Single-base mismatch detection based on charge transduction through DNA

High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based upon charge transport through DNA films. Double-helical DNA films on gold surfaces have been prepared and used to detect DNA mismatches electrochemically. The signals obtained from redox-active intercalators bound to DNA-modified gold surfaces display a marked sensitivity to the presence of base mismatches within the immobilized duplexes. Differential mismatch detection was accomplished irrespective of DNA sequence composition and mismatch identity. Single-base changes in sequences hybridized at the electrode surface are also detected accurately. Coupling the redox reactions of intercalated species to electrocatalytic processes in solution considerably increases the sensitivity of this assay. Reporting on the electronic structure of DNA, as opposed to the hybridization energetics of single-stranded oligonucleotides, electrochemical sensors based on charge transport may offer fundamental advantages in both scope and sensitivity.     

Sonochemically fabricated microelectrode arrays for biosensors offering widespread applicability: Part I

A novel and patented procedure is described for the sonochemical fabrication of a new class of microelectrode array based sensor with electrode element populations of up to 2 x 10(5) cm(-2). For some years it has been accepted that microelectrode arrays offer an attractive route for lowering minimum limits of detection and imparting stir (convectional mass transport) independence to sensor responses; despite this no commercial biosensors, to date, have employed microelectrode arrays, largely due to the cost of conventional fabrication routes that have not proved commercially viable for disposable devices. Biosensors formed by our sonochemical approach offer unrivalled sensitivity and impart stir independence to sensor responses. This format lends itself for mass fabrication due to the simplicity and inexpensiveness of the approach; in the first instance impedimetric and amperometric sensors are reported for glucose as model systems. Sensors already developed for ethanol, oxalate and a number of pesticide determinations will be reported in subsequent publications.     

Onset of pulsatile pressure causes transiently increased filtration through artery wall

Convective fluid motion through artery walls aids in the transvascular transport of macromolecules. Although many measurements of convective filtration have been reported, they were all obtained under constant transmural pressure. However, arterial pressure in vivo is pulsatile. Therefore, experiments were designed to compare filtration under steady and pulsatile pressure conditions. Rabbit carotid arteries were cannulated and excised from male New Zealand White rabbits anesthetized with pentobarbitol sodium (30 mg/kg i.v. administered). Hydraulic conductance was measured in cannulated excised rabbit carotid arteries at steady pressure. Next, pulsatile pressure trains were applied within the same vessels, and, simultaneously, arterial distension was monitored using Optical coherence tomography (OCT). For each pulse train, the volume of fluid lost through filtration was measured (subtracting volume change due to residual distension) and compared with that predicted from steady pressure measurements. At 60- and 80-mmHg baseline pressures, the experimental filtration volumes were significantly increased compared with those predicted for steady pressure (P < 0.05). OCT demonstrated that the excess fluid volume loss was significantly greater than the volume that would be lost through residual distension (P < 0.05). After 30 s, the magnitude of the excess of fluid loss was reduced. These results suggest that sudden onset of pulsatile pressure may cause changes in arterial interstitial hydration.     

Protective role of ETA endothelin receptors during the acute phase of Trypanosoma cruzi infection in rats

Chagas' disease, caused by Trypanosoma cruzi, has an acute phase characterized by blood-circulating trypomastigotes and amastigote proliferation in several cell types, especially muscle cells. In the chronic phase, around 70% of infected people are asymptomatic (latent form). The remainder develop chagasic cardiomyopathy and/or digestive syndromes. There is evidence for aggravation of the chronic cardiac pathology by endothelin-mediated vasoconstriction. Holtzman rats have proven to be a good model for Chagas' disease acute phase and latent chronic phase. Now, we investigate the effects of prolonged treatment with an endothelin ET(A) receptor antagonist, BSF 461314, during the acute phase on parasitemia, coronary flow, tissue parasitism and the inflammatory process. Using isolated heart in Langendorff's preparation, endothelial dysfunction was observed only in non-treated infected animals. Histoquantitative analyses carried out in heart and diaphragm showed higher tissue parasitism and/or inflammatory process in BSF 461314-treated animals. Our data indicate that endothelin ET(A) receptors contribute to the initial mechanisms of parasite control. Impairment of the endothelium-dependent vasodilatation favors hazardous effects. However, blocking endothelin ET(A) receptors can prevent the latter.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

An association between NQO1 genetic polymorphism and risk of bladder cancer

NAD(P)H:quinone oxidoreductase (NQO1) is a detoxification enzyme that plays a critical role in protecting cells against chemically induced oxidative stress, cytotoxicity, mutagenicity, and carcinogenicity. NQO1 protects cells from oxidative damage by preventing the generation of reactive oxygen species and reducing certain environmental carcinogens, such as nitroaromatic compounds, heterocyclic amines, and possible cigarette smoke condensate. A C-->T single nucleotide polymorphism in exon 6 was shown to reduce NQO1 enzyme activity, which may diminish the protection provided by NQO1. Therefore, we hypothesized that people with the variant allele genotypes of NQO1 are at higher risk for bladder cancer. In an ongoing case-control study, the NQO1 genotypes were successfully identified by polymerase chain reaction restriction fragment length polymorphism in 265 bladder cancer patients and 261 control subjects matched for age, sex, and ethnicity. The frequency of the variant NQO1 allele was 18% for controls and 21% for cases. The variant allele genotypes of NQO1 were associated with a higher risk of bladder cancer in Caucasians (odds ratio (OR)=1.51; 95% confidence interval (CI)=1.01-2.25). Further analysis in Caucasians showed an elevated bladder cancer risk in men (OR=1.75; 95% CI=1.08-2.85) but not in women (OR=1.16; 95% CI=0.57-2.37). In addition, the variant allele genotypes were associated with higher bladder cancer risk in ever smokers (OR=1.78; 95% CI=1.06-3.00), but not in never smokers (OR=1.19; 95% CI=0.65-2.20). These results suggest that the NQO1 genetic polymorphism modulates bladder cancer risk, especially in men and ever smokers.     

Numerical chromosomal abnormalities in rat epididymal spermatozoa following chronic cyclophosphamide exposure

Chronic, low-dose treatment of male rats with cyclophosphamide, a chemotherapeutic agent, is known to affect progeny outcome adversely in a dose-dependent and time-specific manner, resulting in increased pre- and postimplantation loss as well as malformations. Concern exists regarding the genetic quality of mature gametes exposed to cyclophosphamide during mitosis and meiosis. The goal of the present study was to determine the effect of chronic cyclophosphamide treatment during spermatogenesis on the frequency of numerical chromosomal anomalies in epididymal spermatozoa. Male rats were treated with either saline or cyclophosphamide (6 mg kg-1 day-1) for 6 or 9 wk, and cauda epididymal spermatozoa were collected. The rat sperm Y-4 fluorescence in situ hybridization assay was used to assess the induction of spermatozoal disomy, nullisomy, and diploidy involving chromosomes Y and 4. The overall frequency of numerically abnormal spermatozoa was elevated approximately 2-fold (P < 0.001) after 9 wk of cyclophosphamide treatment. Exposure for 9 wk, but not for 6 wk, significantly increased the frequency of spermatozoa with chromosome 4 disomy (P < 0.02) and nullisomy (P < 0.05), but disomy Y and diploidy were not significantly increased with treatment compared to corresponding controls. Independent of treatment, only 27% of aneuploid spermatozoa presented with morphological abnormalities, but all diploid spermatozoa were approximately twice the size of normal cells. Thus, cyclophosphamide disrupts meiotic events before pachynema during spermatogenesis, emphasizing the potential for adverse progeny outcomes following genotoxic damage.     

A function for subtelomeric DNA in Saccharomyces cerevisiae

The subtelomeric DNA sequences from chromosome I of Saccharomyces cerevisiae are shown to be inherently poor substrates for meiotic recombination. On the basis of these results and prior observations that crossovers near telomeres do not promote efficient meiosis I segregation, we suggest that subtelomeric sequences evolved to prevent recombination from occurring where it cannot promote efficient segregation.