in the postradiation period. This elimination can be regarded either as a result of selective degradation of mitochondria carrying mutant DNA copies or as a result of cell death being continued in tissues of irradiated animals.     

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples

Background  

Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C₄(RP)-LC protein separation) followed by C₁₈(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.     

New producers of biologically active compounds—fungal strains of the genus <Emphasis Type="Italic">Penicillium</Emphasis> isolated from permafrost

Screening of producers of secondary metabolites was carried out among 25 fungal strains of Penicillium genus isolated from permafrost in Arctic and Antarctic regions and Kamchatka. Nearly 50% of the investigated strains synthesize biologically active substances of alkaloid nature: ergot alkaloids, diketopiperazinees, and quinoline derivatives. A large group of the identified metabolites belongs to mycotoxins. A strain of Penicillium waksmanii was found producing epoxyagroclavine-I and quinocitrinines. The main physiological and biochemical characteristics of this producer were investigated.     

Detection of large deletions of mitochondrial DNA in tissues of mice exposed to X-rays

Large mtDNA deletions in mouse brain and spleen cells, induced by X-radiation at doses of 2 and 5 Gy were studied within four weeks after the exposure of animals to X-rays. Variations in the content of extracellular (deletion) mtDNA were examined in the blood plasma of mice irradiated with 5 Gy in the same postirradiation times. Ionizing radiation was shown to effectively induce large mtDNA deletions at the doses chosen. The level of deletion mtDNA was dependent on dose and postirradiation time.     

Survivin monomer plays an essential role in apoptosis regulation

Survivin was initially described as an inhibitor of apoptosis and attracted growing attention as one of the most tumor-specific genes in the human genome and a promising target for cancer therapy. Lately, it has been shown that survivin is a multifunctional protein that takes part in several crucial cell processes. At first, it was supposed that survivin functions only as a homodimer, but now data indicate that many processes require monomeric survivin. Moreover, recent studies reveal a special mechanism regulating the balance between monomeric and dimeric forms of the protein. In this paper we studied the mutant form of survivin that was unable to dimerize and investigated its role in apoptosis. We showed that survivin monomer interacts with Smac/DIABLO and X-linked inhibitor of apoptosis protein (XIAP) both in vitro and in vivo. Due to this feature, it protects cells from caspase-dependent apoptosis even more efficiently than the wild-type survivin. We also identified that mutant monomeric survivin prevents apoptosis-inducing factor release from the mitochondrial intermembrane space, protecting human fibrosarcoma HT1080 cells from caspase-independent apoptosis. On the other hand, our results indicate that only wild-type survivin, but not the monomer mutant form, enhances tubulin stability in cells. These findings suggest that survivin partly performs its functions as a monomer and partly as a dimer. The mechanism of dimer-monomer balance regulation may also work as a "switcher" between survivin functions and thereby explain remarkable functional diversities of this protein.     

Physiological and biochemical characteristics of fungi of the genus Penicillium as producers of ergot alkaloids and quinocitrinins

Four cultures of fungi of the genus Penicillium belonging to Furcatum Pitt subgenus, such as P. citrinum Thom, 1910; P. corylophilum Dierckx, 1901; P. fellutanum Biourge, 1923; and P. waksmanii Zaleski, 1927, produced the ergot alkaloids, namely, agroclavine-I, and epoxyagroclavine-I; their N-N-dimers, such as dimer of epoxyagroclavine-I and the mixed dimer of epoxyagroclavine-I and agroclavine-I; and also quinoline metabolites, namely, quinocitrinin A and quinocitrinin B. Physiological and biochemical characteristics of the producers were studied. Optimal conditions for the biosynthesis of metabolome components were determined. Zinc additive to the medium stimulated the biosynthesis of the ergot alkaloids in all cases; citrinin production was increased only in P. citrinum, and that was suppressed in P. corylophinum, P. fellutanum, and P. waksmanii. This testifies that genes of the biosynthesis pathways are located in the different clusters of the producers.     

Finding the best antibody dilution by repeated immunostaining of the same tissue section

One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution.