A split-GFP tool reveals differences in the sub-mitochondrial distribution of wt and mutant alpha-synuclein

Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by dopaminergic neuronal loss that initiates in the substantia nigra pars compacta and by the formation of intracellular inclusions mainly constituted by aberrant \u3b1-synuclein (\u3b1-syn) deposits known as Lewy bodies. Most cases of PD are sporadic, but about 10% are familial, among them those caused by mutations in SNCA gene have an autosomal dominant transmission. SNCA encodes \u3b1-syn, a small 140-amino acids protein that, under physiological conditions, is mainly localized at the presynaptic terminals. It is prevalently cytosolic, but its presence has been reported in the nucleus, in the mitochondria and, more recently, in the mitochondria-associated ER membranes (MAMs). Whether different cellular localizations may reflect specific \u3b1-syn activities is presently unclear and its action at mitochondrial level is still a matter of debate. Mounting evidence supports a role for \u3b1-syn in several mitochondria-derived activities, among which maintenance of mitochondrial morphology and modulation of complex I and ATP synthase activity. \u3b1-syn has been proposed to localize at the outer membrane (OMM), in the intermembrane space (IMS), at the inner membrane (IMM) and in the mitochondrial matrix, but a clear and comparative analysis of the sub-mitochondrial localization of WT and mutant \u3b1-syn is missing. Furthermore, the reasons for this spread sub-mitochondrial localization under physiological and pathological circumstances remain elusive. In this context, we decided to selectively monitor the sub-mitochondrial distribution of the WT and PD-related \u3b1-syn mutants A53T and A30P by taking advantage from a bimolecular fluorescence complementation (BiFC) approach. We also investigated whether cell stress could trigger \u3b1-syn translocation within the different mitochondrial sub-compartments and whether PD-related mutations could impinge on it. Interestingly, the artificial targeting of \u3b1-syn WT (but not of the mutants) to the mitochondrial matrix impacts on ATP production, suggesting a potential role within this compartment

HSP60 activity on human bronchial epithelial cells

HSP60 has been implicated in chronic inflammatory disease pathogenesis, including chronic obstructive pulmonary disease (COPD), but the mechanisms by which this chaperonin would act are poorly understood. A number of studies suggest a role for extracellular HSP60, since it can be secreted from cells and bind Toll-like receptors; however, the effects of this stimulation have never been extensively studied. We investigated the effects (pro- or anti-inflammatory) of HSP60 in human bronchial epithelial cells (16-HBE) alone and in comparison with oxidative, inflammatory, or bacterial challenges. 16-HBE cells were cultured for 1-4 h in the absence or presence of HSP60, H2O2, lipopolysaccharide (LPS), or cytomix. The cell response was evaluated by measuring the expression of IL-8 and IL-10, respectively, pro- and anti-inflammatory cytokines involved in COPD pathogenesis, as well as of pertinent TLR-4 pathway mediators. Stimulation with HSP60 up-regulated IL-8 at mRNA and protein levels and down-regulated IL-10 mRNA and protein. Likewise, CREB1 mRNA was up-regulated. H2O2 and LPS up-regulated IL-8. Experiments with an inhibitor for p38 showed that this mitogen-activated protein kinase could be involved in the HSP60-mediated pro-inflammatory effects. HSP60 showed pro-inflammatory properties in bronchial epithelial cells mediated by activation of TLR-4-related molecules. The results should prompt further studies on more complex ex-vivo or in-vivo models with the aim to elucidate further the role of those molecules in the pathogenesis of COPD

Exceptionally potent human monoclonal antibodies are effective for prophylaxis and therapy of tetanus in mice

Human monoclonal antibodies were used here to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate them as a safe preventive and therapeutic substitute of hyperimmune sera for tetanus in mice. By screening memory B cells of immune donors, we selected two monoclonal antibodies specific for tetanus neurotoxin with exceptionally high neutralizing activities, which were extensively characterized both structurally and functionally. We found that these antibodies interfere with the binding and translocation of the neurotoxin into neurons by interacting with two epitopes, whose definition pinpoints crucial events in the cellular pathogenesis of tetanus. This information explains the unprecedented neutralization ability of these antibodies, which were found to be exceptionally potent in preventing experimental tetanus when injected in mice long before the neurotoxin. Moreover, their Fab derivatives neutralized tetanus neurotoxin in post-exposure experiments, suggesting their potential therapeutic use via intrathecal injection. As such, these human monoclonal antibodies, as well as their Fab derivatives, meet all requirements for being considered for prophylaxis and therapy of human tetanus and are ready for clinical trials

Phospho-p38 MAPK expression in COPD patients and asthmatics and in challenged bronchial epithelium

Background: The role of mitogen-activated protein kinases (MAPK) in regulating the inflammatory response in the airways of patients with chronic obstructive pulmonary disease (COPD) and asthmatic patients is unclear. Objectives: To investigate the expression of activated MAPK in lungs of COPD patients and in bronchial biopsies of asthmatic patients and to study MAPK expression in bronchial epithelial cells in response to oxidative and inflammatory stimuli. Methods: Immunohistochemical expression of phospho (p)-p38 MAPK, p-JNK1 and p-ERK1/2 was measured in bronchial mucosa in patients with mild/moderate (n = 17), severe/very severe (n = 16) stable COPD, control smokers (n = 16), control non-smokers (n = 9), in mild asthma (n = 9) and in peripheral airways from COPD patients (n = 15) and control smokers (n = 15). Interleukin (IL)-8 and MAPK mRNA was measured in stimulated 16HBE cells. Results: No significant differences in p-p38 MAPK, p-JNK or p-ERK1/2 expression were seen in bronchial biopsies and peripheral airways between COPD and control subjects. Asthmatics showed increased submucosal p-p38 MAPK expression compared to COPD patients (p 2O2), cytomix (tumour necrosis factor-\u3b1 + IL-1\u3b2 + interferon-\u3b3) and lipopolysaccharide (LPS) upregulated IL-8 mRNA at 1 or 2 h. p38 MAPK\u3b1 mRNA was significantly increased after H2O2 and LPS treatment. JNK1 and ERK1 mRNA were unchanged after H2O2, cytomix or LPS treatments. Conclusion: p-p38 MAPK expression is similar in stable COPD and control subjects but increased in the bronchi of mild asthmatics compared to stable COPD patients. p38 MAPK mRNA is increased after bronchial epithelial challenges in vitro. These data together suggest a potential role for this MAPK in Th2 inflammation and possibly during COPD exacerbations

Impaired Mitochondrial ATP Production Downregulates Wnt Signaling via ER Stress Induction

Wnt signaling affects fundamental development pathways and, if aberrantly activated, promotes the development of cancers. Wnt signaling is modulated by different factors, but whether the mitochondria! energetic state affects Wnt signaling is unknown. Here, we show that sublethal concentrations of different compounds that decrease mitochondrial ATP production specifically downregulate Wnt/beta-catenin signaling in vitro in colon cancer cells and in vivo in zebrafish reporter lines. Accordingly, fibroblasts from a GRACILE syndrome patient and a generated zebrafish model lead to reduced Wnt signaling. We identify a mitochondria-Wnt signaling axis whereby a decrease in mitochondria! ATP reduces calcium uptake into the endoplasmic reticulum (ER), leading to endoplasmic reticulum stress and to impaired Wnt signaling. In turn, the recovery of the ATP level or the inhibition of endoplasmic reticulum stress restores Wnt activity. These findings reveal a mechanism that links mitochondria! energetic metabolism to the control of the Wnt pathway that may be beneficial against several pathologie

Impaired Mitochondrial ATP Production Downregulates Wnt Signaling via ER Stress Induction

Wnt signaling affects fundamental development pathways and, if aberrantly activated, promotes the development of cancers. Wnt signaling is modulated by different factors, but whether the mitochondria! energetic state affects Wnt signaling is unknown. Here, we show that sublethal concentrations of different compounds that decrease mitochondrial ATP production specifically downregulate Wnt/beta-catenin signaling in vitro in colon cancer cells and in vivo in zebrafish reporter lines. Accordingly, fibroblasts from a GRACILE syndrome patient and a generated zebrafish model lead to reduced Wnt signaling. We identify a mitochondria-Wnt signaling axis whereby a decrease in mitochondria! ATP reduces calcium uptake into the endoplasmic reticulum (ER), leading to endoplasmic reticulum stress and to impaired Wnt signaling. In turn, the recovery of the ATP level or the inhibition of endoplasmic reticulum stress restores Wnt activity. These findings reveal a mechanism that links mitochondria! energetic metabolism to the control of the Wnt pathway that may be beneficial against several pathologie

Notulae to the Italian flora of algae, bryophytes, fungi and lichens: 7

In this contribution, new data concerning algae, bryophytes, fungi, and lichens of the Italian flora are presented. It includes new records and confirmations for the algae genus Chara, the bryophyte genera Cephalozia, Conardia, Conocephalum, Didymodon, Sphagnum, Tetraplodon, and Tortula, the fungal genera Endophyllum, Gymnosporangium, Microbotryum, Phragmidium, and Pluteus, and the lichen genera Candelariella, Cladonia, Flavoplaca, Lichenothelia, Peltigera, Placolecis, Rinodina, Scytinium, and Solenopsora