Effects of cesium on in vitro myoblast differentiation: An electron microscopic study

Summary This paper describes the microscopic evidence supporting a cesium-induced delay in the fusion of chick embryo myoblast membranes during in vitro myogenic differentiation. We have recently demonstrated that the sharp decrease in the conductivity and permittivity of the membranes of these myogenic cells at the time of fusion is delayed 30 h by the addition of cesium to the culture medium (Santini et al., Biochim. Biophys. Acta 945:56–64; 1988). We report here that this delay in fusion is substantiated by direct microscopic observation and that cesium also induces ultrastructural changes in the myoblast cells themselves. Possible mechanisms by which cesium may cause both the delay in fusion as well as the ultrastructural changes observed are discussed. This investigation was partially supported by an Italian Consiglio Nazionale delle Ricerche grant 85.00.304.02 (to P. L. I.).     

Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8+ Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8+ CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines

Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4⁺ and CD8⁺ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4⁺ and CD8⁺ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8⁺ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4⁺ and CD8⁺ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.Human papillomavirus (HPV) infection represents the most important risk factor for the development of cervical cancer. Although more than 100 distinct HPV genotypes have been described, and at least 20 are associated with cervical cancer, HPV type 16 (HPV-16) is by far the most frequently detected in cervical neoplasia regardless of the geographical origin of the patients (4). In the last few years significant advances have been made in the development of candidate prophylactic vaccine against cervical cancer and HPV-related infections. In several large prospective randomized studies, virus-like particles consisting of the HPV-16 and HPV-18 major capsid protein L1 (L1-VLPs) have shown promise in protecting young healthy females against persistent infection with HPV-16 and HPV-18 and their associated cervical intraepithelial neoplasia (reviewed in reference 12). These data strongly suggest that the implementation of large-scale L1-VLP-based prophylactic vaccinations have the potential to dramatically reduce worldwide cervical cancer rates in the years to come.Unfortunately, because HPV infection is endemic in humans and there is a long latency from HPV infection to the development of invasive cervical cancer in women, even if prophylactic L1-based vaccinations are implemented on a worldwide scale today it would take decades to perceive any significant benefit. Consistent with this view, an estimated 5 million cervical cancer deaths will occur in the next 20 years due to existing HPV infections (4, 12). Thus, the current development of therapeutic vaccines for protection against persistent HPV infections, cervical cancer, and its precursor lesions remains an area of great interest.Although the interactions between the host immune system and HPV-infected cells are still not completely understood, several lines of evidence suggest that protection against HPV-related infections by L1-VLP-based vaccines is likely conferred by the generation of high levels of neutralizing antibodies (12, 38). Nevertheless, a potential crucial role of L1-specific T-cell responses and the involvement of T cells in mediating the production of neutralizing antibodies and antiviral effect in infected hosts has been previously hypothesized (8, 24). This point may be particularly noteworthy in patients harboring HPV-infected cervical lesions because several studies have demonstrated the critical importance of both cytotoxic (CD8⁺) and helper (CD4⁺) T cells in achieving clinical responses (1, 5, 16-18, 20, 23). However, limited information is currently available to evaluate whether cell-mediated immune responses to L1-VLP may have any significant therapeutic effect in cervical cancer patients harboring HPV-16 positive tumors. Furthermore, to our knowledge, no direct comparison of the therapeutic efficacy of L1 and E7-specific immune responses against naturally HPV-16-infected cervical cancer have been yet reported in human patients.In the present study we have analyzed and quantified by highly sensitive real-time PCR (RT-PCR) the RNA levels of E6, E7, and L1 in flash-frozen biopsy specimens obtained from HPV-16-infected cervical carcinomas and in short- and long-term primary cultures of HPV-16-positive cervical tumors. In addition, we have studied the kinetics of expression of these genes and proteins during the establishment of HPV-16-positive primary tumors in vitro. Finally, using completely autologous systems of naturally infected HPV-16-positive human tumors, we have carefully studied the phenotype and function of L1-specific CD4⁺ and CD8⁺ T-lymphocyte responses generated by VLP-loaded dendritic cells (DCs) and compared their therapeutic potential to those elicited by DC loaded with the E7 oncoprotein.     

Sarcopenic obesity and inflammation in the InCHIANTI study.

The aging process is often paralleled by decreases in muscle and increases in fat mass. At the extreme these two processes lead to a condition known as "sarcopenic obesity" (Roubenoff R. Ann NY Acad Sci 904: 553-557, 2000). Research suggests that inflammatory cytokines produced by adipose tissue, especially visceral fat, accelerate muscle catabolism and thus contribute to the vicious cycle that initiates and sustains sarcopenic obesity. We tested the hypothesis that obesity and poor muscle strength, hallmarks of sarcopenic obesity, are associated with high circulating levels of proinflammatory cytokines in a random sample of the residents of two municipalities in the Chianti geographic area (Tuscany, Italy). The study sample consisted of 378 men and 493 women 65 yr and older with complete data on anthropometrics, handgrip strength, and inflammatory markers. Participants were cross-classified according to sex-specific tertiles of waist circumference and grip strength and according to a cut point for obesity of body mass index > or =30 kg/m(2). After adjusting for age, sex, education, smoking history, physical activity, and history of comorbid diseases, components of sarcopenic obesity were associated with elevated levels of IL-6, C-reactive protein, IL-1 receptor antagonist, and soluble IL-6 receptor (P < 0.05). Our findings suggest that global obesity and, to a greater extent, central obesity directly affect inflammation, which in turn negatively affects muscle strength, contributing to the development and progression of sarcopenic obesity. These results suggest that proinflammatory cytokines may be critical in both the development and progression of sarcopenic obesity.     

Roles of Long-range Electrostatic Domain Interactions and K+ in Phosphoenzyme Transition of Ca2+-ATPase

Sarcoplasmic reticulum Ca²⁺-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca²⁺ transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K⁺ is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.     

Indole derivatives as dual-effective agents for the treatment of neurodegenerative diseases: Synthesis,biological evaluation,and molecular modeling studies

Several indole derivatives, that were highly potent ligands of GluN2B-subunit-containing N-methyl-d-aspartate (NMDA) receptor, also demonstrated antioxidant properties in ABTS method. In particular, the 2-(4-benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethanone (1) proved to be a dual-effective neuroprotective agent. With the aim to increase the antioxidant properties we added a catechol moiety onto piperidine moiety. The designed hybrid derivative 3,4-dihydroxy-N-[1-[2-(5-hydroxy-1H-indol-3-yl)-2-oxoethyl]piperidin-4-yl]benzamide (10) was the most effective antioxidant agent (>94.1 ± 0.1% of inhibition at 17 μM) and showed GluN2B/NMDA receptor affinity at low micromolar concentration (IC₅₀ 0.66 μM). By means of computational studies we explored the effect of the presence of this antioxidant fragment during the recognition process to binding pocket.     

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.     

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.