Nitric oxide and cyclic GMP are messengers in the indole acetic acid-induced adventitious rooting process

This report describes part of the signaling pathway and some of the molecules involved in the auxin-induced adventitious root formation in cucumber (Cucumis sativus). Previous results showed that nitric oxide (NO) mediates the auxin response during adventitious root formation (Pagnussat et al., 2002). To determine the order of action of indole acetic acid (IAA) and NO within the signal transduction pathway and to elucidate the target molecules that are downstream of NO action, cucumber hypocotyl cuttings were submitted to a pretreatment leading to endogenous auxin depletion. The auxin depletion treatment provoked a 3-fold reduction of the root number in comparison to the nondepleted explants. The NO-donor sodium nitroprusside was able to promote adventitious rooting in auxin-depleted explants, whereas the specific NO scavenger cPTIO prevented the effect of sodium nitroprusside. The endogenous NO level was monitored in both control and auxin-depleted explants using a NO-specific fluorescent probe. The NO level was 3.5-fold higher in control (nondepleted) explants than in auxin-depleted ones. The exogenous application of IAA restored the NO concentration to the level found in nondepleted explants. Because NO activates the enzyme guanylate cyclase (GC), we analyzed the involvement of the messenger cGMP in the adventitious root development mediated by IAA and NO. The GC inhibitor LY83583 reduced root development induced by IAA and NO, whereas the cell-permeable cGMP derivative 8-Br-cGMP reversed this effect. The endogenous level of cGMP is regulated by both the synthesis via GC and its degradation by the phosphodiesterase activity. When assayed, the phosphodiesterase inhibitor sildenafil citrate was able to induce adventitious rooting in both nondepleted and auxin-depleted explants. Results indicate that NO operates downstream of IAA promoting adventitious root development through the GC-catalyzed synthesis of cGMP.     

Differential diagnosis between cutaneous lymphoma and pseudolymphoma

Differential diagnosis of cutaneous lymphoproliferative disorders represents one of the most vexing problems in dermatology and dermatopathology. For nearly a century the diagnosis has been based only upon clinicopathologic correlation. Immunohistochemical and molecular techniques developed during the last 3 decades added new criteria for the differentiation of these diseases. The purpose of this articles is to summarize the criteria for the differential diagnosis of benign vs. malignant lymphoid infiltrates of the skin. In this context, a proper classification of cutaneous lymphoproliferative disorders can be achieved only by a synthesis of clinical, histopathologic, immunophenotypic and molecular criteria, and in some cases only follow-up data allow a precise diagnosis to be made.     

Effect of conjugation methodology on the immunogenicity and protective efficacy of meningococcal group C polysaccharide-P64k protein conjugates

Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.     

PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and -independent effects

The G protein-coupled, receptor-activated phosphoinositide 3-kinase gamma (PI3Kgamma) mediates inflammatory responses and negatively controls cardiac contractility by reducing cAMP concentration. Here, we report that mice carrying a targeted mutation in the PI3Kgamma gene causing loss of kinase activity (PI3KgammaKD/KD) display reduced inflammatory reactions but no alterations in cardiac contractility. We show that, in PI3KgammaKD/KD hearts, cAMP levels are normal and that PI3Kgamma-deficient mice but not PI3KgammaKD/KD mice develop dramatic myocardial damage after chronic pressure overload induced by transverse aortic constriction (TAC). Finally, our data indicate that PI3Kgamma is an essential component of a complex controlling PDE3B phosphodiesterase-mediated cAMP destruction. Thus, cardiac PI3Kgamma participates in two distinct signaling pathways: a kinase-dependent activity that controls PKB/Akt as well as MAPK phosphorylation and contributes to TAC-induced cardiac remodeling, and a kinase-independent activity that relies on protein interactions to regulate PDE3B activity and negatively modulates cardiac contractility.     

Cytotoxicity of copper ions released from metal

The aim of this work is to contribute to the elucidation of the cytotoxic process caused by the copper ions released from the biomaterials. Clonal cell lines UMR106 were used in the experiments. Copper ions were obtained from two different sources: copper salts and metal dissolution. Experiments carried out with constant ion concentrations (copper salts) were compared with those with concentrations that vary with time and location (dissolution of the metal). Present results and others previously reported could be interpreted through mathematical models that describe: (1) the variation of concentration of copper ions with time and location within a biofilm and (2) the variation of the killing rate with the concentration of the toxic ion and time. The large number of dead cells found near the copper sample with an average ion concentration below the toxic limit could be interpreted bearing in mind that these cells should be exposed to a local concentration higher than this limit. A logarithmic dependence between the number of cells and exposure time was found for nearly constant ion concentrations. Apparent discrepancies, observed when these results and those of different researchers were contrasted, could be explained considering the dissimilar experimental conditions such as the source of the ions and their local concentration at real time.     

Size and shape modifications in Sardinian adolescent girls

In females, menarche is the defining moment of puberty, the period of life when the greatest body changes occur. In the present study, the metric and morphological variations associated with sexual maturation are defined in 155 Sardinian girls (10-17 years) and the role of some potentially influential variables is discussed: age, age at menarche and time since menarche. We studied thirty-eight anthropometric variables, the fat-free mass and the fat mass estimated by Bioelectrical Impedance Analysis. Statistical analyses were performed to evaluate the difference between pre- and post-menarcheal girls of the same age (Student's t-test) and to evaluate the different role played by the variables (principal components analysis, cluster analysis, multiple regression). The results demonstrate that the body dimensions of the adolescent girls mainly increase in concomitance with sexual maturation. The age at menarche influences the fat mass but not the distribution of visceral and subcutaneous fat. The time since menarche has also no effect on the distribution of subcutaneous fat.     

Respiratory inhibition of isolated mammalian mitochondria by salivary antifungal peptide histatin-5

Histatin-5 is a peptide secreted in the human saliva, which possesses powerful antifungal activity. Previous studies have shown that this peptide exerts its candidacidal activity, through the inhibition of both mitochondrial respiration and the formation of reactive oxygen species. The purpose of the present study was to investigate the biological consequences of histatin-5 action on mammalian mitochondria to verify if the toxic mechanism exerted on mitochondria from Candida albicans is an exclusive for fungal cells. Moreover, hypothesising that the damage exerted on mitochondria may induce programmed cellular death pathways, we evaluated two main markers of apoptosis: the mitochondrial membrane potential (DeltaPsi) and the release of cytochrome c. The results obtained show that exposure of isolated mammalian mitochondria to histatin-5 determines: (i) a large inhibition of the respiratory chain at the level of complex I, (ii) a slight decrease in the mitochondrial membrane potential, and (iii) no release of cytochrome c.     

Cathepsin D triggers Bax activation,resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis

Activated human T lymphocytes exposed to apoptotic stimuli targeting mitochondria (i.e. staurosporine), enter an early, caspase-independent phase of commitment to apoptosis characterized by cell shrinkage and peripheral chromatin condensation. We show that during this phase, AIF is selectively released from the intermembrane space of mitochondria, and that Bax undergo conformational change, relocation to mitochondria, and insertion into the outer mitochondrial membrane, in a Bid-independent manner. We analyzed the subcellular distribution of cathepsins (Cat) B, D, and L, in a search for caspase-independent factors responsible for Bax activation and AIF release. All were translocated from lysosomes to the cytosol, in correlation with limited destabilization of the lysosomes and release of lysosomal molecules in a size selective manner. However, only inhibition of Cat D activity by pepstatin A inhibited the early apoptotic events and delayed cell death, even in the presence of bafilomycin A1, an inhibitor of vacuolar type H+-ATPase, which inhibits acidification in lysosomes. Small interfering RNA-mediated gene silencing was used to inactivate Cat D, Bax, and AIF gene expression. This allowed us to define a novel sequence of events in which Cat D triggers Bax activation, Bax induces the selective release of mitochondrial AIF, and the latter is responsible for the early apoptotic phenotype.