Apoptosis initiation and angiogenesis inhibition: melanoma targets for nanosecond pulsed electric fields

Many effective anti-cancer strategies target apoptosis and angiogenesis mechanisms. Applications of non-ionizing, nanosecond pulsed electric fields (nsPEFs) induce apoptosis in vitro and eliminate cancer in vivo; however in vivo mechanisms require closer analysis. These studies investigate nsPEF-induced apoptosis and anti-angiogenesis examined by fluorescent microscopy, immunoblots, and morphology. Six hours after treatment with one hundred 300 ns pulses at 40 kV/cm, cells transiently expressed active caspases indicating that caspase-mediated mechanisms. Three hours after treatment transient peaks in Histone 2AX phosphorylation coincided with terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells and pyknotic nuclei, suggesting caspase-independent mechanisms on nuclei/DNA. Large DNA fragments, but not 180 bp fragmentation ladders, were observed, suggesting incomplete apoptosis. Nevertheless, tumor weight and volume decreased and tumors disappeared. One week after treatment, vessel numbers, vascular endothelial growth factor (VEGF), platelet derived endothelial cell growth factor (PD-ECGF), CD31, CD35 and CD105 were decreased, indicating anti-angiogenesis. The nsPEFs activate multiple melanoma therapeutic targets, which is consistent with successes of nsPEF applications for tumor treatment in vivo as a new cancer therapeutic modality.     

Pol32 is required for Pol zeta-dependent translesion synthesis and prevents double-strand breaks at the replication fork

POL32 encodes a non-essential subunit of Polδ and plays a role in Polδ processivity and DNA repair. In order to understand how Pol32 is involved in these processes, we performed extensive genetic analysis and demonstrated that POL32 is required for Polζ-mediated translesion synthesis, but not for Polη-mediated activity. Unlike Polζ, inactivation of Pol32 does not result in decreased spontaneous mutagenesis, nor does it limit genome instability in the absence of the error-free postreplication repair pathway. In contrast, inactivation of Pol32 results in an increased rate of replication slippage and recombination. A genome-wide synthetic lethal screen revealed that in the absence of Pol32, homologous recombination repair and cell cycle checkpoints play crucial roles in maintaining cell survival and growth. These results are consistent with a model in which Pol32 functions as a coupling factor to facilitate a switch from replication to translesion synthesis when Polδ encounters replication-blocking lesions. When Pol32 is absent, the S-phase checkpoint complex Mrc1–Tof1 becomes crucial to stabilize the stalled replication fork and recruit Top3 and Sgs1. Lack of any of the above activities will cause double strand breaks at or near the replication fork that require recombination as well as Rad9 for cell survival.     

The heterodimeric primase from the euryarchaeon Pyrococcus abyssi: a multifunctional enzyme for initiation and repair?

We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase α–primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg²⁺ and Mn²⁺. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair.     

Nucleotide Content Gradients in Maternally and Paternally Inherited Mitochondrial Genomes of the Mussel <Emphasis Type="Italic">Mytilus</Emphasis>

Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerate sites is well correlated with the site’s time of exposure to the single-strand state, as predicted from the asymmetrical model of mtDNA replication. Here we examine whether the same explanation may hold for the regional variation in nucleotide content in the maternal and paternal mtDNAs of the mussel Mytilus galloprovincialis. The origin of replication of the heavy strand (O_H) of these genomes has been previously established. A systematic search of the two genomes for sequences that are likely to act as the origin of replication of the light strand (O_L) suggested that the most probable site lies within the ND3 gene. By adopting this O_L position we calculated times of exposure for 0_FD (nondegenerate), 2_FD (twofold degenerate), and 4_FD (fourfold degenerate) sites of the protein-coding part of the genome and for the rRNA, tRNA and noncoding parts. The presence of thymine and absence of guanine at 4_FD sites was highly correlated with the presumed time of exposure. Such an effect was not found for the 2_FD sites, the rRNA, the tRNA, or the noncoding parts. There was a trend for a small increase in cytosine at 0_FD sites with exposure time, which is explicable as the result of biased usage of 4_FD codons. The same analysis was applied to a recently sequenced mitochondrial genome of Mytilus trossulus and produced similar results. These results are consistent with the asymmetrical model of replication and suggest that guanine oxidation due to single-strand exposure is the main cause of regional variation of nucleotide content in Mytilus mitochondrial genomes. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. David Pollock]     

MRN复合物的结构及功能研究进展

MRN复合物包括MRE11、NBS1、RAD50,此复合物中的MRE11或NBS1缺失或突变会导致人的共济失调一毛细血管扩张样疾病、Nijmegen断裂综合征。MRN复合物在DNA双链损伤修复、同源重组、非同源重组、端粒长度维持、细胞检验点激活、保证DNA复制的顺利进行,以及维持基因组的稳定性等方面都起到了重要的作用。从以上几个方面简要综述MRN复合物的研究进展。     

DNA asymmetric strand bias affects the amino acid composition of mitochondrial proteins.

Variations in GC content between genomes have been extensively documented. Genomes with comparable GC contents can, however, still differ in the apportionment of the G and C nucleotides between the two DNA strands. This asymmetric strand bias is known as GC skew. Here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. We compared orthologous genes between animal mitochondrial genomes that show large differences in GC and AT skews. Specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative GC skew and a positive AT skew, to those of flatworms, which show the opposite skews for both GC and AT base pairs. We found that the mammalian proteins are highly enriched in amino acids encoded by CA-rich codons (as predicted by their negative GC and positive AT skews), whereas their flatworm orthologs were enriched in amino acids encoded by GT-rich codons (also as predicted from their skews). We found that these differences in mitochondrial strand asymmetry (measured as GC and AT skews) can have very large, predictable effects on the composition of the encoded proteins.     

Beta-strand recombination in tricalbin evolution and the origin of synaptotagmin-like C2 domains

Two protein families involved in membrane traffic, tricalbins and synaptotagmins, contain several copies of C2 domains and are related based on their sequence and domain architecture. Paradoxically, tricalbin and synaptotagmin C2 domains belong to different structural types with apparent circular permutation of terminal beta-strands. To understand whether a topological switch took place, we analyzed tricalbin and synaptotagmin-like C2 domains using two-dimensional structural analysis. We found that yeast tricalbins contain five to six C2 domains. One of these C2 domains possesses many features of synaptotagmin-like C2 domains and also carries a conserved C-terminal strand that is similar to its structural equivalent in synaptotagmin-like C2 domains, suggesting a structural permutation event. Indeed, among higher eukaryotes, animal tricalbins have evolved a C2 domain with synaptotagmin-like topology indicating that the structural conversion has taken place. Investigation of plant synaptotagmins, however, proves that they are direct tricalbin orthologs. Our analysis shows that beta-strand recombination is a possible evolutionary mechanism to generate new structural topologies with altered functional properties.     

Biological protection of hardwood logs destined for panel manufacturing using <Emphasis Type="Italic">Gliocladium roseum</Emphasis> against biodegradation

The objective of the present study was to develop a biological technology that would protect logs destined for oriented strand board (OSB) manufacturing from biodegradation. Aspen, red maple, and yellow birch trees were felled in one summer and the logs either debarked or not debarked, and either treated or not treated with a biological product of Gliocladium roseum. The logs were piled in different treatment groups and stored in a yard for 5 months and 1 year before evaluation. The results showed that all untreated logs, with or without bark, were seriously degraded by moulds, stain and decay fungi after a summer storage period of 5 months. The logs with bark were more degraded than the debarked logs, and the log ends were more degraded than the middle sections. After 5 months, 55–83% of the surface area of the wood discs was degraded in untreated logs. The biological treatment was effective, and only 4–16% of the surface area of the wood discs in treated logs was infected by various fungi. Strands cut from untreated logs consisted of 50–75% grey- or blue-stained strands, whereas those cut from biologically treated logs consisted of 10–25% such strands. Panels made using biologically treated logs had a lower thickness swelling and water absorption values compared to panels made using freshly cut logs and untreated stored logs. The other physical and mechanical properties of the various panels made in this test were comparable. In terms of mould resistance, all panels made from fungal-treated logs had a better mould resistance than those made from freshly cut and untreated logs.     

Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer

Breast cancer is the most common malignancy in women. Radiotherapy is frequently used in patients with breast cancer, but some patients may be more susceptible to ionizing radiation, and increased exposure to radiation sources may be associated to radiation adverse events. This susceptibility may be related to deficiencies in DNA repair mechanisms that are activated after cell-radiation, which causes DNA damage, particularly DNA double strand breaks. Some of these genetic susceptibilities in DNA-repair mechanisms are implicated in the etiology of hereditary breast/ovarian cancer (pathologic mutations in the BRCA 1 and 2 genes), but other less penetrant variants in genes involved in sporadic breast cancer have been described. These same genetic susceptibilities may be involved in negative radiotherapeutic outcomes. For these reasons, it is necessary to implement methods for detecting patients who are susceptible to radiotherapy-related adverse events. This review discusses mechanisms of DNA damage and repair, genes related to these functions, and the diagnosis methods designed and under research for detection of breast cancer patients with increased radiosensitivity.