DNA fork displacement rates in Bloom's syndrome fibroblasts

DNA fork displacement rates were measured in three lines of Bloom's syndrome cells and in a normal diploid fibroblast line. Fork displacement rates in Bloom's cells were approx. 55–65% of the rate in normal fibroblasts.     

Effects of heavy metals Cd2+, Pb2+ and Zn2+ on DNA damage of loach Misgurnus anguillicaudatus

The effects of heavy metals Cd2+, Pb2+ and Zn2+ at 0.05, 0.5 and 5.0 mg/L level and their interactions at 0.5 mg/L level on DNA damage in hepatopancreas of loach Misgurnus anguillicaudatus for 1-35 days exposure were examined by single cell gel electrophoresis (SCGE). For each test group, 20 loaches with similar body size (5.17-7.99g; 11.79-13.21 cm) were selected and kept in aquaria with dechlori-nated water at (22±1)℃ and fed a commercial diet every 48 h. According to the percentage of damaged DNA with tail and its TL/D (tail length to diameter of nucleus) value, the relationship between DNA damage degree and heavy metal dose and exposure time was determined. Results showed that the percentage of damaged DNA and the TL/D value were increased with the prolonged exposure time. The highest percentage (84.85%) of damaged DNA was shown in 5.0 mg/L Zn2+ group after 28 days exposure and the biggest TL/D value (2.50) in all treated groups after 35 days exposure. During the first treated week, the damnification of DNA was mainly recognized as the first level, after that time, the third damaged level was mostly observed and the percentage of damaged DNA was beyond 80%. The joint toxic effects among Cd2+, Pb2+ or Zn2+ revealed much complexity, but it generally displayed that the presence of Cd2+ could enhance the genotoxicity of Pb2+ or Zn2+. In conclusion, the results suggested that there was a significant time-and dose-depended relationship between the heavy metal and DNA damage in hepatopancreas of loach, and SCGE could represent a useful means to evaluate the genotoxicity of environmental contamination on aquatic organisms.     

Genetic analysis of the Hispano-Breton heavy horse

Hispano‐Breton (HB) is a horse breed with a recent mixed ancestry. It was developed in the 1930s by crossing local mares with Breton draught horses imported from France. Nowadays it is considered to be in a vulnerable situation due to census decline. To genetically characterize the breed and to set up the basis for a conservation programme, we have employed two types of molecular markers: a 347‐bp D‐loop mitochondrial DNA (mtDNA) fragment and 13 microsatellite loci. A representative sample of 53 HB individuals was analysed together with a sample of 40 Pura Raza Española horses for comparison. Both types of markers revealed a high level of genetic diversity in the HB breed, emphasizing the importance of its conservation. The construction of a phylogenetic network with mtDNA sequences including various Iberian breeds and European heavy horses provided an overall picture of the ubiquitous appearance of HB matrilines with respect to other breeds and revealed the singularity of certain HB maternal lineages. Despite the high allelic richness found in HB horses, microsatellite analysis evidenced a certain degree of inbreeding as a consequence of the type of management generally used for local breeds.     

Normal response of Fanconi's anemia cells to high concentrations of O2 as determined by alkaline elution

In this investigation, normal and Fanconi's anemia fibroblasts were exposed to high concentrations of oxygen and the effects of this treatment on DNA were analyzed by alkaline elution. No DNA single-strand breaks were detected in either cell type with up to 20 h incubation in high (50–95%) concentrations of O₂. No evidence of DNA damage by O₂ could be detected with an endonuclease preparation from Micrococcus luteus. Cells which have been treated with various DNA-damaging agents in the presence of the polymerase inhibitor cytosine arabinoside have been shown to accumulate DNA single-strand breaks during DNA excision repair. When cells were treated with the polymerase inhibitor in 50 or 95% O₂, a low level of DNA single-strand breaks accumulated in both cell types. However, no significant differences in the frequency of DNA single-strand breaks were detected between normal and Fanconi's anemia cells after exposure to high O₂.     

Serial deletion constructs of human cardiac myosin heavy chain genes generated by PCR amplification

Summary Serial deletion constructs derived from the 5-flanking regions of the human cardiac - and -myosin heavy chain genes were generated by polymerase chain reaction (PCR) amplifications. Generation of different length chimeric constructs were based on the complete sequence of the human cardiac myosin heavy chain genes [1, 2]. The primers were synthesized with HindIII and BamH₁ sites and were linked to any designed nucleotide of the 5 flanking sequence of the myosin heavy chain gene(s). Following the PCR amplification and the site-directed mutagenesis, the PCR products were verified by DNA sequencing and subsequently ligated to the chloramphenical acetyltransferase (pBLCAT₃) reporter gene which was restricted with Hind III and BamH₁. Neonatal rat cardiocytes were used to assay the promotor activity (i.e. CAT activity) of different lengths of the chimeric constructs of the gene.     

Effects of heavy metals Cd2+, Pb2+ and Zn2+ on DNA damage of loach Misgurnus anguillicaudatus

The effects of heavy metals Cd²⁺, Pb²⁺ and Zn²⁺ at 0.05, 0.5 and 5.0 mg/L level and their interactions at 0.5 mg/L level on DNA damage in hepatopancreas of loach Misgurnus anguillicaudatus for 1–35 days exposure were examined by single cell gel electrophoresis (SCGE). For each test group, 20 loaches with similar body size (5.17–7.99 g; 11.79–13.21 cm) were selected and kept in aquaria with dechlorinated water at (22±1)°C and fed a commercial diet every 48 h. According to the percentage of damaged DNA with tail and its TL/D (tail length to diameter of nucleus) value, the relationship between DNA damage degree and heavy metal dose and exposure time was determined. Results showed that the percentage of damaged DNA and the TL/D value were increased with the prolonged exposure time. The highest percentage (84.85%) of damaged DNA was shown in 5.0 mg/L Zn²⁺ group after 28 days exposure and the biggest TL/D value (2.50) in all treated groups after 35 days exposure. During the first treated week, the damnification of DNA was mainly recognized as the first level, after that time, the third damaged level was mostly observed and the percentage of damaged DNA was beyond 80%. The joint toxic effects among Cd²⁺, Pb²⁺ or Zn²⁺ revealed much complexity, but it generally displayed that the presence of Cd²⁺ could enhance the genotoxicity of Pb²⁺ or Zn²⁺. In conclusion, the results suggested that there was a significant time-and dose-depended relationship between the heavy metal and DNA damage in hepatopancreas of loach, and SCGE could represent a useful means to evaluate the genotoxicity of environmental contamination on aquatic organisms. __________ Translated from Acta Hydrobiologica Sinica, 2006, 30(4): 399–403 [译自: 水生生物学报]     

Functional role of the Werner syndrome RecQ helicase in human fibroblasts

Werner syndrome is an autosomal recessive human genetic instability and cancer predisposition syndrome that also has features of premature aging. We focused on two questions related to Werner syndrome protein (WRN) function in human fibroblasts: Do WRN‐deficient fibroblasts have a consistent cellular phenotype? What role does WRN play in the recovery from replication arrest? We identified consistent cell proliferation and DNA damage sensitivity defects in both primary and SV40‐transformed fibroblasts from different Werner syndrome patients, and showed that these defects could be revealed by acute depletion of WRN protein. Mechanistic analysis of the role of WRN in recovery from replication arrest indicated that WRN acts to repair damage resulting from replication arrest, rather than to prevent the disruption or breakage of stalled replication forks. These results identify readily quantified cell phenotypes that result from WRN loss in human fibroblasts; delineate the impact of cell transformation on the expression of these phenotypes; and define a mechanistic role for WRN in the recovery from replication arrest.     

RECQL4-deficient cells are hypersensitive to oxidative stress/damage: Insights for osteosarcoma prevalence and heterogeneity in Rothmund-Thomson syndrome

Rothmund-Thomson syndrome (RTS) is a heterogeneous disease, associated with increased prevalence of osteosarcoma in very young patients with a mutated RECQL4 gene. In this study, we tested the ability of RECQL4 deficient fibroblasts, derived from a RTS patient to recover from hydrogen peroxide (H(2)O(2))-induced oxidative stress/damage. Immunoperoxidase staining for 8-oxo-deoxyguanosine (8-oxo-dG) formation in RTS and normal human fibroblasts were compared to assess DNA damage. We determined DNA synthesis, cell growth, cell cycle distribution, and viability in RTS and normal human fibroblasts before and after H(2)O(2) treatment. H(2)O(2) induces 8-oxo-dG formation in both RTS and normal fibroblasts. In normal human fibroblasts, RECQL4 was predominantly localized to cytoplasm; nuclear translocation and foci formation occurred in response to oxidant stimulation. After recovery from oxidant exposure, viable RTS fibroblasts showed irreversible growth arrest compared to normal fibroblasts. DNA synthesis decreased significantly in treated RTS cells, with concomitant reduction of cells in the S-phase. These results suggest that enhanced oxidant sensitivity in RECQL4 deficient fibroblasts derived from RTS patients could be attributed to abnormal DNA metabolism and proliferation failure. The ramifications of these findings on osteosarcoma prevalence and heterogeneity in RTS are discussed.     

重金属污染影响植食性昆虫的研究进展

重金属污染是世界各国面临的最为棘手的问题之一,对生态系统和食品安全构成了严重威胁。作为生态系统中食物链和食物网的重要环节,植食性昆虫是环境中重金属迁移、积累的重要媒介,其因重金属污染而受到的影响引起了大家的关注。本文综述了从2007至2018年重金属污染对植食性昆虫影响的研究进展。昆虫受重金属胁迫的研究途径有人工饲料添加、野外田间暴露、“土壤-植物-昆虫”食物链传递以及体外注射等。积累在植食性昆虫体内的过量重金属可导致其存活率、繁殖力和种群增长率降低,生长发育迟缓。重金属污染对植食性昆虫的生理生化毒性包括细胞超微结构破坏和DNA损伤,体内能量物质含量降低,酶活性、基因表达改变等。植食性昆虫会通过重金属硫蛋白、解毒酶活性的诱导等机制抵御重金属的毒害,从而对低浓度、长期重金属暴露产生生态适应性,甚至提高对其他逆境(如农药等)的耐受性。     

Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB)s often require end processing prior to joining during their repair by non-homologous end joining (NHEJ). Although the yeast proteins, Pol4, a Pol X family DNA polymerase, and Rad27, a nuclease, participate in the end processing reactions of NHEJ, the mechanisms underlying the recruitment of these factors to DSBs are not known. Here we demonstrate that Nej1, a NHEJ factor that interacts with and modulates the activity of the NHEJ DNA ligase complex (Dnl4/Lif1), physically and functionally interacts with both Pol4 and Rad27. Notably, Nej1 and Dnl4/Lif1, which also interacts with both Pol4 and Rad27, independently recruit the end processing factors to in vivo DSBs via mechanisms that are additive rather than redundant. As was observed with Dnl4/Lif1, the activities of both Pol4 and Rad27 were enhanced by the interaction with Nej1. Furthermore, Nej1 increased the joining of incompatible DNA ends in reconstituted reactions containing Pol4, Rad27 and Dnl4/Lif1, indicating that the stimulatory activities of Nej1 and Dnl4/Lif1 are also additive. Together our results reveal novel roles for Nej1 in the recruitment of Pol4 and Rad27 to in vivo DSBs and the coordination of the end processing and ligation reactions of NHEJ.     

Biological cost of tolerance to heavy metals in the mosquito Anopheles gambiae

The global rate of heavy metal pollution is rapidly increasing in various habitats. Anopheles malaria vector species (Diptera: Culicidae) appear to tolerate many aquatic habitats with metal pollutants, despite their normal proclivity for ‘clean’ water (i.e. low levels of organic matter). Investigations were conducted to establish whether there are biological costs for tolerance to heavy metals in Anopheles gambiae Giles sensu stricto and to assess the potential impact of heavy metal pollution on mosquito ecology. Anopheles gambiae s.s. were selected for cadmium, copper or lead tolerance through chronic exposure of immature stages to solutions of the metals for three successive generations. Biological costs were assessed in the fourth generation by horizontal life table analysis. Tolerance in larvae to cadmium (as cadmium chloride, CdCl₂), copper [as copper II nitrate hydrate, Cu(NO₃)₂ 2.5 H₂O] and lead [as lead II nitrate, Pb(NO₃)₂], monitored by changes in LC₅₀ concentrations of the metals, changed from 6.07 µg/L, 12.42 µg/L and 493.32 µg/L to 4.45 µg/L, 25.02 µg/L and 516.69 µg/L, respectively, after three generations of exposure. The metal‐selected strains had a significantly lower magnitude of egg viability, larval and pupal survivorship, adult emergence, fecundity and net reproductive rate than the control strain. The population doubling times were significantly longer and the instantaneous birth rates lower in most metal‐selected strains relative to the control strain. Our results suggest that although An. gambiae s.s. displays the potential to develop tolerance to heavy metals, particularly copper, this may occur at a significant biological cost, which can adversely affect its ecological fitness.     

Blood screening for heavy metals and organic pollutants in cancer patients exposed to toxic waste in southern Italy: A pilot study

In Italy, in the eastern area of the Campania region, the illegal dumping and burning of waste have been documented, which could potentially affect the local population's health. In particular, toxic waste exposure has been suggested to associate with increased cancer development/mortality in these areas, although a causal link has not yet been established. In this pilot study, we evaluated blood levels of toxic heavy metals and persistent organic pollutants (POPs) in 95 patients with different cancer types residing in this area and in 27 healthy individuals. While we did not find any significant correlation between the blood levels of POPs and the provenance of the patients, we did observe high blood concentrations of heavy metals in some municipalities, including Giugliano, where many illegal waste disposal sites have previously been documented. Our results showed that patients with different cancer types from Giugliano had higher blood levels of heavy metals than healthy controls. Despite the obvious limitations of this exploratory study, our preliminary observations encourage further research assessing the possible association between exposure to hazardous waste, increased blood metals, and increased risk of cancer.     

Immunoglobulin heavy chain constant and heavy chain variable region genes in phylogenetically diverse species of bony fish

Summary Genomic DNA from 18 phylogenetically diverse species of bony fish was hybridized with probes specific for the channel catfish immunoglobulin heavy chain constant (CH) gene, as well as with immunoglobulin heavy chain variable (VH) probes specific for five channel catfish VH gene families. The results showed that CH probes strongly hybridized only to genomic fragments from other catfish species. In contrast, restricted DNA from most other species hybridized with at least two channel catfish VH probes. In those species whose DNA hybridized with multiple VH probes, the restriction pattern of hybridizing fragments was probe-dependent. These studies suggest that (1) the CH gene defined in channel catfish appears to share similarity only with CH genes in other catfish species, (2) families of VH genes appear to have diverged in early phylogenetic lineages of teleosts, and (3) VH genes similar to those defined in catfish appear to be widely represented in phylogenetically diverse species of teleosts.     

Genetic diversity of heavy metal-tolerant populations in Silene paradoxa L. (Caryophyllaceae): a chloroplast microsatellite analysis

Eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in uncontaminated soil in central Italy were studied. Genetic diversity was estimated using five polymorphic chloroplast microsatellite loci (cpSSR), identifying 27 different chloroplast haplotypes. The effective number of alleles, the haplotypic diversity and a stepwise mutational model-based parameter (DSH2) were computed. The effective number of alleles observed within populations from copper mine deposits was 20% that of the serpentine neighbouring populations, suggesting the occurrence of a founder effect. Moreover, 13 of the 27 different haplotypes scored were exclusive to only one population, indicating genetic isolation for all tolerant populations. Even the copper-tolerant populations appeared to have evolved independently. Finally, analysis of molecular variance (AMOVA) of the cpSSR markers gave statistical significance to the grouping of populations according to their geographical location. This study demonstrates that cpSSR markers could be a useful complementary tool to isoenzymes or random amplified polymorphic DNA markers for elucidating the pattern of genetic differentiation in heavy metal-tolerant populations.     

Elevation of nucleotide pyrophosphatase activity in skin fibroblasts from patients with Lowe's syndrome

Undersulfation observed in the glycosaminoglycans synthesized by cultured skin fibroblasts from a Lowe's syndrome patient[Fukui, S. etal. (1981) J. Biol. Chem. 256, 10313–10318] was found to be caused by elevated degradation of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). The enzyme involved in this degradation was then identified as an enzyme of nucleotide pyrophosphatase (EC 3.6.1.9) nature, cleaving the phosphosulfate linkage. The specific activities were 8 – 24 (mU/mg protein) in patients' fibroblasts, in contrast to 3 in normal and 5 – 14 in heterozygote cells. A possibility is discussed that the elevation of nucleotide pyrophosphatase activity is the primary genetic defect in Lowe's syndrome.     

DNA repair in Bloom's syndrome fibroblasts after UV irradiation or treatment with mitomycin C

Sensitivities to UV and mitomycin C (MC) of fibroblasts obtained from 3 Japanese patients with Bloom's syndrome (BS) were studied. One BS strain was more sensitive to UV than normal cells only in colony-forming ability. Other responses to UV, such as unscheduled DNA synthesis, host-cell reactivation and removal of UV-endonuclease-susceptible sites, were normal in all 3 strains. These BS strains were more sensitive to MC than were normal cells. However, the amounts of unscheduled DNA synthesis after treatment with MC in BS cells did not differ from those in normal cells.     

水环境中重金属铜对异育银鲫肠道微生物的影响

【目的】了解水环境中重金属铜对异育银鲫肠道微生物组成及多样性的影响。【方法】采用试剂盒提取异育银鲫肠道总DNA,然后对总DNA进行16S r RNA进行扩增,构建异育银鲫肠道微生物16S r RNA基因克隆文库,最后进行数据分析。【结果】厚壁菌门、变形菌门和拟杆菌门为异育银鲫肠道中主要的细菌类群,在不同浓度的重金属铜胁迫处理后,厚壁菌门的含量明显降低。稀释性曲线、Venn图和多样性指数分析结果表明,重金属铜胁迫处理后异育银鲫肠道微生物多样性明显降低。【结论】重金属铜会使异育银鲫肠道微生物组成及多样性降低。此结果为研究重金属污染对异育银鲫健康状况的影响及异育银鲫养殖过程中病害的诊断奠定基础。     

Pesticides and heavy metals in Danish streambed sediment

The role of streambed sediment as a sink for pesticides and heavy metals was investigated in 30 Danish lowland streams. The investigated streams drain catchments varying in hydrology, topography, soil type and land use. The <250 m newly accumulated fraction of the uppermost 1–2 cm layer of streambed sediment was analysed for 19 old and modern pesticides and 9 heavy metals. DDE was present in the sediment of all the streams. Of the herbicides, fungicides and insecticides currently in use, the most frequently detected was diuron (50.0%), fenpropimorph (66.7%) and lambda-cyhalothrin (6.7%), respectively. The pesticides detected in the highest concentration were fenpropimorph (1700 ng g^–1), propiconazole (130 ng g^–1) and isoproturon (110 ng g^–1). The heavy metals are listed in order of increasing median concentration: Cd (0.80 g g^–1), Co (9.1 g g^–1), As (12.0 g g^–1), Ni (19.0 g g^–1), Cr (19.2 g g^–1), Pb (19.7 g g^–1), Cu (20.1 g g^–1), V (28.5 g g^–1), Zn (103 g g^–1). The average number of pesticides detected in the 27 streams draining predominantly agricultural catchments was (3.7±2.0) being higher (p=0.077) than in the three streams draining non-agricultural catchments (1.7±0.6). Pesticides were significantly related to catchment size, soil type and hydrological regime. Several heavy metals (Cr, Cu, Pb, V and Zn) were related to urban activity and soil type.     

Repair of bleomycin-damaged DNA by human fibroblasts

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.     

5-bromodeoxyuridine labeling of monomeric and catenated circular mitochondrial DNA in HeLa cells

Bromouracil labeling of the mitochondrial DNA in exponentially growing HeLa cells produces two hybrid mitochondrial DNA species, with density shifts of 41.9 and 54.0 mg/ml relative to unlabeled mitochondrial DNA, as well as heavy mitochondrial DNA, with a shift of 95.3 mg/ml. The two hybrid species result from the difference in thymine composition of the complementary strands of mitochondrial DNA. In addition, mitochondrial DNA with a density intermediate between the hybrid and unlabeled species was found. This quarter heavy mitochondrial DNA represents 25% (w/w) of the total DNA after eight hours of labeling, and forms two peaks with shifts of 20.6 and 27.0 mg/ml relative to unlabeled mitochondrial DNA. 70% (w/w) of the quarter heavy mitochondrial DNA is in catenated forms, while 30% (w/w) is monomeric. Degradation of the catenanes by shearing of purified quarter heavy mitochondrial DNA results in the appearance of hybrid and unlabeled mitochondrial DNA bands, demonstrating that the quarter heavy catenanes contain both hybrid and unlabeled submolecules. The implications of the structure of the quarter heavy catenanes on the mechanism of formation of catenanes are discussed.