Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches

Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these "silent" variations can have a significant impact on protein expression and function and should no longer be considered "silent". Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.     

Preventive and therapeutic effects of SkQ1-containing Visomitin eye drops against light-induced retinal degeneration

The human retina is constantly affected by light of varying intensity, this being especially true for photoreceptor cells and retinal pigment epithelium. Traditionally, photoinduced damages of the retina are induced by visible light of high intensity in albino rats using the LIRD (light-induced retinal degeneration) model. This model allows study of pathological processes in the retina and the search for retinoprotectors preventing retinal photodamage. In addition, the etiology and mechanisms of retina damage in the LIRD model have much in common with the mechanisms of the development of age-related retinal disorders, in particular, with age-related macular degeneration (AMD). We have studied preventive and therapeutic effects of Visomitin eye drops (based on the mitochondria-targeted antioxidant SkQ1) on albino rat retinas damaged by bright light. In the first series of experiments, rats receiving Visomitin for two weeks prior to illumination demonstrated significantly less expressed atrophic and degenerative changes in the retina compared to animals receiving similar drops with no SkQ1. In the second series, the illuminated rats were treated for two weeks with Visomitin or similar drops without SkQ1. The damaged retinas of the experimental animals were repaired much more effectively than those of the control animals. Therefore, we conclude that Visomitin SkQ1-containing eye drops have pronounced preventive and therapeutic effects on the photodamaged retina and might be recommended as a photoprotector and a pharmaceutical preparation for the treatment of AMD in combination with conventional medicines.     

Receptive Fields of Visually Sensitive Neurons of the Extrastriate Associative Area 21b of the Cat Cerebral Cortex

Organization of the receptive fields (RFs) of neurons of the extrastriate associative region 21b of the cerebral cortex was studied in cats. Most neurons under study (63%) were “monocular,” while 37% of the cells were “binocular” units. Among 178 neurons examined in detail, heterogeneous RF functional organization was typical of about 76% of the units; point-to-point testing of the entire RF area by stationary stimuli resulted in the generation of various types of responses (on, off, or on-off). The rest of the neurons (24%) generated homogeneous responses. The dimension, form, and functional organization of RFs of the neurons under study depended to a certain extent on the parameters of visual stimuli used for the measurements. Examination of the influence of the visual space, which surrounded the RF, on responses of the neurons evoked by stimulation of the RF per se showed that darkening of the visual space adjacent to the RF inhibited neuronal responses to moving stimuli; in some cases the responses were totally suppressed. Analysis of spatial overlapping of the RF sequentially recorded in the course of each insertion of the electrode showed that the density of distribution of the overlapping RF areas of neighboring neurons with the RF of the examined neuron is irregular, and that the RF is of a mosaic nature. We hypothesize that the visual space surrounding the RF plays a significant role in the formation of responses of visually sensitive neurons to presentation of moving stimuli. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 223–234, May–June, 2005.     

Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.

The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.     

Determinants of avian malaria prevalence in mountainous Transcaucasia

Deforestation, urban development, and global climate change can lead to dramatic changes of ecological communities and increase prevalence of infectious diseases at higher latitudes and altitudes. Identification of factors responsible for the prevalence of parasites is of crucial importance to understand the dynamics of parasite distribution in a changing environment. Mountain areas are especially suitable for studies of factors governing parasite distribution and prevalence due to heterogeneity of landscapes, climatic regimes, and other biotic and abiotic conditions. We examined 903 avian blood smears collected in mountains of Transcaucasia for prevalence of Haemoproteus and Plasmodium. We found that the haemoparasites prevalence differed among bird species and localities, highlighting the environmental components affecting disease distribution. The prevalence of both Haemoproteus and Plasmodium was significantly higher in males, adults, and migratory species than in females, juveniles, and resident species. Geographic Information System (GIS) and linear regression analyses revealed that elevation and monthly average precipitation were strongly correlated with proportion of infected birds with Plasmodium, indicating that the prevalence increased with increase of monthly average temperature and elevation. Birds from forested and high grassed areas were also more infected with avian haemosporidia. Our study provides baseline data for modelling of parasites distribution under global climate change scenarios, which is of great importance for monitoring and management of communities and environment for conservation and human health.     

Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

Early Embryonic Chromosome Instability Results in Stable Mosaic Pattern in Human Tissues

The discovery of copy number variations (CNV) in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations.     

Dynamic Spatial Organization of Receptive Fields of Neurons in the 21a Cortical Area

We studied changes in the spatial parameters of receptive fields (RFs) of visually sensitive neurons in the associative area 21a of the cat cortex under conditions of presentation of moving visual stimuli. The results of experiments demonstrated that these parameters are dynamic and depend, from many aspects, on the pattern of the stimulus used for their estimation. Angular lengths of the horizontal and vertical axes of the RFs measured in the case of movement of the visual stimuli exceeded many times those determined by presentation of stationary blinking stimuli. As is supposed, a visual stimulus, when moving along the field of vision, activates a certain number of the neurons synaptically connected with the examined cell and possessing RFs localized along the movement trajectory. As a result, such integrated activity of the neuronal group can change the excitation threshold and discharge frequency of the studied neuron. It seems probable that correlated directed activation of the neuronal groups represents a significant neurophysiological mechanism providing dynamic modifications of the RF parameters of visually sensitive neurons in the course of processes of visual perception and identification of moving objects within the field of vision.     

Lysophosphatidic acid and lipopolysaccharide bind to the PIP2-binding domain of gelsolin

The binding of the gelsolin P2 peptide (residues 150-169) with lysophosphatidic acid (LPA) and lipopolysaccharide (LPS) was investigated by isothermal titration calorimetry. P2 binds to LPS with higher affinity than to LPA. For the interaction of 1-oleoyl-LPA with P2 in the absence of salt, K(d) and deltaH degrees were 920 nM and -2.07 kcal/mol, respectively, at pH 7.4 and 25 degrees C. For the interaction of lipopolysaccharide (LPS) from P. aeruginosa with P2 under the same conditions, K(d) was 177 nM and deltaH degrees was -7.6 kcal/mol.