Plutonium production and particles incorporation into the human body.

Plutonium is one of the most toxic radioactive substances known. The isotope 239Pu gained attention when it had become known as a potential explosive material for atomic bombs. This paper describes the main problems encountered during the early years of operation of the first plutonium production plant in the former Soviet Union, the Mayak Production Association (Mayak PA). Mayak PA caused severe radioactive contamination of the environment and exposure personnel and population living in the vicinity areas to high radiation doses. The authors focus on key findings of large-scale studies on the internal dosimetry of workers for use in assessment of radiological risks from exposure to plutonium. This work presents an overview of the important issues for inhalation dose assessments such as generation of plutonium particles, plutonium intake, dissolution of plutonium particles, distribution of plutonium in humans, related exposures and health effects. Understanding the relationship between health effects, radiation dose and route of exposure helps quantify the health risks associated with occupational exposure in the nuclear industry and validate the radiation protection standards used in the Russian Federation and worldwide.

THE STRUCTURE OF DNA CHROMOCENTRES IN MOUSE IN SILICO AND IN SITU. LINE AND ERV FRAGMENTS ARE AN OBLIGATORY COMPONENTS OF DNA CHROMOCENTRES BESIDES TANDEM REPEATS.

Constitutive heterochromatin in higher eukaryotes is located in the primary chromosomal strangulation (centromeres and pericentromeres) and subtelomeric regions of chromosomes. In house mouse constitutive chromatin formes structures in the nuclei of cells, brightly colored with DAPI. It is known that the constitutive heterochromatin is enriched with large tandem repeats, however, its exact composition is unknown. In this paper chromocenters were studied using several approaches: cloning and sequencing by Sanger, as well as by high-throughput sequencing. We have shown that DNA components of chromocenters and constitutive chromatin are full-length endogenous retroviruses (ERV) and a 2 kbp fragment of the 3R-end of the LINE.

The first transcriptome and genetic linkage map for Asian arowana.

Asian arowana or dragonfish (Scleropages formosus) is an important fish species due to its unusual breeding biology and high economic value in the ornamental fish markets. In the present study, we aimed to (i) create the first transcriptome by Roche 454 pyrosequencing of Asian arowana brain and gonad samples; (ii) identify differentially expressed genes between the two sexes and develop microsatellite (SSR) markers; and (iii) construct a first-generation SSR-based genetic linkage map. A total of over 1.3 million reads were obtained from the brain and gonad of adult Asian arowana individuals through pyrosequencing. These reads were assembled into 16,242 contigs that were further grouped into 13,639 isogroups. BLASTX annotation identified a total of 8316 unique proteins from this data set. Many genes with sexually dimorphic expression levels and some putatively involved in sex development were identified. A total of 316 EST-SSRs and 356 new genomic-SSRs were developed by screening through the current transcriptome data set and SSR-enriched genomic libraries. The first genetic linkage map of the species was constructed based on these markers. Linkage analysis allowed for mapping of 308 markers to 28 linkage groups (LGs), ranging in size from 14.9 to 160.6 cM. The potentially sex-associated gene sox9 was mapped to LG4 on the consensus linkage map. Pairwise putative conserved syntenies between the Asian arowana, zebrafish, and three-spined stickleback were also established. These resources will help the conservation of the species through better understanding of its phylogenetics, genomics and biology, and comparative genome analysis within the Osteoglossidae family.

Solid cancer incidence other than lung, liver and bone in Mayak workers: 1948-2004.

BACKGROUND: Cancer incidence in the Mayak Production Association (PA) cohort was analysed to investigate for the first time whether external gamma-ray and internal plutonium exposure are associated with raised incidence of solid cancers other than lung, liver and bone (other solid cancers). METHODS: The cohort includes 22,366 workers of both sexes who were first employed between 1948 and 1982. A total of 1447 cases of other solid cancers were registered in the follow-up period until 2004. The Poisson regression was used to estimate the excess relative risk (ERR) per unit of cumulative exposure to plutonium and external gamma-ray. RESULTS: A weak association was found between cumulative exposure to external gamma-ray and the incidence of other solid cancers (ERR/Gy=0.07; 95% confidence intervals (CIs): 0.01-0.15), but this association lost its significance after adjusting for internal plutonium exposure. There was no indication of any association with plutonium exposure for other solid cancers. Among 16 individual cancer sites, there was a statistically significant association with external exposure for lip cancer (ERR/Gy=1.74; 95% CI: 0.37; 6.71) and with plutonium exposure for pancreatic cancer (ERR/Gy=1.58; 95% CI; 0.17; 4.77). CONCLUSION: This study of Mayak workers does not provide evidence of an increased risk of other solid cancers. The observed increase in the risk of cancer of the lip and pancreas should be treated with caution because of the limited amount of relevant data and because the observations may be simply due to chance.

Radiation risk of malignant neoplasms in organs of main deposition for plutonium in the cohort of Mayak workers with regard to histological types.

This paper presents the results of analyses of the incidence of malignant neoplasms in lung, liver, and bone and associated connective tissues among Mayak nuclear workers exposed to both internally incorporated plutonium and to external gamma radiation. The study cohort included 22,373 individuals employed at the reactors and radiochemical and plutonium production facilities of the Mayak nuclear complex during 1948-1982 and followed up to the end of 2004. All analyses were carried out by Poisson regression, and the doses used were derived using a recently available update of organ doses, Mayak doses-2008. There was clear evidence for the linear association between internal plutonium dose and the risk of lung cancer. For males, there was evidence of a significant internal plutonium dose response for all histological types of lung cancer evaluated (adenocarcinoma, squamous-cell, and other epithelial); the estimated excess relative risk (ERR)/Gy for adenocarcinoma was the largest (ERR/Gy = 32.5; 95% CI: 16.3; 71.9), about 11-fold higher than that for squamous-cell lung cancer (ERR/Gy = 3.1; 95% CI: 0.3; 9.1). The relationship between liver cancer risk and plutonium exposure was best described by a linear-quadratic (LQ) function, but the LQ effect was diminished after restricting internal doses <2 Gy. Hepatocellular cancer was the most frequently observed type of liver cancer associated with internal plutonium exposure, and hemangiosarcomas were exclusively observed only at high internal plutonium doses (>4 Gy). For malignant neoplasms of bone and associated connective tissues, the trend was not statistically significant in relation to internal plutonium dose, but a statistically significantly higher risk (RR=13.7; 95% CI= 3.0; 58.5) was found among unmonitored female plutonium workers who were employed in the most hazardous plutonium production facility commissioned prior to 1950.

[Ocular and orbital blood flow in patients with proliferative vitreoretinopathy associated with rhegmatogenous retinal detachment].

Features of blood flow in ophthalmic artery, central retinal artery, posterior short ciliary arteries were studied using color Doppler mapping (CDM) in 40 patients (40 eyes) with primary rhegmatogenous retinal detachment (RRD) before and after surgery depending on postoperative course. The features of hemodynamic changes were revealed in patients with favorable postoperative course, progression of local and diffuse proliferative vitreoretinopathy (PVR). The risk factors of PVR after surgery were identified. CDM is valuable for predicting of postoperative course after RRD surgery.

[Satellite DNA as a phylogenetic marker: case study of three genera of the Murinae subfamily].

Satellite DNA (satDNA) represent tens percent of any of the vertebrate genome. Still, a complete set of sat-DNA fragments is not determined for either species. It is known that some genus with species-specific modifications possess a satDNA characteristic for the genus. So, satDNA was used as a phylogenetic marker in some cases when precise satDNA fragment was cloned. We used the probe of the whole pericentromeric region and 4 cloned satDNA fragments of Mus musculus in order to consider probes value for phylogenesis of 3 Murinae genera. Fluorescent in situ hybridization (FISH) revealed similar pattern on metaphase spreads inside genus Mus, though some difference was noted. None of the satDNA fragment gave signal in the centromeric region on chromosomes from genera Sylvaemus and Apodemus. These data are in agreement with those on satDNA fragments in the genome determined by dot-blot hybridization: M musculus satDNA fragments are absent in the genomes of both remote genera while they are present in the genomes of the genera Mus, though in different amounts. SatDNA of each genera should be cloned for the phylogenetic purposes.

[Mouse centromeric tandem repeats in silico and in situ].

The search for all sequences containing centromeric (CEN) minor satellite (MiSat) or pericentromeric (peri-CEN) mouse major satellite (MaSat) was conducted in the whole genome shotgun (WGS) database. The sequences were checked for the presence of the known dispersed repeats using the Censor software. The presence of tandem repeats was tested using Tandem Repeat Finder (TRF). Monotonous MiSat and MaSat arrays and MaSat to MiSat array transitions were detected. Moreover, two other types of contacts were revealed: (1) MiSat transition to fragments of retroelements LINE and IAP (ERV family, intracisternal A-type particles), mainly to ORF2 and 5'-LTR containing elements; (2) MaSat transition to two tandem repeats with monomers 21 bp and 31 bp in size. The presence of the MiSat/IAP transition could be checked experimentally. The common DNA motif among the IAP fragments close to MiSat was isolated. IAP-specific primers were constructed and the fragments obtained in PCR with LAP and MiSat primers compiled the plasmid vector library. Clone n51 with the maximum length of the possible insertion (approximately no. 800 bp) was selected from the library. FISH on extended chromatin fibers (fiberFISH) carried out on the n51 clone demonstrated that the main signal definitely belonged to CEN. However, the signals on the chromosome arms were also detected that could be due to the partial homology of n51 to the dispersed repeats. The duplicated fiberFISH with MiSat and n51 allowed to measure the distances between the fragments. The previously obtained MS3 sequence has some homology to IAP and CEN localization. Accordingly, the regular associations of MiSat with IAP retroelements were shown in silico and in situ. Together with the published data, the present findings suggest that retroelements or their fragments may be essential components of the normal centromere of higher eukaryotes.

[Modifications in major satellite methylation in the nucleus of a two-cell mouse embryo dependent on developmental conditions].

The study of the degree of DNA methylation in the nucleus, in particular of the major satellite in two-cell mouse embryos developing in the maternal organism, in standard cultural media M16 used for cultivation of mouse embryos and M2 media used for manipulations with embryos in the air was conducted. Two-cell embryos nucleus aged 44-46 hours after chorionic hormone injection were investigated. The revealed results are evidence for the dependence of the major satellite Ts methylation level of the developmental conditions of embryos. The methylation level of the nucleus DNA was shown to increase with a deterioration of environmental conditions. It was reported, that in the case of cultivation in M2 media not suitable for long cultivation, the DNA Ts methylation level, MaSat in particular, was higher compared to other embryo groups. Accordingly, not only a significant number of genes but also repeats of satellite DNA are involved in epigenetic regulation.

[Localization of satellite DNA and associated protein in respect to nucleolar precursor bodies in one- and two-cell mouse embryos].

Nucleolar precursor bodies (NPB) are characteristic structures in the nuclei of one- and two cell mouse embryos. The alignment of centromeric (CEN) and pericentromeric (periCEN) chromosome regions to the chromatin layer surrounding NPB is known. Mus musculus 4 satellite DNA (satDNA) types are known to be located in CEN region--mouse minor satellite (MiSat) and mouse satellite 3 (MS3); and periCEN region--mouse major satellite (MaSat) and mouse satellite (MS4). We determined the localization of 4 types of mouse satDNA CEN and periCEN regions and associated proteins: RNA-helicase p68, SMC3, Rad21 subunits of the cohesin complex and SYCP3 subunit of the synaptonemal complex (SC). Partially flattened nuclei of the one- and two-cell embryos and embryos treated with ocadaic acids (OA) were used. Different satDNA fragments revealed distinct domains at the surface of NPB: periCEN MaSat was always localized in NPB more internally covering almost entire surface of NPB while CEN MiSat, MS3 and periCEN MS4 showed more peripheral localization. All 4 satDNA did not cover the entire areas of the NPB, indicating the presence of other DNA sequence involved in its formation. RNA-helicase p68 and components of multiprotein cohesin and synaptonemal complexes are the necessary components of NPB. Our results support the opinion that NPB serve as a precursor of chromocenters.

[Heterochromatin and centromere structure paradox].

Centromere (CEN) is the structure responsible for the chromatid association, chromosome attachment to the spindle, and correct position in the plate. The only DNA found in the mammalian CEN belongs to the satellite DNA--high repeated tandem repeats. Mounting evidence indicates that both types of chromatin (CEN and peri-CEN) are required for proper centromere function. CEN, peri-CEN and peritelomeric regions remain white spots at the chromosome maps appeared after reading genomes of human, mouse, and rat. SatDNA is considered to be species-specific. Library hypothesis regards heterochromatin as the library of different satDNA one fragments of which became spread and fixed in species fixation. We have analyzed database Chromosome Unknown (ChrUn) and found several new classes of mouse tandem repeats. The features of these classes are similar with the ones from rat ChrUn, as well as their distributions according to GC-richness. We believe that similar fragments' structure, i. e. intermingling of fragments with different curvature rather than their primary sequence will help to solve the paradox, when CEN or peri-CEN fragments from different animals have nothing in common, but bind the same sets of proteins.

Cholesterol-induced stimulation of postinflammatory liver fibrosis.

We studied the effect of high-cholesterol diet and factors inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase on the development of liver fibrosis in C57Bl/6 mice with CCl4- or zymosan-induced hepatitis. Feeding a high-cholesterol diet led to a sharp increase in collagen content in the liver tissue of animals with CCl4-induced or zymosan-induced hepatitis. Atorvastatin and calcitriol produced less pronounced fibrogenic effects. Mevalonate partially prevented the development of cholesterol-induced fibrogenesis. High-cholesterol diet led to accumulation of oxysterols, cholesterol esters, and triglycerides and increased the expression of transforming growth factor-beta1 mRNA in liver tissue. Cholesterol-induced potentiation of the fibrogenic response is probably associated with transforming growth factor-beta1 induction due to accumulation of lipids and oxysterols in the liver.

Evidence for the existence of satellite DNA-containing connection between metaphase chromosomes.

Physical connections between mitotic chromosomes have been reported previously. It was assumed that the interchromosome connection was based on the DNA-protein thread. However, the data about DNA sequences and protein component in the thread is fragmentary. We demonstrated on the mouse cultured cell line and prematurely condensed chromosomes that: (a) all four mouse satellite DNA fragments (major and minor satellite, mouse satellite 3 (MS3) and mouse satellite 4 (MS4)) were involved in the thread formation; (b) MS4 was involved in the thread to the least extent among all the other fragments; (c) telomere was never a member of the thread; (d) the thread was synthesized at a late G(2) phase; (e) RNA helicase p68 and CENP-B were among the protein components of the interchromosome connection. It was shown by FACS analysis that in mouse and human cell lines: (1) the flow karyotype spectrums were never free from chromosome aggregates; (2) chromosome association did not depend on the chromosome length and each chromosome was free to associate with the other.

Telomere and TRF2/MTBP localization in respect to satellite DNA during the cell cycle of mouse cell line L929.

The mouse Mus musculus chromosomes are all acrocentric; each centromere (CEN) is adjacent to a telomere. The aim of the current work is to find out if at least half of the mouse telomeres (Tel) always follow satellite DNA sequences and if membrane telomere binding protein TRF2/MTBP is always in association with the Tel during the cell cycle. FISH, immunoFISH and confocal microscopy were used. During the cell cycle, Tel undergo extensive movement and rearrangement. Most Tel tend to aggregate into large conglomerates in G0/G1. Aggregates colocalize with major satellite (MaSat) and minor satellite (MiSat) to a lesser extent. Tel aggregates are embedded into the MaSat granules at G0/G1. A number of single Tel signals underline the nuclear envelope. In prometaphase, during the metaphase plate formation, half of the Tel, together with CEN, are arranged in a circle and half of the long arms form four clusters. Most of the Tel hybridization signals are colocalized with TRF2/MTBP throughout all stages of the cell cycle, although it is possible to find some telomeres that are not covered with the protein. A prominent shift of TRF2/MTBP signals in respect to the Tel signals is visible in the prophase. The biochemical features of TRF2/MTBP make it possible for the protein to be responsible for Tel clustering.

Telomere-binding TRF2/MTBP localization during mouse spermatogenesis and cell cycle of the mouse cells L929.

Observations of the organization and distribution of telomeres (Tel) in somatic tissues still remain controversial. The Tel topography revealed by modern microscopy shows them to be associated with the nuclear envelope (NE) in a wide variety of eukaryotic cells, although not at the Rabl orientation (peripheral position at one pole of the nucleus at prophase). We used two cell types that have different nuclear architectures. The cell line L929 shows lack of any rigid Tel architecture in the nucleus. In contrast, spermatozoa have a precise architecture established during spermiogenesis. We observed Tel and membrane Tel binding protein (MTBP/TRF2) position by immunoFISH in L929 cells and by immunofluorescence and immunogold electron microscopy, using antibodies against Membrane Tel Binding Protein (MTBP/TRF2), during different stages of spermiogenesis. At all stages of the L929 cell cycle, MTBP/TRF2 is co-localized with Tel. The only Tel order found in this cell type is similar to the Rabl-orientation, probably due to fast divisions. In the mouse pachytene spermatocytes, the membrane structures abut on the synaptonemal complex (SC) attachment sites contain MTBP/TRF2. In fully formed spermatozoa and during spermiogenesis, apart from the expected MTBP/TRF2 position at the nuclear periphery, MTBP/TRF2 unexpectedly localized at the acrosomal membrane that is adjacent to the nucleus. The difference in the MTBP/TRF2 distribution in the oocyte and spermatozoa leads to the suggestion that the MTBP/TRF2 location might reflect preparation for fertilization events. The Tel distribution is not static in cultured cells throughout the cell cycle or during spermatogenesis. When the Tel are attached to the NE, as during SC formation, MTBP/TRF2 is the member of the protein complex, which appears to be responsible for this attachment.

[The mammalian centromere organization]. / Organizatsiia tsentromera mlekopitaiushchikh.

Routine and recently obtained data on the pattern and functions of the mammalian centromeres and kinetochores have been reviewed. Several problems of kinetochore formation (centromere recognition, anaphase checkpoint) are specially discussed, in addition to the role played by centromere DNA in the interphase nucleus consideration.

Cancer mortality risk among workers at the Mayak nuclear complex.

At present, direct data on risk from protracted or fractionated radiation exposure at low dose rates have been limited largely to studies of populations exposed to low cumulative doses with resulting low statistical power. We evaluated the cancer risks associated with protracted exposure to external whole-body gamma radiation at high cumulative doses (the average dose is 0.8 Gy and the highest doses exceed 10 Gy) in Russian nuclear workers. Cancer deaths in a cohort of about 21,500 nuclear workers who began working at the Mayak complex between 1948 and 1972 were ascertained from death certificates and autopsy reports with follow-up through December 1997. Excess relative risk models were used to estimate solid cancer and leukemia risks associated with external gamma-radiation dose with adjustment for effects of plutonium exposures. Both solid cancer and leukemia death rates increased significantly with increasing gamma-ray dose (P < 0.001). Under a linear dose-response model, the excess relative risk for lung, liver and skeletal cancers as a group (668 deaths) adjusted for plutonium exposure is 0.30 per gray (P < 0.001) and 0.08 per gray (P < 0.001) for all other solid cancers (1062 deaths). The solid cancer dose-response functions appear to be nonlinear, with the excess risk estimates at doses of less than 3 Gy being about twice those predicted by the linear model. Plutonium exposure was associated with increased risks both for lung, liver and skeletal cancers (the sites of primary plutonium deposition) and for other solid cancers as a group. A significant dose response, with no indication of plutonium exposure effects, was found for leukemia. Excess risks for leukemia exhibited a significant dependence on the time since the dose was received. For doses received within 3 to 5 years of death the excess relative risk per gray was estimated to be about 7 (P < 0.001), but this risk was only 0.45 (P = 0.02) for doses received 5 to 45 years prior to death. External gamma-ray exposures significantly increased risks of both solid cancers and leukemia in this large cohort of men and women with occupational radiation exposures. Risks at doses of less than 1 Gy may be slightly lower than those seen for doses arising from acute exposures in the atomic bomb survivors. As dose estimates for the Mayak workers are improved, it should be possible to obtain more precise estimates of solid cancer and leukemia risks from protracted external radiation exposure in this cohort.

[Position of the satellite DNA relative to heterochromatin in the interphase nuclei in cultured cells]. / Polozhenie satellitnykh DNK po otnosheniiu k geterokhromatinu v interfaznykh iadrakh kletok v kul'ture.

It is considered that centromeric (CEN) regions play the leading role in the formation of chromocentres predominantly consisting of satellite DNA. Cloned mouse and human satellite DNA sequences from CEN and periGEN regions were used in order to trace their positions relative to chromocentres. Methods of fluorescent in situ hybridization (FISH) and immunoFISH with antibodies against CENP-B, known is a marker of prekinetochore, were employed. Peripheral position of the signals was observed at the chromocentres, but a combined signal of GEN and periGEN satellite DNAs never covered the whole brightly DAPI stained regions. A reasonable amount of the chromocentre body is not a satellite DNA. We suppose that the matrix-associated regions (MAR) of structural genes and, probably, the heavy methylated coding sequences of the genes, which are not expressed in the given cell type, are included in the chromocentres in addition to satellite DNAs.

[Signet ring cell cancer of the breast]. / Perstnevidnokletochnyi rak molochnykh zhelez.

A rare case of mammary signet-cell carcinoma which was not diagnosed at life is described. In a woman of 28, an enlargement of the right uterine appendages with pleural and lung metastasis were clinically noted and this was interpreted as ovarian carcinoma with pleural and lung metastasis. Histological examination of ovarian tumour performed during the necropsy allowed one to suggest the metastatic signet-cell carcinoma. No tumours were found macroscopically or histologically in the organs of the alimentary canal. Fibro-cystic mastopathy and no nodes were found macroscopically in the mammary glands. Lobular invasive carcinoma consisting of signet-cell elements containing mucus and carcinomatous lymphangoitis were found histologically. Metastasis to the left axillary, perigastral and pancreatic lymph nodes, lungs, carcinomatosis of pleura were revealed. It is suggested, due to its clinical course and histological structure, to distinguish this form of carcinoma as a separate variety of the lobular invasive carcinoma of the mammary gland.