Role of the impairment of oxidative metabolism in pathogenesis of lower urinary tract symptoms with benign prostatic hyperplasia and their treatment by alpha1-adrenoblocker alfuzosin

The role of impairment of general oxidative and energy metabolism in pathogenesis of lower urinary tract symptoms (LUTS) in patients with benign prostatic hyperplasia (BPH) and their correction by (1-adrenoblocker alfuzosin was studied. One group of patients (N = 126) was examined by standard methods for determination of the severity of LUTS by IPSS and mean effective volume of urinary bladder (MEVUB). In the second group (N = 29) in addition to functional examinations, metabolic indicators in blood were measured: antioxidant activity (AOA) and succinate dehydrogenase activity (SDA). Severity of LUTS depends greatly on the MEVUB. It was the first to show a practically complete correlation between LUTS, AOA and SDA. Severity of LUTS exactly correlates with indicators of oxidative and energy metabolism. In patients with more heavy LUTS, lowest AOA and SDA values were found. In the course of effective treatment, both phenomena developed an improvement of clinical symptoms and a rise of biochemical parameters. Close correlation between functional and metabolic phenomena is evidence of an essential role of metabolic mechanisms in the pathogenesis of LUTS with BPH. This opens perspectives to use antioxidants and energy metabolism activators for correction of UB dysfunction in patients with BPH.     

DNA damage in frog erythrocytes after in vitro exposure to a high peak-power pulsed electromagnetic field

Till the present time, the genotoxic effects of high peak-power pulsed electromagnetic fields (HPPP EMF) on cultured cells have not been studied. We investigated possible genotoxic effects of HPPP EMF (8.8 GHz, 180 ns pulse width, peak power 65 kW, repetition rate 50 Hz) on erythrocytes of the frog Xenopus laevis. We used the alkaline comet assay, which is a highly sensitive method to assess DNA single-strand breaks and alkali-labile lesions. Blood samples were exposed to HPPP EMF for 40 min in rectangular wave guide. The specific absorption rate (SAR) calculated from temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The temperature rise in the blood samples at steady state was 3.5 +/- 0.1 degrees C. The data show that the increase in DNA damage after exposure of erythrocytes to HPPP EMF was induced by the rise in temperature in the exposed cell suspension. This was confirmed in experiments in which cells were incubated for 40 min under the corresponding temperature conditions. The results allow us to conclude that HPPP EMF-exposure at the given modality did not cause any a-thermal genotoxic effect on frog erythrocytes in vitro.     

The increase in the variability of microsatellite-associated repeats in the genome of gamma-irradiated male mice is tissue specific

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to measure the level of polymorphism of microsatellite (MCS)-associated repeating sequences of spleen, lung, and brain DNA in the F1 progeny of male BALB/c mice exposed to acute gamma-radiation at doses of 50 cGy and 200 cGy 15 days before mating with unirradiated females. The variability of MCS-associated sequences in the genome of brain and lung cells was higher as compared to the spleen cells of the progeny of unirradiated males. In the progeny of irradiated males, a 20% increase in MCS polymorphism of spleen DNA was found as an increase in the frequency of "non-parent" bands in DNA-fingerprints as against to the progeny of unirradiated males. Significant changes in this parameter were revealed for brain tissue and not for lung tissue only in the progeny of males exposed to 200 cGy. The results suggest a tissue-specific character of transmission of radiation-induced alterations in the genome of germ cells of male parents to the somatic cells of the progeny.     

Preservation of native properties of mitochondria in rat liver homogenate

A protocol is developed for preparation of concentrated rat liver homogenate preserving assemblies of mitochondria in isotonic KCl under 0 and 15 degrees C. Assemblies preserve ability for self-organization during storage in homogenate. All key energy functions of mitochondria can be investigated in such a homogenate. Oxidative phosphorylation and membrane potential are stable for 5-7 h and can be still observed on the next day. Substrate-level phosphorylation is better pronounced for mitochondria in KCl than in sucrose medium while Ca2+ capacity is greater and lipid peroxidation is much lower. Sucrose addition impairs these functions. The rate of phosphorylating respiration is lower in large assemblies and higher in small. Transition from large to small assemblies corresponds to the transition from quiescent state of animal to adrenaline induced active state. The proposed method is particularly convenient for clinical investigations with small bioptates.     

Ability of ELISA and a toxin neutralization assay to detect changes in immunogenicity of a recombinant Bacillus anthracis protective antigen vaccine upon storage

We examined the capability of a mouse immunogenicity assay to detect improper storage of a recombinant protective antigen (rPA)-based anthrax vaccine formulated with an aluminum adjuvant, using ELISA and a toxin neutralization assay (TNA) to measure the antibody response to rPA. The vaccine was stored at 4 °C, room temperature (RT) or 37 °C for one, four and eight weeks and used for immunization, along with freshly prepared vaccine. Results showed that, contrary to ELISA, TNA is suitable to detect a loss of immunogenicity of the rPA vaccine following its exposure to RT for a period of eight weeks and to 37 °C for a period as short as 1 week.     

Levels of Circulating DNA in Blood Serum and DNA Damage in Leukocytes of Healthy Donors of Different Genders and Ages

We studied the levels of extracellular nuclear and mitochondrial DNA of blood serum and DNA damage in leukocytes of healthy donors of different sex and age groups. The baseline levels of DNA damage in leukocytes and serum DNA levels were shown to vary greatly among different donors. The baseline level of DNA damage in leukocytes was not associated with the presence of chronic deceases or an occupational health risk for elderly donors. It was found that extracellular DNA concentrations were generally higher in men than in women. There is a tendency towards an increase in the relative mitochondrial DNA copy number determined by ΔCt in women but not in men: the relative mtDNA copy number in elderly individuals varies significantly in both sexes, possibly due to age-related physiological changes. It is necessary to consider the gender and age of patients when using an indicator such as the level of extracellular DNA of blood serum for diagnosis and monitoring.

     

Identifying a high fraction of the human genome to be under selective constraint using GERP++

Computational efforts to identify functional elements within genomes leverage comparative sequence information by looking for regions that exhibit evidence of selective constraint. One way of detecting constrained elements is to follow a bottom-up approach by computing constraint scores for individual positions of a multiple alignment and then defining constrained elements as segments of contiguous, highly scoring nucleotide positions. Here we present GERP++, a new tool that uses maximum likelihood evolutionary rate estimation for position-specific scoring and, in contrast to previous bottom-up methods, a novel dynamic programming approach to subsequently define constrained elements. GERP++ evaluates a richer set of candidate element breakpoints and ranks them based on statistical significance, eliminating the need for biased heuristic extension techniques. Using GERP++ we identify over 1.3 million constrained elements spanning over 7% of the human genome. We predict a higher fraction than earlier estimates largely due to the annotation of longer constrained elements, which improves one to one correspondence between predicted elements with known functional sequences. GERP++ is an efficient and effective tool to provide both nucleotide- and element-level constraint scores within deep multiple sequence alignments.     

Ranking non-synonymous single nucleotide polymorphisms based on disease concepts

As the number of non-synonymous single nucleotide polymorphisms (nsSNPs) identified through whole-exome/whole-genome sequencing programs increases, researchers and clinicians are becoming increasingly reliant upon computational prediction algorithms designed to prioritize potential functional variants for further study. A large proportion of existing prediction algorithms are ‘disease agnostic’ but are nevertheless quite capable of predicting when a mutation is likely to be deleterious. However, most clinical and research applications of these algorithms relate to specific diseases and would therefore benefit from an approach that discriminates between functional variants specifically related to that disease from those which are not. In a whole-exome/whole-genome sequencing context, such an approach could substantially reduce the number of false positive candidate mutations. Here, we test this postulate by incorporating a disease-specific weighting scheme into the Functional Analysis through Hidden Markov Models (FATHMM) algorithm. When compared to traditional prediction algorithms, we observed an overall reduction in the number of false positives identified using a disease-specific approach to functional prediction across 17 distinct disease concepts/categories. Our results illustrate the potential benefits of making disease-specific predictions when prioritizing candidate variants in relation to specific diseases. A web-based implementation of our algorithm is available at http://fathmm.biocompute.org.uk.     

Cytoplasmic superoxide dismutase activity is a sensitive indicator of the antioxidant status of the rat liver and brain

Several parameters of the cytoplasmic enzymatic antioxidant system of the liver and brain of the rat have been investigated under conditions of immobilization stress and of an antioxidant preparation in the diet of animals. These included superoxide dismutase (SOD) and glutathione reductase (GR) activities and nonspecific NADPH oxidation. Only changes in the activity of SOD both in the liver and brain were revealed. In the liver of animals that receive no preparation, a decrease in the activity of SOD after 30-min immobilization and its restoration after a 360-min immobilization were observed. In the brain, the activity of SOD decreased only in preconditioned animals after 30 and 360 min of exposure to stress. In addition, the activity of SOD in the brain of preconditioned animals, both stressed and unstressed, was lower than in the corresponding groups of control animals. It is probable that, under the conditions of immobilization stress, the level of reactive oxygen species (ROS) and as a consequence the activity of SOD decrease. The intake of an antioxidant preparation under these conditions seems to be not correct.