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The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the target cells, which activates the Ca 2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca 2+ ] i and NO production. The current study assessed whether and how glutamate drives Ca 2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca 2+ ] i , which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca 2+ oscillations were triggered by rhythmic endogenous Ca 2+ mobilization and maintained over time by extracellular Ca 2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca 2+ release was mediated by InsP 3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca 2+ entry mediated Ca 2+ entry during ongoing Ca 2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca 2+ signals. Of note, glutamate induced Ca 2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca 2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.

miR-2861 endorsing osteoblast differentiation through the overexpression of Runt-related transcription factor 2 (RUNX2) protein has been recently described. In this study we evaluated: the performance of living construct, composed by human Periodontal Ligament Stem Cells (hPDLSCs) and 3D scaffold (EXg), and the behaviour of miR-2861/RUNX2 expression pathway on the osteogenic commitment. Human PDLSCs were seeded with and without EXg scaffold and cultured under basal and osteogenic conditions. Morphological features, adhesiveness and differentiation abilities were analysed using scanning electron and confocal laser scanning microscopy. Time-course of RUNX2, ALP, OPN and miR-2861 were evaluated through RT-PCR analysis. Our results highlighted that the osteogenic differentiation was mostly obvious in the hPDLSCs, grown onto 3D scaffold in presence of osteoinductive medium. Moreover, the overexpression of miR-2861 and RUNX2 in hPDLSCs cultured in presence of EXg under osteogenic and standard conditions was demonstrated. In synthesis, the increased expression of miR-2861/RUNX2 provides new insights regarding miRNA signaling network in the presence of scaffold providing an additional method to evaluate the performance of biomaterial in bone regeneration.

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2–activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2–activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2–activated NK cells. Moreover, IL-2–induced interferon-γ production appears also to be dependent, at least in part, on iNOS induction.

Background The aim of the study was to identify p53 gene mutations by FAMA (fluorescence-assisted mismatch analysis) in colorectal cancers. Patients and methods Analytical scanning of the p53 gene (exons 5–9) was performed in colon cancer samples from 44 consecutive patients by FAMA. FAMA is a semiautomatic scanning approach based on the chemical cleavage of the mismatch in fluorescently labeled heteroduplex DNA, obtained from the combination of a normal and a mutated allele. FAMA has already shown optimal levels of diagnostic accuracy and sensitivity in detecting gene mutations (nucleotide substitutions, insertions/deletions) both at the germline and somatic level. The peculiar feature of FAMA is its ability to detect and localize mutations, by a redundant pattern of signals due to fluorescent DNA fragments generated by chemical cleavage. Moreover, previous data have demonstrated that normal contaminating DNA from stromal cells in the sample does not affect the sensitivity of the procedure, leading to the identification of the mutation even when the ratio mutant/normal allele is 10%. Results Eighteen mutations (12 missense, one nonsense, two deletions, three nucleotide substitutions at the level of the splice-junctions) and two polymorphisms were detected by FAMA in 17 patients (39%) and then confirmed by automated sequence analysis. Six of 18 mutations (33%) were not previously reported for colon cancer samples and two of 18 lesions (11%) were identified as novel p53 mutations. Conclusions Analytical scanning of the p53 gene by FAMA in DNA from colon cancer samples provides a sensitive, accurate and specific diagnostic procedure for routine clinical application.

CONTEXT: Myocardial infarction (MI) and ischemic stroke are thought to be caused by matrix digestion by metalloproteinases (MMPs) leading to rupture of atherosclerotic plaques. Production of macrophage MMP-2 and MMP-9 is induced by cyclooxygenase 2 (COX-2) and prostaglandin E(2) synthesis. Although COX-2 expression may be genetically determined, the relation between COX-2 polymorphisms and the risk of MI and stroke is unclear. OBJECTIVE: To investigate the relationship between the -765G-->C polymorphism of the COX-2 gene and clinically evident plaque rupture. DESIGN, SETTING, AND PARTICIPANTS: Prospective, matched case-control study conducted between March 2002 and October 2003 among 864 patients with first MI or atherothrombotic ischemic stroke and 864 hospitalized controls. The groups were matched for age, sex, body mass index, smoking, hypertension, hypercholesterolemia, and diabetes. The -765G-->C variant of the COX-2 gene was genotyped by restriction endonuclease digestion of polymerase chain reaction products. MAIN OUTCOME MEASURES: Presence of the -765G-->C polymorphism of the COX-2 gene; COX-2, MMP-2, and MMP-9 expression and activity in plaques and in peripheral monocytes; urinary 6-keto PGF1alpha (marker of endothelial prostacyclin); and endothelium-dependent and -independent forearm blood flow vasodilation. RESULTS: The prevalence of -765GC was 2.41 times higher among controls than among cases (43.3% vs 17.9%; PC polymorphism did not affect endothelial prostacyclin biosynthesis or endothelium-dependent vasodilation in vivo. In subgroup analyses (n = 224 cases), serum high-sensitivity C-reactive protein was significantly lower in patients carrying the -765C allele (mean [SD], 0.78 [0.1] vs 2.56 [0.4] mg/L; P =.04). CONCLUSIONS: We found that the -765G-->C polymorphism of the COX-2 gene is associated with a decreased risk of MI and stroke. Detection of this genotype may be useful for predicting genetic risk of MI and stroke.

The occurrence of mutations in the p53 tumor suppressor gene is a specific and recurring genetic event in solid tumors. P53 plays a pivotal role in multiple cellular processes such as cell growth control, DNA repair and programmed cell death. Genotoxic damage, also induced by chemotherapy or radiotherapy, induces p53 overexpression in order to control the rate of proliferating damaged cells, thus triggering the mismatch repair or apoptotic pathways. P53 inactivation determines a condition of genetic instability, justifying the subsequent susceptibility to acquire mutations of different other genes. P53 mutations are associated with worse prognosis and with chemo/radioresistance, due to the inability to trigger p53-dependent programmed cell death. Molecular diagnostic strategies show 32% p53 mutations in breast cancer. The analysis of the p53 gene performed by FAMA (Fluorescence Assisted Mismatch Analysis) in high-risk breast cancer patients withor = 10 involved axillary nodes may help identify a subset of very high risk BC patients (vHR-BC) with poorer prognosis and a subset with better prognosis, potentially responsive to medical treatments. The accurate evaluation of the p53 status can predict prognosis and sensitivity to chemotherapy, thus representing the first step toward better definition of therapeutic strategies according to the molecular characterization of the individual patient.