DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

Dasatinib Inhibits CXCR4 Signaling in Chronic Lymphocytic Leukaemia Cells and Impairs Migration Towards CXCL12

Chemokines and their ligands play a critical role in enabling chronic lymphocytic leukaemia (CLL) cells access to protective microenvironmental niches within tissues, ultimately resulting in chemoresistance and relapse: disruption of these signaling pathways has become a novel therapeutic approach in CLL. The tyrosine kinase inhibitor dasatinib inhibits migration of several cell lines from solid-organ tumours, but effects on CLL cells have not been reported. We studied the effect of clinically achievable concentrations of dasatinib on signaling induced by the chemokine CXCL12 through its'' receptor CXCR4, which is highly expressed on CLL cells. Dasatinib pre-treatment inhibited Akt and ERK phosphorylation in CLL cells upon stimulation with CXCL12. Dasatinib also significantly diminished the rapid increase in actin polymerisation observed in CLL cells following CXCL12 stimulation. Moreover, the drug significantly inhibited chemotaxis in a transwell assay, and reduced the percentage of cells able to migrate beneath a CXCL12-expressing murine stromal cell line. Dasatinib also abrogated the anti-apoptotic effect of prolonged CXCL12 stimulation on cultured CLL cells. These data suggest that dasatinib, akin to other small molecule kinase inhibitors targeting the B-cell receptor signaling pathway, may redistribute CLL cells from protective tissue niches to the peripheral blood, and support the investigation of dasatinib in combination strategies.     

Evaluation of some immune functions in a patient affected by common variable immunodeficiency using luminescent techniques

Common variable immunodeficiency is a primary immunodeficiency characterized by a failure of antibody synthesis, whose fundamental immunologic abnormality is still unknown. In our study, we evaluated some immune functions using chemiluminescence in a 32-year-old woman affected by common variable immunodeficiency. In particular, we showed an impairment of her lymphomonocyte proliferative response which was evaluated using a method based on the bioluminescent measurement of ATP. Besides, we found a reducton of her lymphomonocyte IL2 and IL4 production: the IL4 production was evaluated through an ELISA method, whereas the IL2 activity was determined by its ability to support the IL2-dependent murine T-cell line (CTLL) proliferation which was established through a method based on the bioluminescent measurement of ATP. Finally, we evaluated both yeast-induced and fMLP-induced polymorphonuclear and monocyte oxidative metabolism through a luminol-amplified chemiluminescence; these functions were within normal values. Therefore, in our patient affected by common variable immunodeficiency, we demonstrated an impairment of cellular immunity, which might contribute to the pathogenesis of the disease. © 1997 John Wiley & Sons, Ltd.     

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.     

Antinociceptive and gastroprotective effects of inhaled and orally administered Lavandula hybrida Reverchon "Grosso" essential oil

In this study the antinociceptive and the gastroprotective effects of orally administered or inhaled Lavandula hybrida Reverchon "Grosso" essential oil, and its principal constituents linalool and linalyl acetate were evaluated in rodents. Either when orally administered (100 mg/kg) or inhaled for 60 min lavender essential oil significantly reduced the acetic acid-writhing response in a naloxone-sensitive manner. In the hot plate test, analgesic activity observed after oil inhalation was inhibited by naloxone, atropine, mecamylamine pretreatment suggesting the involvement of opioidergic as well as cholinergic pathways. Regardless of the administration route and the experimental model used both linalool and linalyl acetate did not produce significant analgesic response. Oral or inhalatory treatment with analgesic doses of essential oil did not affect mice spontaneous locomotor activity. Concerning the gastric effects, lavender oil, linalool and linalyl acetate oral administration protected against acute ethanol-induced gastric ulcers but did not prevent indomethacin-induced lesions indicating no interference with arachidonic acid metabolic cascade. In conclusion, besides this gastroprotection, lavender oil reveals an interesting analgesic activity mainly relevant after inhalation, at doses devoid of sedative side effect, suggesting the interest for potential application of this oil in aromatherapy.     

Interaction between opiates and hypothalamic dopamine on prolactin release.

Opiate stimulation of prolactin (PRL) release appears to involve a hypothalamic mechanism(s). The present study utilized both central acting drugs and direct measurement of hypothalamic dopamine (DA) to investigate this problem. Administration of L-dopa, the precursor of DA; piribedil, a DA agonist; or amineptine, a DA reuptake inhibitor, each decreased serum PRL concentrations. Morphine sulfate (MS) and haloperidol (HAL) significantly increased serum PRL levels. L-dopa and piribedil reversed the stimulatory effect of MS on serum PRL concentrations by increasing dopamine activity. MS blocked the inhibitory effects of amineptine on serum PRL release, possibly by decreasing the concentration of DA available for reuptake. Injection of subeffective doses of HAL concurrently with a subeffective dose of MS increased serum PRL concentrations, by an additive inhibitory action on dopaminergic activity. β-endorphin, an endogenous opioid peptide, decreased the rate of DA turnover in the median eminence, and increased serum PRL levels approximately 10 - fold. These observations indicate that opiates stimulate PRL release by decreasing DA activity in the median eminence.     

Intergenic spacers of rRNA genes in three species of theCynareae (Asteraceae)

Intergenic spacers of the rRNA genes of three species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Onopordum acanthium, andO. illyricum were cloned in the plasmid pGEM-7zf(+). Detailed restriction mapping and partial sequencing of the IGSs were carried out. The structural analysis showed a clear diversity betweenCynara andOnopordum, while a high degree of homology was found between the twoOnopordum spp. In all three species a fragment of about 450 bp from the 5 end of 18S to the Acc I site with a high sequence homology was present. Nucleotide sequences upstream from the above mentioned Acc I site show a gradual decrease of homology betweenCynara andOnopordum.     

TGFbeta protects mesoangioblasts from apoptosis via sphingosine kinase-1 regulation

Mesoangioblasts are vessel-derived progenitor cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. Here we examined the role of transforming growth factor-beta (TGFbeta), a pleiotropic cytokine that plays a major role in development and specifically induces smooth muscle differentiation of mesoangioblasts, in the regulation of death and survival of these cells. TGFbeta exerts a marked anti-apoptotic action in mesoangioblasts with a mechanism involving regulation of sphingosine kinase 1 (SphK1), one of the isoforms responsible for S1P formation. Treatment with the cytokine efficaciously protected mesoangioblasts from apoptosis induced by serum starvation or staurosporine treatment assessed by various means such as activation of caspase-3, determination of cytoplasmic histone-associated-DNA-fragments and PE-AnnexinV staining. The protective action of TGFbeta from staurosporine-induced apoptosis was strongly reduced when the SphK activity was inhibited by drugs, when SphK1 but not SphK2 was downregulated by specific siRNA and when a SphK1 dominant negative mutant was overexpressed. Staurosporine treatment induced down-regulation of both SphK isoforms and TGFbeta rescued SphK1 but not SphK2 expression. Interestingly, TGFbeta strongly enhanced SphK activity during staurosporine-induced cell death. Both TGFbeta-induced SphK1 up-regulation and TGFbeta anti-apoptotic action were found to be dependent on p42/44 MAPK activation.     

Synthesis and BZR affinity of pyrazolo[1,5-a]pyrimidine derivatives. Part 1: study of the structural features for BZR recognition

Examination of the pharmacophoric points of the pyrazolo[1,5-a]pyrimidine derivatives, ligands for BZR, previously published led us to the design of a novel class of 3,6-diaryl-4,7-dihydro-pyrazolo[1,5-a]pyrimidin-7-ones and to determine the groups involved in the BZR recognition.