Frequency in hypertensives of alleles for a RFLP associated with the renin gene

The genetic basis of primary hypertension is not known. Renin is important in blood pressure and volume control and a HindIII restriction fragment length polymorphism (RFLP) is present within the human renin gene locus. To examine whether there is a relationship between this RFLP and primary hypertension, DNA and renin analyses were performed on leukocytes and plasma from hypertensive and normotensive individuals. In hypertensives the frequencies of alleles for the HindIII RFLP were found to be 0.55 and 0.45, compared with 0.60 and 0.40 in the total population of 231 subjects examined, a difference that was not statistically significant. There also appeared to be no significant difference in renin activity in plasma for hypertensive patients of each genotype, nor in their pre- or post-treatment blood pressures. We thus conclude that, within the limits of the present study, the suspected genetic abnormalities associated with primary hypertension in man do not appear to be related to a HindIII RFLP in the renin gene.

Well-trained, healthy triathletes experience no adverse health risks regarding oxidative stress and DNA damage by participating in an ultra-endurance event

Also physical exercise in general is accepted to be protective, acute and strenuous exercise has been shown to induce oxidative stress. Enhanced formation of free radicals leads to oxidation of macromolecules and to DNA damage. On the other hand ultra-endurance events which require strenuous exercise are very popular and the number of participants is continuously increasing worldwide. Since only few data exists on Ironman triathletes, who are prototypes of ultra-endurance athletes, this study was aimed at assessing the risk of oxidative stress and DNA damage after finishing a triathlon and to predict a possible health risk. Blood samples of 42 male athletes were taken 2 days before, within 20 min after the race, 1, 5 and 19 days post-race. Oxidative stress marker increased only moderately after the race and returned to baseline after 5 days. Marker of DNA damage measured by the SCGE assay with and without restriction enzymes as well as by the sister chromatid exchange assay did either show no change or deceased within the first day after the race. Due to intake during the race and the release by the cells plasma concentrations of vitamin C and α-tocopherol increased after the event and returned to baseline 1 day after. This study indicates that despite a temporary increase in some oxidative stress markers, there is no persistent oxidative stress and no DNA damage in response to an Ironman triathlon in trained athletes, mainly due to an appropriate antioxidant intake and general protective alterations in the antioxidant defence system.

Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje

Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns—they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.

Meta-analyses identify 13 loci associated with age at menopause and highlight DNA repair and immune pathways

To newly identify loci for age at natural menopause, we carried out a meta-analysis of 22 genome-wide association studies (GWAS) in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 loci newly associated with age at natural menopause (at P < 5 x 10(-8)). Candidate genes located at these newly associated loci include genes implicated in DNA repair (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG and PRIM1) and immune function (IL11, NLRP11 and PRRC2A (also known as BAT2)). Gene-set enrichment pathway analyses using the full GWAS data set identified exoDNase, NF-kappaB signaling and mitochondrial dysfunction as biological processes related to timing of menopause.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease. © 2011 Elsevier Ltd. All rights reserved.

Reactive Oxygen Species and Reactive Nitrogen Species in Epigenetic Modifications

For the normal homeostasis of a cell, there must be a balance between radical oxygen species/radical nitrogen species (ROS/RNS) production and the neutralization of these species by antioxidant scavenging. In times of stress, this balance is not maintained, and the result is oxidative stress. This stress can affect many pathways in the body and result in pathological consequences. Recent evidence suggests that ROS/RNS can affect the epigenetic regulation of genes by affecting the function of histone and DNA modifying enzymes, thus affecting phenotypic changes within the cellular environment. In the following chapter, we provide a broad overview of how oxidative stress induced by ROS/RNS can affect epigenetics, and using lung disease as our model we link the connection between these processes.

Differentiation of Rice Tungro Bacilliform Virus Strains by Restriction Analysis and DNA Hybridization

Rice tungro bacilliform virus (RTBV) is one of the two viruses that cause tungro disease. Four RTBV strains maintained in the greenhouse for 4 years, G1, G2, Ic, and L, were differentiated by restriction fragment length polymorphism (RFLP) analysis of the native viral DNA. Although strains G1 and Ic had identical restriction patterns when cleaved with Pst1, BamHI, EcoRI, and EcoRV, they can be differentiated from strains G2 and L by EcoRI and EcoRV digestion. These same endonucleases also differentiate strain G2 from strain L. When total DNA extracts from infected plants were used instead of viral DNA, and digested with EcoRV, identical restriction patterns for each strain (G2 and L) were obtained from roots, leaves, and leaf sheaths of infected plants. The restriction patterns were consistent from plant to plant, in different varieties, and at different times after inoculation. This technique can be used to differentiate RTBV strains and determine the variability of a large number of field samples.

A genetic analysis of serotonergic biosynthetic and metabolic enzymes in migraine using a DNA pooling approach

Migraine is a common debilitating primary headache disorder with significant mental, physical and social health implications. The brain neurotransmitter 5-hydroxytryptamine (5-HT; serotonin) is involved in nociceptive pathways and has been implicated in the pathophysiology of migraine. With few genetic studies investigating biosynthetic and metabolic enzymes governing the rate of 5-HT activity and their relationship to migraine, it was the objective of this study to assess genetic variants within the human tryptophan hydroxylase (TPH), amino acid decarboxylase (AADC) and monoamine oxidase A (MAOA) genes in migraine susceptibility. This objective was undertaken using a high-throughput DNA pooling experimental design, which proved to be a very accurate, sensitive and specific method of estimating allele frequencies for single nucleotide polymorphism, insertion deletion and variable number tandem repeat loci. Application of DNA pooling to a wide array of genetic loci provides greater scope in the assessment of population-based genetic association study designs. Despite the application of this high-throughput genotyping method, negative results from the two-stage DNA pooling design used to screen loci within the TPH, AADC and MAOA genes did not support their role in migraine susceptibility.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

DNA technology for the detection of common genetic variants that predispose to thrombophilia

With the identification of common single locus point mutations as risk factors for thrombophilia, many DNA testing methodologies have been described for detecting these variations. Traditionally, functional or immunological testing methods have been used to investigate quantitative anticoagulant deficiencies. However, with the emergence of the genetic variations, factor V Leiden, prothrombin 20210 and, to a lesser extent, the methylene tetrahydrofolate reductase (MTHFR677) and factor V HR2 haplotype, traditional testing methodologies have proved to be less useful and instead DNA technology is more commonly employed in diagnostics. This review considers many of the DNA techniques that have proved to be useful in the detection of common genetic variants that predispose to thrombophilia. Techniques involving gel analysis are used to detect the presence or absence of restriction sites, electrophoretic mobility shifts, as in single strand conformation polymorphism or denaturing gradient gel electrophoresis, and product formation in allele-specific amplification. Such techniques may be sensitive, but are unwielding and often need to be validated objectively. In order to overcome some of the limitations of gel analysis, especially when dealing with larger sample numbers, many alternative detection formats, such as closed tube systems, microplates and microarrays (minisequencing, real-time polymerase chain reaction, and oligonucleotide ligation assays) have been developed. In addition, many of the emerging technologies take advantage of colourimetric or fluorescence detection (including energy transfer) that allows qualitative and quantitative interpretation of results. With the large variety of DNA technologies available, the choice of methodology will depend on several factors including cost and the need for speed, simplicity and robustness. © 2000 Lippincott Williams & Wilkins.

Krüppel-associated box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1 (HP1- ) mobilization and DNA repair in heterochromatin

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair.

Chromatin modifications involved in the DNA damage response to double strand breaks

In eukaryotes, genomic DNA is tightly compacted into a protein-DNA complex known as chromatin. This dense structure presents a barrier to DNA-dependent processes including transcription, replication and DNA repair. The repressive structure of chromatin is overcome by ATP-dependent chromatin remodelling complexes and chromatin-modifying enzymes. There is now ample evidence that DNA double-strand breaks (DSBs) elicit various histone modifications (such as acetylation, deacetylation, and phosphorylation) that function combinatorially to control the dynamic structure of the chromatin microenvironment. The role of these mechanisms during transcription and replication has been well studied, while the research into their impact on regulation of DNA damage response is rapidly gaining momentum. How chromatin structure is remodeled in response to DNA damage and how such alterations influence DSB repair are currently significant questions. This review will summarise the major chromatin modifications and chromatin remodelling complexes implicated in the DNA damage response to DSBs.

Association and linkage analyses of restriction fragment length polymorphisms for the human renin and antithrombin III genes in essential hypertension

Essential hypertension is a highly hereditable disorder in which genetic influences predominate over environmental factors. The molecular genetic profiles which predispose to essential hypertension are not known. In rats with genetic hypertension, there is some recent evidence pointing to linkage of renin gene alleles with blood pressure. The genes for renin and antithrombin III belong to a conserved synteny group which, in humans, spans the q21.3-32.3 region of chromosome I and, in rats, is linkage group X on chromosome 13. The present study examined the association of particular human renin gene (REN) and antithrombin III gene (AT3) polymorphisms with essential hypertension by comparing the frequency of specific alleles for each of these genes in 50 hypertensive offspring of hypertensive parents and 91 normotensive offspring of normotensive parents. In addition, linkage relationships were examined in hypertensive pedigrees with multiple affected individuals. Alleles of a REN HindIII restriction fragment length polymorphism (RFLP) were detected using a genomic clone, λHR5, to probe Southern blots of HindIII-cut leucocyte DNA, and those for an AT3 Pstl RFLP were detected by phATIII 113 complementary DNA probe. The frequencies of each REN allele in the hypertensive group were 0.76 and 0.24 compared with 0.74 and 0.26 in the normotensive group. For AT3, hypertensive allele frequencies were 0.49 and 0.51 compared with normotensive values of 0.54 and 0.46. These differences were not significant by χ2 analysis (P > 0.2). Linkage analysis of a family (data from 16 family members, 10 of whom were hypertensive), informative for both markers, without an age-of-onset correction, and assuming dominant inheritance of hypertension, complete penetrance and a disease frequency of 20%, did not indicate linkage of REN with hypertension, but gave a positive, although not significant, logarithm of the odds for linkage score of 0.784 at a recombination fraction of 0 for AT3 linkage to hypertension. In conclusion, the present study could find no evidence for an association of a REN HindIII RFLP with essential hypertension or for a linkage of the locus defined by this RFLP in a family segregating for hypertension. In the case of an AT3 Pstl RFLP, although association analysis was negative, linkage analysis suggested possible involvement (odds of 6:1 in favour) of a gene located near the 1q23 locus with hypertension in one informative family.

Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding β-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense ∼22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.