The role of DNA damage response proteins at telomeres--an "integrative" model

Telomeres are specialized structures at chromosome ends which play the key role in chromosomal end protection. There is increasing evidence that many DNA damage response proteins are involved in telomere maintenance. For example, cells defective in DNA double strand break repair proteins including Ku, DNA-PKcs, RAD51D and the MRN (MRE11/RAD51/NBS1) complex show loss of telomere capping function. Similarly, mouse and human cells defective in ataxia telangiectasia mutated (ATM) have defective telomeres. A total of 14 mammalian DNA damage response proteins have, so far, been implicated in telomere maintenance. Recent studies indicate that three more proteins, namely BRCA1, hRad9 and PARP1 are involved in telomere maintenance. The involvement of a wide range of DNA damage response proteins at telomeres raises an important question: do telomere maintenance mechanisms constitute an integral part of DNA damage response machinery? A model termed the "integrative" model is proposed here to argue in favour of telomere maintenance being an integral part of DNA damage response. The "integrative" model is supported by the observation that a telomeric protein, TRF2, is not confined to its local telomeric environment but it migrates to the sites of DNA breakage following exposure of cells to ionizing radiation. Furthermore, even if telomeres are maintained in a non-canonical way, as in the case of Drosophila, DNA damage response proteins are still involved in telomere maintenance suggesting integration of telomere maintenance mechanisms into the DNA damage response network.     

Optimization and validation of the direct HPLC method for the determination of moxifloxacin in plasma

Moxifloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo-[3,4-b]pyridin-6-yl]-4-oxo-3-quinolinecarboxylic acid hydrochloride) is new, fourth generation fluoroquinolone with broaden spectrum of antibacterial activity. In the present work simple and rapid RP-HPLC method for the direct determination of moxifloxacin in human plasma is described. Separation of moxifloxacin from plasma components was achieved on Supelco LC-Hisep shielded hydrophobic phase column. The mobile phase consisted of acetonitrile and 0.25mol/dm(3) Na(3)PO(4) (pH 3) in a volume percent ratio (5:95, v/v) and was delivered at a rate of 1mL/min. Fluorescence detection was employed with excitation at 290nm and emission at 500nm. Ofloxacin was used as internal standard and sodium dodecylsulfate solution was used as a displacing agent. Sample preparation was simplified and involved only addition of displacing agent and internal standard and dilution with water. The separation conditions were optimized by the response surface method in two factor space, i.e. the dependence of the retention time on volume percent of acetonitrile and on pH of aqueous phase was optimized. The method was fully validated and validation parameters were: linearity range 3-1300microg/L; correlation coefficient, 0.99986; mean recovery, 92.5%; limit of quantification, 3.0microg/L and limit of detection, 1.0microg/L. Method was applied for the determination of moxifloxacin in human plasma after single or repeated oral doses of 400mg Avelox tablets. The proposed method proved to be rapid and accurate and can be successfully used in pharmacokinetic studies and routine clinical practice.     

A microsatellite-based genetic linkage map of the waterflea, Daphnia pulex: On the prospect of crustacean genomics

We describe the first genetic linkage map for Daphnia pulex using 185 microsatellite markers, including 115 new markers reported in this study. Our approach was to study the segregation of polymorphisms in 129 F2 progeny of one F1 hybrid obtained by crossing two genetically divergent lineages of Daphnia isolated from two Oregon populations. The map spanned 1206 Kosambi cM and had an average intermarker distance of 7 cM. Linkage groups ranged in size from 7 to 185 cM and contained 4 to 27 markers. The map revealed 12 linkage groups corresponding to the expected number of chromosomes and covers approximately 87% of the genome. Tests for random segregation of alleles at individual loci revealed that 21% of the markers showed significant transmission ratio distortion (primarily homozygote deficiency) likely due to markers being linked to deleterious recessive alleles. This map will become the anchor for the physical map of the Daphnia genome and will serve as a starting point for mapping single and quantitative trait loci affecting ecologically important phenotypes. By mapping 342 tentative orthologous gene pairs (Daphnia/Drosophila) into the Daphnia linkage map, we facilitate future comparative projects.     

Filamin-a and rheological properties of cultured melanoma cells

Here we report the rheological properties of cultured hsFLNa (filamin-A)-expressing (FIL+) and hsFLNa-deficient (FIL-) melanoma cells. Using magnetic twisting cytometry over a wide range of probing frequencies, and targeting either cortical or deeper cytoskeletal structures, we found that differences in stiffness of FIL+ versus FIL- cells were remarkably small. When probed through deep cytoskeletal structures, FIL+ cells were, at most, 30% stiffer than FIL- cells, whereas when probed through more peripheral cytoskeletal structures FIL- cells were not different except at very high frequencies. The loss tangent, expressed as an effective cytoskeletal temperature, was systematically greater in FIL- than FIL+ cells, but these differences were small and showed that the FIL+ cells were only slightly closer to a solidlike state. To quantify cytoskeletal remodeling, we measured spontaneous motions of beads bound to cortical cytoskeletal structures and found no difference in FIL+ versus FIL- cells. Although mechanical differences between FIL+ and FIL- cells were evident both in cortical and deeper structures, these differences were far smaller than expected based on measurements of the rheology of purified actin-filamin solutions. These findings do not rule out an important contribution of filamin to the mechanical properties of the cortical cytoskeleton, but suggest that effects of filamin in the cortex are not exerted on the length scale of the probe used here. These findings would appear to rule out any important contribution of filamin to the bulk mechanical properties of the cytoplasm, however. Although filamin is present in the cytoplasm, it may be inactive, its mechanical effects may be small compared with other crosslinkers, or mechanical properties of the matrix may be dominated by an overriding role of cytoskeletal prestress.     

Length-dependent prediction of protein intrinsic disorder

Background  

Due to the functional importance of intrinsically disordered proteins or protein regions, prediction of intrinsic protein disorder from amino acid sequence has become an area of active research as witnessed in the 6th experiment on Critical Assessment of Techniques for Protein Structure Prediction (CASP6). Since the initial work by Romero et al. (Identifying disordered regions in proteins from amino acid sequences, IEEE Int. Conf. Neural Netw., 1997), our group has developed several predictors optimized for long disordered regions (>30 residues) with prediction accuracy exceeding 85%. However, these predictors are less successful on short disordered regions (≤30 residues). A probable cause is a length-dependent amino acid compositions and sequence properties of disordered regions.     

Parasitofauna of freshwater fishes in the Serbian open waters: a checklist of parasites of freshwater fishes in Serbian open waters

As a result of freshwater fish parasitofauna investigations, throughout the past 75 years, the number of parasitic species is presented. This paper reviews the history, current state, and tendencies of the fish parasitofauna studies in Serbian open waters. Up to now, in total 170 parasitic species have been reported. Systematic parasitofauna investigations have been carried out on 54 freshwater fish species in waters of the Danube River Basin in Serbia, also in hill-mountanious watercourses, lakes and salmonid fishpond of Sjenicko-Pesterska plateau (south-west region of Serbia), Homolje area (east Serbia), and in Vlasina lake (south-east Serbia). This data review pointed that it is necessary to continue the investigation on fish parasites in Serbia. The data on freshwater fish parasites are important for evaluation of general influence on the community structure.     

Platelet dysfunction as the presenting feature of atypical myelodysplastic syndrome with monosomy 7, normal blood counts and no bleeding tendency

A 71-year-old male patient with atypical myelodysplastic syndrome showing monosomy 7 is described. He presented with severe foot pains, trophic skin and nail changes, loss of distal pulses, all compatible with peripheral arterial occlusive disease. He had completely normal blood counts and no bleeding tendency. Prolonged bleeding time was disclosed by chance, during routine haemostatic studies. An acquired platelet dysfunction was considered, with prolonged bleeding time and large platelets that failed to aggregate in response to arachidonic acid and that had impaired response to collagen and adrenaline. The bone marrow was hypercellular, with numerous dysplastic megakaryocytes and two other slightly dysplastic myeloid lines. Cytogenetic analyses of the bone marrow cells showed a mosaic karyotype: 46,XY/45,XY,-7. On angiography, bilateral thrombosis of the iliac, superficial femoral and popliteal arteries was disclosed. The patient was prepared with platelet transfusions. Arterial thrombectomy and amputation of the left calf were performed. Ten months later, his blood counts showed mild pancytopenia. He died at home. The authors discuss some clinical and pathogenetical aspects of such presentations of myelodysplastic syndromes.