The role of nonhomologous end joining and homologous recombination in the clonogenic bystander effects of mammalian cells after exposure to counted 10 MeV protons and 4.5 MeV α-particles of the PTB microbeam

We have studied the dependence of clonogenic bystander effects on defects in the pathways of DNA double-strand break (DSB) repair and on linear energy transfer (LET). The single-ion microbeam of the Physikalisch-Technische Bundesanstalt (PTB) was used to irradiate parental Chinese hamster ovary cells or derivatives deficient in nonhomologous end joining (NHEJ) or homologous recombination (HR) in the G1-phase of the cell cycle. Cell nuclei were targeted with 10 MeV protons (LET = 4.7 keV/μm) or 4.5 MeV α-particles (LET = 100 keV/μm). During exposure, the cells were confluent, allowing signal transfer through both gap junctions and diffusion. When all cell nuclei were targeted with 10 MeV protons, approximately exponential survival curves were obtained for all three cell lines. When only 10% of all cell nuclei were targeted, a significant bystander effect was observed for parental and HR-deficient cells, but not for NHEJ-deficient cells. For all three cell lines, the survival data after exposure of all cell nuclei to 4.5 MeV α-particles could be fitted by exponential curves. When only 10% of all cell nuclei were targeted, significant bystander effects were obtained for parental and HR-deficient cells, whereas for NHEJ-deficient cells a small, but significant, bystander effect was observed only at higher doses. The data suggest that bystander cell killing is a consequence of un- or misrejoined DSB which occur in bystander cells during the S-phase as a result of the processing of oxidative bistranded DNA lesions. The relative contributions of NHEJ and HR to the repairing of DSB in the late S/G2-phase may affect clonogenic bystander effects.     

Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

Dynamics of Newly Established Elk Populations

Abstract: The dynamics of newly established elk (Cervus elaphus) populations can provide insights about maximum sustainable rates of reproduction, survival, and increase. However, data used to estimate rates of increase typically have been limited to counts and rarely have included complementary estimates of vital rates. Complexities of population dynamics cannot be understood without considering population processes as well as population states. We estimated pregnancy rates, survival rates, age ratios, and sex ratios for reintroduced elk at Theodore Roosevelt National Park, North Dakota, USA; combined vital rates in a population projection model; and compared model projections with observed elk numbers and population ratios. Pregnancy rates in January (early in the second trimester of pregnancy) averaged 54.1% (SE = 5.4%) for subadults and 91.0% (SE = 1.7%) for adults, and 91.6% of pregnancies resulted in recruitment at 8 months. Annual survival rates of adult females averaged 0.96 (95% CI = 0.94-0.98) with hunting included and 0.99 (95% CI = 0.97-0.99) with hunting excluded from calculations. Our fitted model explained 99.8% of past variation in population estimates and represents a useful new tool for short-term management planning. Although we found no evidence of temporal variation in vital rates, variation in population composition caused substantial variation in projected rates of increase (Λ = 1.20-1.36). Restoring documented hunter harvests and removals of elk by the National Park Service led to a potential rate of Λ = 1.26. Greater rates of increase substantiated elsewhere were within the expected range of chance variation, given our model and estimates of vital rates. Rates of increase realized by small elk populations are too variable to support inferences about habitat quality or density dependence.     

Induction of DNA double-strand breaks by 1H and 4He lons in primary human skin fibroblasts in the LET range of 8 to 124 keV/microm.

Yields of DNA double-strand breaks were determined in primary human skin fibroblasts exposed to 1H and 4He ions at various linear energy transfers (LETs) and to 15 MeV electrons as the reference radiation. The values obtained for the relative biological effectiveness (RBE) were 2.03, 1.45 and 1.36 for 1H ions at LETs of 35, 23 and 7.9 keV/microm, respectively, and 1.2, 1.18, 1.38 and 1.31 for 4He ions at LETs of 124, 76, 35 and 27 keV/microm, respectively. The data were obtained using pulsed-field gel electrophoresis of DNA released from cells using the chromosomes of the yeast Saccharomyces cerevisiae as length markers and fitting the experimental mass distributions of fragmented DNA to those obtained by computer simulation of the random breakage of human chromosomes. The RBE values for induction of DSBs in mammalian cells cannot be fitted to a common RBE-LET relationship for electrons and 1H, 4He and light ions. Comparison of the RBEs for mammalian cells with the corresponding RBEs obtained for yeast cells shows similar RBEs of electrons for yeast and mammalian cells; however, for 4He and light ions in the LET range of 100 to 1000 keV/microm, the RBEs for yeast are significantly higher compared with mammalian cells. These characteristics of the RBE-LET relationships for yeast and mammalian cells are attributed to the fraction of small DNA fragments induced by particles when traversing the higher-order chromatin structures which are different to some extent in these two cell types.     

Chromosome aberrations induced in human lymphocytes by 3.45 MeV alpha particles analyzed by premature chromosome condensation.

Premature chromosome condensation (PCC) experiments using human lymphocytes with centromere staining have shown that after exposure to 3.45 MeV alpha-particle radiation, the full number of dicentric chromosomes appears when the cell fusion protocol is applied immediately after irradiation. In this case, the time available for repair and misrepair of DNA damage is only about 30 min. The number of dicentrics does not change with a further increase in the time available for chromatin rearrangement. This fast response confirms the expectation based on our previous experiments using PCC with 150 kV X rays in which the alpha component of the yield of dicentrics was found to appear when the cell fusion protocol was applied immediately after irradiation, whereas the beta component was delayed by several hours. The time constant for rejoining of the excess acentric chromosome fragments is found to be donor-specific and not to differ for alpha particles and X rays, but alpha-particle radiation leaves a larger fraction of the excess acentric fragments unrejoined. The RBEs of the 3.45 MeV alpha-particle radiation compared to 150 kV X rays, evaluated for the alpha component for the yield of dicentrics and for the yield of unrepaired acentric fragments, have almost equal values of about 4. This is consistent with data in the literature on chromosome aberrations observed in metaphase that show the equality of the RBE values for production of dicentrics and acentric fragments. Our experimental results concerning the fast kinetics of the alpha component of the yield of exchange-type chromosome aberrations are not consistent with Lea's pairwise lesion interaction model, and they support the proposed alternative mechanism of lesion-nonlesion interaction between chromatin regions carrying clustered DNA damage and intact chromatin regions.     

Production, purification, and characterization of a Mg2+-responsive porphobilinogen synthase from Pseudomonas aeruginosa.

During tetrapyrrole biosynthesis the metalloenzyme porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid to form the pyrrole porphobilinogen. Pseudomonas aeruginosa PBGS was synthesized in Escherichia coli, and the enzyme was purified as a fusion protein with glutathione S-transferase (GST). After removal of GST, a molecular mass of 280 000 +/- 10 000 with a Stokes radius of 57 A was determined for native PBGS, indicating a homooctameric structure of the enzyme. Mg2+ stabilized the oligomeric state but was not essential for octamer formation. Alteration of N-terminal amino acids changed the oligomeric state and reduced the activity of the enzyme, revealing the importance of this region for oligomerization and activity. EDTA treatment severely inhibited enzymatic activity which could be completely restored by the addition of Mg2+ or Mn2+. At concentrations in the micromolar range Co2+, Zn2+, and Ni2+ partially restored EDTA-inhibited enzymatic activity while higher concentrations of Zn2+ inhibited the enzyme. Pb2+, Cd2+, and Hg2+ did not restore activity. A stimulatory effect of monovalent ions was observed. A Km of 0.33 mM for ALA and a maximal specific activity of 60 micromol h-1 mg-1 at the pH optimum of 8.6 in the presence of Mg2+ and K+ were found. pH-dependent kinetic studies were combined with protein modifications to determine the structural basis of two observed pKa values of approximately 7.9 (pKa1) and 9.5 (pKa2). These are postulated respectively as ionization of an active site lysine residue and of free substrate during catalysis. Some PBGS inhibitors were characterized. Finally, we succeeded in obtaining well-ordered crystals of P. aeruginosa PBGS complexed with the substrate analogue levulinic acid.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.     

Pseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions.

Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions. These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.     

Influence of non-homology between recombining DNA sequences on double-strand break repair in Saccharomyces cerevisiae

In this paper we study the influence of non-homology between plasmid and chromosomal DNA on the efficiency of recombinational repair of plasmid double-strand breaks and gaps in yeast. For this purpose we used different combinations of plasmids and yeast strains carrying various deletions within the yeast LYS2 gene. A 400 by deletion in plasmid DNA had no effect on recombinational plasmid repair. However, a 400 by deletion in chromosomal DNA dramatically reduced the efficiency of this repair mechanism, but recombinational repair of plasmids linearized by a double-strand break with cohesive ends still remained the dominant repair process. We have also studied the competition between recombination and ligation in the repair of linearized plasmids. Our experimental evidence suggests that recombinational repair is attempted but aborted if only one recombinogenic end with homology to chromosomal DNA is present in plasmid DNA. This situation results in a decreased probability of non-recombinational (i.e. ligation) repair of linearized plasmid DNA.