Molecular characterization of the first satellite DNA with CENP-B and CDEIII motifs in the bat Pipistrellus kuhli

The centromere is an essential structure in the chromosomes of all eukariotes and is central to the mechanism that ensures proper segregation during mitosis and meiosis. The comparison of DNA sequence motifs, organization and kinetocore components from yeast to man is beginning to indicate that, although centromeres are highly variable DNA elements, a conserved pattern of sequence arrangement and function is emerging. We have identified and characterized the first satellite DNA (P.k.SAT) from microbat species Pipistrellus kuhli. The presence of mammalian CENP-B box and yeast CDEIII box could indicate the participation of P.k.SAT in centromere organization.     

The application of oligonucleotide probes and microarrays for the identification of freshwater diatoms

Diatoms are major contributors to global carbon fixation and constitute a significant portion of biofilms found in lotic ecosystems. Despite their widespread abundance and the fact that extensive studies have been performed on morphological features of frustules, molecular tools for the identification of diatoms are not commonly available. This study focuses on the development of oligonucleotide probes for the detection of diatom species relevant to water quality assessment. The selected panel of diatoms covers all the species found in water of varying quality from the rivers of central-East Apennine (Italy). Small subunit rRNA-targeted probes were applied to a microarray platform as well as to a new technique termed Primer–Probe, with the aim of obtaining a molecular tool suitable for accurate identification of both single and mixed species diatom populations. The Primer–Probe technique together with dot-blot assays proved to be ideal for the preliminary screening of a large set of DNA oligonucleotides designed by ARB software. It was shown that microarrays, as a promising technology for rapid and simultaneous detection of a wide range of species-specific genetic markers, can be adapted to monitor changes within a diatom community. It is suggested that microarrays will provide a molecular basis for microbial identification to support standard microscopy techniques used by ecologists and environmental scientists for monitoring water quality.     

Metabolic complications of HIV-1 antiretroviral therapy: the lipodystrophy syndrome

The lipodystrophy syndrome is one of the complications reported with increased frequency in patients with HIV-1 infection receiving antiretroviral therapy. The wide range of prevalence estimates may be due to differing definitions, methods and patient populations. We described the various pathogenic theories and the morphological and metabolic alterations associated with this syndrome. Even if no effective treatment exists, a correct lifestyle, adequate diet and physical exercise seem to be very important. Moreover drug therapies should be used with care to avoid potentially harmful interactions with antiretroviral agents. Ideally, the future effort to define the mechanism of lipodystrophy would be multidisciplinary and would involve not only experts in AIDS research but also nutritionists, endocrinologists and cardiologists.     

Block of mitochondrial apoptotic pathways in lizard ovarian follicle cells as an adaptation to their nurse function

Pyriforms are ovarian follicle nurse cells that undergo apoptosis at the end of previtellogenesis and are completely eliminated by the epithelium. This event is accompanied by the active transfer of organelles and macromolecules to the oocyte via an intercellular bridge. Since it would be a nonsense for damaged mitochondria to reach the oocyte, we have postulated that pyriform cells have adapted their apoptotic machinery to prevent mitochondrial degradation. To verify this hypothesis, we have studied mitochondrial morphology and functionality during follicle cell regression. Cytological and biochemical evidence indicates that mitochondria in pyriforms maintain their size, organization and membrane potential. This clearly indicates that they are not involved in apoptosis signalling/progression. This block would favour both the oocyte, by increasing the pool of organelles available from follicle cells, and also the regressing pyriforms, by maintaining the energy resources required for completion of their nurse function. The block is probably attributable to an over-expression of Bcl-2 and might be carried out by sequestering cytochrome c inside the organelles. As demonstrated by in vitro experiments, the mitochondrial apoptosis pathway can be activated by stress induction, such as serum deprivation, but not following physiological pro-apoptotic signalling, such as treatment with gonadotrophin-releasing hormone. These studies were supported by a grant from the MIUR (PRIN project: Molecular responses of embryonic, differentiated and tumoral cells exposed to cadmium intoxication).     

Nuclear Organization in the Spinal Cord Depends on Motor Neuron Lamination Orchestrated by Catenin and Afadin Function

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Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.     

CXC chemokine receptor 4 is expressed in uveal malignant melanoma and correlates with the epithelioid-mixed cell type

PURPOSE: Although relatively rare, uveal melanoma is the most common ocular tumor of adults. Up to half of uveal melanoma patients die of metastatic disease. CXCR4, a chemokine receptor, is a prognostic factor in cutaneous melanoma involved in angiogenesis and metastasis formation. The aim of this study was to evaluate the expression of CXCR4 in uveal melanoma. METHODS: CXCR4 was detected by immunohistochemistry in 44 samples of uveal melanoma. Staining was categorized into three semiquantitative classes based on the rate of stained (positive) tumor cells: absence of staining, <50% of cell (+) and >50% (++). Correlations between CXCR4 expression, data on patient and tumor features were studied by contingency tables and the chi2 test. Time-to-event curves were studied using the Kaplan-Meier method. Univariate analysis was performed using the log-rank test. Ninety-five percent confidence intervals (95% CI) of hazard ratios were also reported. RESULTS: Staining for CXCR4 protein was absent in 18 tumors (40.9%), present in <50% of cells in 19 (43.2%) and in >50% of cells in 7 (15.9%) tumors. CXCR4 expression correlated to the epithelioid-mixed cell type (P=0.030). No statistically significant relation emerged between CXCR4 expression, largest tumor diameter (LTD) and extracellular matrix patterns as evaluated through histological patterns stained with periodic acid-Schiff (PAS). Events occurred in 2 out of 18 patients (11.1%) with negative tumors (2 deaths), in 3 out of 19 patients (15.8%) with <50% of positive tumor cells (2 deaths and 1 occurrence of metastases) and in 1 out of 7 patients (14.3%) with >50% of positive tumor cells (1 occurrence of metastases). The cell type (P=0.0457) but not CXCR4 showed prognostic value at univariate analysis. CONCLUSION: This study shows that CXCR4 is commonly expressed in uveal melanoma and correlates with cell type a well-established prognostic factor.     

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: Dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors—Part II

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones have been identified as highly potent 5-HT_1A/B/D receptor antagonists with and without additional SerT activity and a high degree of selectivity over hERG potassium channels. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.