General assessment of copy number variation in normal and tumor tissues of the domestic dog (Canis lupus familiaris)

In recent years, characterization of a copy number variation (CNV) of the genomic DNA has provided evidence for the relationship of this type of genetic variation with the occurrence of a broad spectrum of diseases, including cancer lesions. Copy number variants (CNVs) also occur in the genomes of healthy individuals as a result of abnormal recombination processes in germ cells and have a hereditary character contributing to the natural genetic diversity. Recent image analysis methods and advanced computational techniques allow for identification of CNVs using SNPs genotyping microarrays based on the analysis of signal intensity observed for markers located in the specific genomic regions. In this study we used CanineHD BeadChip assay (Illumina) to identify both natural and cancer-induced CNVs in the genomes of different dog breeds and in different cancer types occurring in this species. The obtained results showed that structural aberrations are a common phenomenon arising during a tumor progression and are more complex and widespread in tumors of mesenchymal tissue origin than in epithelial tissue originating tumors. The tumor derived CNVs, in comparison to healthy samples, were characterized by larger sizes of regions, higher number of amplifications, and in some cases encompassed genes with potential effect on tumor progression.     

Postglacial history of vegetation,human activity and lake-level changes at Jezioro Linówek in northeast Poland,based on multi-proxy data

We present the postglacial history of vegetation, human activities and changes in lake level in the context of climate change in northeast Poland from ~14,000 cal. b.p. to the present day. The palaeoecological reconstruction is based on the results of high-resolution plant macrofossil analyses as well as records from pollen, Cladocera and radiocarbon dating. Climate fluctuations and human activity have caused many changes in vegetation development in Jezioro Linówek and in the vicinity of this lake. The Early Holocene warming that occurred at ~9500 b.c. caused an increase in Betula and the colonisation of Linówek by Potamogeton lucens, Nymphaea alba and Chara sp. At ~2300 b.c., climate cooling was accompanied by the spread of Picea abies and the appearance of Potamogeton alpinus and Nuphar pumila in the lake. The first traces of farming in the form of Cerealia pollen have been dated back to ~2100 b.c. The cultivation of Triticum began at ~250 b.c., Secale at ~a.d. 550, and Fagopyrum at ~a.d. 1720. The rapid increase in human activity at ~a.d. 1700 and the simultaneous loss of woodland is associated with the establishment of villages in the area and is expressed by the decline of tree curves. In Linówek, which was formed ~14,000 cal. b.p., three periods of high water level occurred (12000–9400, 7000–4000 and 1450 b.c.–a.d. 650), and two periods of low water level (9400–7100 and 3700–1700 b.c.). The changes of water level correspond well with other sites in central and northern Europe.     

Molecular determinants for antibody binding on group 1 house dust mite allergens

House dust mites produce potent allergens, Der p 1 and Der f 1, that cause allergic sensitization and asthma. Der p 1 and Der f 1 are cysteine proteases that elicit IgE responses in 80% of mite-allergic subjects and have proinflammatory properties. Their antigenic structure is unknown. Here, we present crystal structures of natural Der p 1 and Der f 1 in complex with a monoclonal antibody, 4C1, which binds to a unique cross-reactive epitope on both allergens associated with IgE recognition. The 4C1 epitope is formed by almost identical amino acid sequences and contact residues. Mutations of the contact residues abrogate mAb 4C1 binding and reduce IgE antibody binding. These surface-exposed residues are molecular targets that can be exploited for development of recombinant allergen vaccines.     

Artifacts in magnetic resonance imaging and computed tomography caused by dental materials

Background

Artifacts caused by dental restorations, such as dental crowns, dental fillings and orthodontic appliances, are a common problem in MRI and CT scans of the head and neck. The aim of this in-vitro study was to identify and evaluate the artifacts produced by different dental restoration materials in CT and MRI images.

Methods

Test samples of 44 materials (Metal and Non-Metal) commonly used in dental restorations were fabricated and embedded with reference specimens in gelatin moulds. MRI imaging of 1.5T and CT scan were performed on the samples and evaluated in two dimensions. Artifact size and distortions were measured using a digital image analysis software.

Results

In MRI, 13 out of 44 materials produced artifacts, while in CT 41 out of 44 materials showed artifacts. Artifacts produced in both MRI and CT images were categorized according to the size of the artifact.

Significance

Metal based restoration materials had strong influence on CT and less artifacts in MRI images. Rare earth elements such as Ytterbium trifluoride found in composites caused artifacts in both MRI and CT. Recognizing these findings would help dental materials manufacturers and developers to produce materials which can cause less artifacts in MRI and CT images.     

The insulin-suppressive effect of resveratrol - an in vitro and in vivo phenomenon

Resveratrol, a naturally occurring phytoalexin, is known to exert numerous beneficial effects in the organism. Literature data indicate that this compound may, among other effects, play a role in prevention of diabetes and diabetic complications. Resveratrol was recently found to affect insulin secretion in vitro and to change blood insulin concentrations. These effects are, however, not fully elucidated. In the present study, 1, 10 and 100microM resveratrol incubated for 90min with pancreatic islets isolated from normal rats failed to affect basal insulin release, but substantially impaired the secretory response to physiological and maximally effective glucose. In depolarized islets exposed to resveratrol, succinate-induced insulin secretion was also diminished. The blockade of somatostatin receptors substantially enhanced insulin secretion induced by 6.7mM glucose and simultaneously suppressed the inhibitory effect of 1microM resveratrol, but at 10 and 100microM, resveratrol was still able to attenuate hormone secretion. Acetylcholine clearly increased the insulin-secretory response to 6.7mM glucose and canceled the inhibitory effect of 1microM resveratrol. However, resveratrol at concentrations 10 and 100microM strongly decreased insulin secretion. The direct activation of protein kinase C totally suppressed the inhibitory influence of 1 and 10microM resveratrol on hormone secretion. However, activation of this enzyme appeared to be insufficient to cancel the insulin-suppressive effect of 100microM resveratrol. These data indicate that resveratrol-induced inhibition of insulin secretion may be partially mitigated by suppression of somatostatin action, activation of protein kinase C or the presence of acetylcholine. The in vivo experiment revealed that resveratrol, administered to normal rats at the dose 50mg/kg body weight, diminished blood insulin concentrations at 30min, without concomitant changes in glycemia. These observations point to the direct insulin-suppressive action of resveratrol in the rat.     

Homozygote for mutation c.1204 + 1G > A of the ARSA gene presents with a late-infantile form of metachromatic leukodystrophy and a rare MRI white matter lesion type

The metachromatic leukodystrophy (MLD)--causing mutation c.1204 + 1G > A damages an intron-exon splice site recognition sequence. This results in a complete loss of enzymatic activity of arylsulfatase A (ARSA) protein molecules. We have found a late-infantile type MLD-patient to be homozygous for this mutation, which was not reported earlier, but is consistent with previous suggestions. Interestingly, the cerebral magnetic resonance imaging (MRI) in this patient displayed linear or punctuate structures radiating in the demyelinated white matter, which resembled the patterns described in Pelizaeus-Merzbacher disease. It should be emphasised that whenever a cerebral MRI demonstrates the "tigroid" or "leopard-skin" demyelination pattern not only Pelizaeus-Merzbacher disease, but also metachromatic leukodystrophy diagnosis should be considered; this suggests the necessity of ARSA activity estimations in patients with such specific MRI patterns.     

Non-homologous DNA end joining repair in normal and leukemic cells depends on the substrate ends

Double-strand breaks (DSBs) are the most serious DNA damage which, if unrepaired or misrepaired, may lead to cell death, genomic instability or cancer transformation. In human cells they can be repaired mainly by non-homologous DNA end joining (NHEJ). The efficacy of NHEJ pathway was examined in normal human lymphocytes and K562 myeloid leukemic cells expressing the BCR/ABL oncogenic tyrosine kinase activity and lacking p53 tumor suppressor protein. In our studies we employed a simple and rapid in vitro DSB end joining assay based on fluorescent detection of repair products. Normal and cancer cells were able to repair DNA damage caused by restriction endonucleases, but the efficiency of the end joining was dependent on the type of cells and the structure of DNA ends. K562 cells displayed decreased NHEJ activity in comparison to normal cells for 5' complementary DNA overhang. For blunt-ended DNA there was no significant difference in end joining activity. Both kinds of cells were found about 10-fold more efficient for joining DNA substrates with compatible 5' overhangs than those with blunt ends. Our recent findings have shown that stimulation of DNA repair could be involved in the drug resistance of BCR/ABL-positive cells in anticancer therapy. For the first time the role of STI571 was investigated, a specific inhibitor of BCR/ABL oncogenic protein approved for leukemia treatment in the NHEJ pathway. Surprisingly, STI571 did not change the response of BCR/ABL-positive K562 cells in terms of NHEJ for both complementary and blunt ends. Our results suggest that the various responses of the cells to DNA damage via NHEJ can be correlated with the differences in the genetic constitution of human normal and cancer cells. However, the role of NHEJ in anticancer drug resistance in BCR/ABL-positive cells is questionable.     

Low variability of the POLG (CAG)n repeat in north Eurasian populations

We investigated the frequency of different repeat-length alleles of the trinucleotide CAG microsatellite repeat in the coding sequence of the nuclear gene for the catalytic subunit of mitochondrial DNA polymerase gamma (POLG) in 12 ethnic groups from northern Eurasia. The population sample consisted of 1,330 individuals from 3 large geographic areas: Europe, Southwest Asia, and Siberia/East Asia. We found that the 10-repeat allele of the POLG gene is the most frequent in all analyzed populations, with a frequency of 88-96%. The heterozygosity level ranges from 22% in Europe to 13.6% in Southwest Asia with the lowest value of 7.4% in Siberia/East Asia. The present study provides evidence of clinal distribution of POLG gene heterozygosity in North Eurasian populations. In general, we found an extremely low variability of the trinucleotide CAG microsatellite repeat, suggesting that purifying selection acts against deleterious alleles, although low mutability of the repeated region cannot be ruled out.     

Protein S attenuates the invasive potential of THP-1 cells by interfering with plasminogen binding on cell surface via a protein C-independent mechanism

Protein S, a cofactor for activated protein C (aPC) to inactivate coagulation factors, also plays a pivotal role in inflammation. Based on our recent findings that aPC and protein S modifies tissue plasminogen activator (tPA)-catalyzed activation of Glu-plasminogen (Glu-plg), we analyzed possible role of protein S in cell-associated plasminogen activation and invasive potential of inflammatory cells. Monocyte-like THP-1 cells, to which both plasminogen and tPA bind, enhanced tPA-catalyzed plasminogen activation, which was partially abolished by protein S but not by aPC. Protein S attenuated both the plasminogen binding to THP-1 cells and associated their invasive potential through Matrigel.     

Yeast as a biosensor for antioxidants: simple growth tests employing a Saccharomyces cerevisiae mutant defective in superoxide dismutase

Mutants of Saccharomyces cerevisiae devoid of Cu,Zn-superoxide dismutase are hypersensitive to a range of oxidants, hyperbaric oxygen and hyperosmotic media, show lysine and methionine auxotrophy when grown under the atmosphere of air and have a shortened replicative life span when compared to the wild-type strain. Ascorbate and other antioxidants can ameliorate these defects, which may be a basis of simple tests sensing the presence of antioxidants. In particular, tests of growth on solid medium (colony formation) in the absence of methionine and/or lysine, or in the presence of 0.8 M NaCl can be useful for detection and semiquantitative estimation of compounds of antioxidant properties. Hypoxic atmosphere was found to increase the sensitivity of detection of antioxidants. The test of abolishment of lysine auxotrophy showed a concentration dependence of the antioxidant effects of cysteine and N-acetylcysteine which, however, lost their protective action at high concentration, in contrast to glutathione which was effective also at higher concentrations.