Real-time polymerase chain reaction.

Real-time PCR is the state-of-the-art technique to quantify nucleic acids for mutation detection, genotyping and chimerism analysis. Since its development in the 1990s, many different assay formats have been developed and the number of real-time PCR machines of different design is continuously increasing. This review provides a survey of the instruments and assay formats available and discusses the pros and cons of each.

Hypoxia and reoxygenation increase H2O2 production in rats.

To test the effect of transition from sustained hypoxia to normoxia on production of reactive oxygen species (ROS) in lungs, the authors measured hydrogen peroxide (H(2)O(2)) output in the expired air of rats breathing hypoxic, normoxic, and hyperoxic gas mixtures at the end of exposure to 72 hours of hypoxia. Twenty-one male Wistar rats (200 to 280 g) were randomly assigned to 1 of 3 groups. First two groups (experimental) were kept for 3 days in normobaric hypoxic chamber (F(1)O(2) 0.

Elasticity of stiff polymer networks.

We study the elasticity of a two-dimensional random network of rigid rods ("Mikado model"). The essential features incorporated into the model are the anisotropic elasticity of the rods and the random geometry of the network. We show that there are three distinct scaling regimes, characterized by two distinct length scales on the elastic backbone.

Increased expression of disintegrin-metalloproteinases ADAM-15 and ADAM-9 following upregulation of integrins alpha5beta1 and alphavbeta3 in atherosclerosis.

Regulation of alphavbeta3 and alpha5beta1 integrin function plays a crucial role in atherosclerosis. Possible regulators of integrin-matrix interactions are integrin-binding ADAMs (proteins with a disintegrin- and metalloproteinase-domain), like ADAM-15 and ADAM-9. Molecular interactions between ADAM-15, alpha5beta1, and alphavbeta3 have been demonstrated.

The role of free radicals in canine counterpart of senile dementia of the Alzheimer type.

The pathogenesis of Alzheimer's disease is still unknown. In recent time oxidative stress has been discussed as an important contributor. In the present study we investigated the role of free radicals in the spontaneous canine model of Alzheimer's disease.

Hydrogen peroxide production by alveolar macrophages is increased and its concentration is elevated in the breath of rats exposed to hypoxia: relationship to lung lipid peroxidation.

Hypoxic exposure triggers a generation of reactive oxygen species that initiate free radical damage to the lung. Hydrogen peroxide is the product of alveolar macrophages detectable in the expired breath. We evaluated the significance of breath H(2)O(2) concentration for the assessment of lung damage after hypoxic exposure and during posthypoxic period.

Validation of an algorithm for automatic quantification of nucleic acid copy numbers by real-time polymerase chain reaction.

Real-time quantitative polymerase chain reaction (PCR) with on-line fluorescence detection has become an important technique not only for determination of the absolute or relative copy number of nucleic acids but also for mutation detection, which is usually done by measuring melting curves. Optimum assay conditions have been established for a variety of targets and experimental setups, but only limited attention has been directed to data evaluation and validation of the results. In this work, algorithms for the processing of real-time PCR data are evaluated for several target sequences (p53, IGF-1, PAI-1, Factor VIIc) and compared to the results obtained by standard procedures.

[Markers of lung disease in expired air].

Many chronic inflammatory pulmonary diseases are associated with inflammation and oxidative stress. A complex interplay between the specific cause of the diseases, the type and intensity of inflammation and oxidative stress, results in the clinical picture and reveals the probability of progression of the specific disease. The presence of inflammation and oxidative stress has been established in interstitial lung diseases, cystic fibrosis, bronchiectasis, adult respiratory distress syndrome, chronic obstructive lung diseases and asthma.

Real-time PCR-based method for the estimation of genome sizes.

The fast and reliable estimation of the genome sizes of various species would allow for a systematic analysis of many organisms and could reveal insights into evolutionary processes. Many methods for the estimation of genome sizes have already been described. The classical methods are based on the determination of the phosphate content in the DNA backbone of total DNA isolated from a defined number of cells or on reassociation kinetics of high molecular weight genomic DNA (c(0)t assay).

Apolipoprotein E epsilon 4 is associated with hippocampal volume reduction in females with alcoholism.

There is evidence that a higher incidence of diverse neurodegenerative diseases is associated with the apolipoprotein E epsilon4 allele (ApoE4). Most recently it has been found that the ApoE4 allele is specifically related to an accelerated hippocampal atrophy in patients with Alzheimer's disease. Therefore, the aim of the present study was to investigate the association between ApoE4 genotypes and brain hippocampal volume reduction in alcoholics by using volumetric high-resolution MR imaging.

SoFAR: software for fully automatic evaluation of real-time PCR data.

Quantitative real-time PCR has proven to be an extremely useful technique in life sciences for many applications. Although a lot of attention has been paid to the optimization of the assay conditions, the analysis of the data acquired is often done with software tools that do not make optimum use of the information provided by the data. Particularly, this is the case for high-throughput analysis, which requires a careful characterization and interpretation of the complete data by suitable software.

HER-2/neu gene copy number quantified by real-time PCR: comparison of gene amplification, heterozygosity, and immunohistochemical status in breast cancer tissue.

Background: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods.

Methods: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status.

Lack of association between hippocampal volume reduction and first-onset alcohol withdrawal seizure. A volumetric MRI study.

Aims And Methods: Magnetic resonance imaging (MRI) of the hippocampus has been extensively studied in both neurological and psychiatric disorders. Furthermore, hippocampal volume reductions on MRI have been reported in patients with chronic alcoholism. The present volumetric MRI study was undertaken to determine whether an association exists between hippocampal volume reduction and first-onset alcohol withdrawal seizure.

Hyperhomocysteinemia as a new risk factor for brain shrinkage in patients with alcoholism.

Chronic alcohol consumption can induce brain atrophy, whereby the exact mechanism of brain damage in alcoholics remains unknown. There is evidence that chronic alcoholism is associated with hyperhomocysteinemia. Homocysteine is an excitatory amino acid which markedly enhances the vulnerability of neuronal cells to excitotoxic and oxidative injury in vitro and in vivo.

The role of carbon dioxide in free radical reactions of the organism.

Carbon dioxide interacts both with reactive nitrogen species and reactive oxygen species. In the presence of superoxide, NO reacts to form peroxynitrite that reacts with CO2 to give nitrosoperoxycarbonate. This compound rearranges to nitrocarbonate which is prone to further reactions.

Detection and quantification of insertion/deletion variations by allele-specific real-time PCR: application for genotyping and chimerism analysis.

The DNA-based quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allele-specific amplification with a real-time PCR-based quantification for rapid allelotyping and chimerism analysis.

Impact of mobility on structure-based drug design for the MMPs.

Structure-based approaches for drug design generally do not incorporate solvent effects and dynamic information to predict inhibitor-binding affinity because of practical limitations. The matrix metalloproteinases (MMPs) have previously been demonstrated to exhibit significant mobility in their active sites. This dynamic characteristic significantly complicates the drug design process based on static structures, which was clearly observed for a class of hydroxamic acids containing a butynyl moiety.

[A plea for a chronobiological approach to diabetes mellitus].

Diabetes mellitus is, in most patients, a multi-metabolic condition disease under fixed standardized conditions. We develop here the advantages of a chronobiological approach, exploring nycthemeral variations in blood pressure, variations in insulin resistance, postprandial changes in blood glucose and postabsorptive variations in blood lipids. Such information can help improve our understanding of the disease, better identify risk and prognosis, and enlighten therapeutic options.

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide.

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q (CoQ).

Ultraviolet light-irradiated collagen III modulates expression of cytoskeletal and surface adhesion molecules in rat aortic smooth muscle cells in vitro.

Systemic and pulmonary hypertension is characterised by structural reconstruction of the vascular wall which includes hypertrophy and hyperplasia of vascular smooth muscle cells (VSMCs) and fibroproduction. We hypothesise that these changes are stimulated by non-enzymatic modification of collagen molecules in the injured vascular wall by radicals. We exposed collagen III to ultraviolet (UV) light irradiation which, as indicated by fluorescence and electrophoretic analyses, resulted in its fragmentation.

Ypsilon Schachtel, a Drosophila Y-box protein, acts antagonistically to Orb in the oskar mRNA localization and translation pathway.

Subcellular localization of mRNAs within the Drosophila oocyte is an essential step in body patterning. Yps, a Drosophila Y-box protein, is a component of an ovarian ribonucleoprotein complex that also contains Exu, a protein that plays an essential role in mRNA localization. Y-box proteins are known translational regulators, suggesting that this complex might regulate translation as well as mRNA localization.

[Von Recklinghausen's disease associated with pancreatic somatostatinoma].

Introduction: Pancreatic somatostatinoma is a rare entity and its association with Von Recklinghausen's disease has only been described on two occasions. We report a new observation, with clinical and evolving status differing from those described in isolated cases of somatostatinoma.

Observation: A 28 year-old man presenting with familial Von Recklinghausen's disease, had suffered for 10 years from paroxysmic abdominal pain.