Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 muM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 muM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.     

A rapid, whole-tissue determination of the functional fraction of PSII after photoinhibition of leaves based on flash-induced P700 redox kinetics

Assaying the number of functional PSII complexes by the oxygen yield from leaf tissue per saturating, single-turnover flash, assuming that each functional PSII evolves one oxygen molecule after four flashes, is one of the most direct methods but time-consuming. The ratio of variable to maximum Chl fluorescence yield (F_v/F_m) in leaves can be correlated with the oxygen yield per flash during a progressive loss of PSII activity associated with high-light stress and is rapid and non-intrusive, but suffers from being representative of chloroplasts near the measured leaf surface; consequently, the exact correlation depends on the internal leaf structure and on which leaf surface is being measured. Our results show that the average F_v/F_m of the adaxial and abaxial surfaces has a reasonable linear correlation with the oxygen yield per flash after varied extents of photoinactivation of PSII. However, we obtained an even better linear correlation between (1) the integrated, transient electron flow (Σ) to P700⁺, the dimeric Chl cation in PSI, after superimposing a single-turnover flash on steady background far-red light and (2) the relative oxygen yield per flash. Leaves of C3 and C4 plants, woody and herbaceous species, wild-type and a Chl- b -less mutant, and monocot and dicot plants gave a single straight line, which seems to be a universal relation for predicting the relative oxygen yield per flash from Σ. Measurement of Σ is non-intrusive, representative of the whole leaf tissue, rapid and applicable to attached leaves; it may even be applicable in the field.     

The anti-ageing molecule sirt1 mediates beneficial effects of cardiac rehabilitation

Background

An exercise-based Cardiac Rehabilitation Programme (CRP) is established as adjuvant therapy in heart failure (HF), nevertheless it is underutilized, especially in the elderly. While the functional and hemodynamic effects of CRP are well known, its underlying molecular mechanisms have not been fully clarified. The present study aims to evaluate the effects of a well-structured 4-week CRP in patients with stable HF from a molecular point of view.

Results

A prospective longitudinal observational study was conducted on patients consecutively admitted to cardiac rehabilitation. In fifty elderly HF patients with preserved ejection fraction (HFpEF), levels of sirtuin 1 (Sirt1) in peripheral blood mononuclear cells (PBMCs) and of its targets, the antioxidants catalase (Cat) and superoxide dismutase (SOD) in serum were measured before (Patients, P) and at the end of the CRP (Rehabilitated Patients, RP), showing a rise of their activities after rehabilitation.Endothelial cells (ECs) were conditioned with serum from P and RP, and oxidative stress was induced using hydrogen peroxide. An increase of Sirt1 and Cat activity was detected in RP-conditioned ECs in both the absence and presence of oxidative stress, together with a decrease of senescence, an effect not observed during Sirt1 and Cat inhibition.

Conclusions

In addition to the improvement in functional and hemodynamic parameters, a supervised exercise-based CRP increases Sirt1 activity and stimulates a systemic antioxidant defence in elderly HFpEF patients. Moreover, CRP produces antioxidant and anti-senescent effects in human endothelial cells mediated, at least in part, by Sirt1 and its target Cat.

     

Inhibition of Cx43 mediates protective effects on hypoxic/reoxygenated human neuroblastoma cells

Olfactory ensheathing cells (OECs), a special population of glial cells, are able to synthesise several trophic factors exerting a neuroprotective action and promoting growth and functional recovery in both in vitro and in vivo models. In the present work, we investigated the neuroprotective effects of OEC‐conditioned medium (OEC‐CM) on two different human neuron‐like cell lines, SH‐SY5Y and SK‐N‐SH (neuroblastoma cell lines), under normoxic and hypoxic conditions. In addition, we also focused our attention on the role of connexins (Cxs) in the neuroprotective processes. Our results confirmed OEC‐CM mediated neuroprotection as shown by cell adherence, proliferation and cellular viability analyses. Reduced connexin 43 (Cx43) levels in OEC‐CM compared to unconditioned cells in hypoxic conditions prompted us to investigate the role of Cx43‐Gap junctions (GJs) and Cx43‐hemichannels (HCs) in hypoxic/reoxygenation injury using carbenoxolone (non‐selective GJ inhibitor), ioxynil octanoato (selective Cx43‐GJ inhibitor) and Gap19 (selective Cx43‐HC inhibitor). We found that Cx43‐GJ and Cx43‐HC inhibitors are able to protect SH‐SY5Y and allow to these cultures to overcome the injury. Our findings support the hypothesis that both OEC‐CM and the inhibition of Cx43‐GJs and Cx43‐HCs offer a neuroprotective effect by reducing Cx43‐mediated cell‐to‐cell and cell‐to‐extracellular environment communications.     

Impaired central hemodynamic response and exaggerated vasoconstriction during muscle metaboreflex activation in heart failure patients

The muscle metaboreflex is enhanced in chronic heart failure (CHF) patients, and this fact has been associated with the early fatigue shown by these patients in response to exercise. In animal studies of CHF, it was found that the limited capacity to enhance ventricular performance is responsible for a functional shift from a cardiac output to a systemic vascular resistance (SVR) increase in the mechanism by which the cardiovascular system raises blood pressure in response to the metaboreflex. However, the existence of this functional shift is still unknown in humans. The present study was undertaken to test the hypothesis that a similar hemodynamic response was also present in humans with CHF. The hemodynamic response to metaboreflex activation obtained through postexercise ischemia was assessed in nine patients with CHF and nine healthy controls (CTL) by means of impedance cardiography. The main results were that 1) the blood pressure rise due to the metaboreflex was similar in the two groups; 2) the CTL group achieved the blood pressure response via cardiac output increase, and the CHF group, via SVR increase; and 3) stroke volume was enhanced in the CTL group and decreased in the CHF group. This study demonstrates that in CHF patients, metaboreflex recruitment causes a functional shift from flow increase to peripheral vasoconstriction in the mechanism through which blood pressure is increased. The incapacity to enhance cardiac performance and stroke volume is probably the primary cause of this cardiovascular alteration.     

Phenotypic characterization and putative virulence factors of human,animal and environmental isolates of <Emphasis Type=Italic>Plesiomonas shigelloides</Emphasis>

Plesiomonas shigelloides (a bacterium widely distributed in aquatic ecosystems causing both intestinal and extra-intestinal diseases) shows a host of putative virulence markers, such as hemolysins, cytotoxins, production of exoenzymes associated with pathogenicity, adhesive ability and vacuolation of cell lines in vitro. Technical difficulties in detecting some of these virulence factors together with scantiness of epidemiological information, due to the lack of routine analysis for P. shigelloides as etiological agent of gastroenteritis, lead to sporadic and occasional finding of these bacteria. All this casts doubt on the real virulence potential of P. shigelloides and fuels a debate about assignment of these bacteria to the list of human pathogens. Here we demonstrated the phenotypic diversity and the putative virulence markers by examining serotype biochemical and virulence properties of 60 strains of P. shigelloides isolated from human, animal and environmental samples in different countries, which showed the unpredictable occurrence of the above properties depending on various locations and diverse sources.     

Regulation of ENaC and CFTR expression with K+ channel modulators and effect on fluid absorption across alveolar epithelial cells

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027-L1037, 2004), we identified an ATP-sensitive K(+) (K(ATP)) channel in alveolar epithelial cells, formed by inwardly rectifying K(+) channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of K(ATP), voltage-dependent K(+) channel KvLQT1, and calcium-activated K(+) (K(Ca)) channel modulators modified Na(+) and Cl(-) currents in alveolar monolayers. In addition, it was shown previously that a K(ATP) opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K(+) channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of K(ATP), KvLQT1, and K(Ca) channels identified by PCR. Glibenclamide and clofilium (K(ATP) and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na(+) current, and forskolin-activated Cl(-) currents, whereas pinacidil, a K(ATP) activator, increased them. Interestingly, K(+) inhibitors or membrane depolarization (induced by valinomycin in high-K(+) medium) decreased alpha-, beta-, and gamma-ENaC and CFTR mRNA. alpha-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na(+) transport through ENaC, we tested the impact of K(+) channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K(+) channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.     

MicroRNAs in durum wheat seedlings under chronic and short-term nitrogen stress

Nitrogen is an essential macronutrient for plant growth and reproduction. In durum wheat, an appropriate nitrogen soil availability is essential for an optimal seed development. miRNAs contribute to the environmental change adaptation of plants through the regulation of important genes involved in stress processes. In this work, nitrogen stress response was evaluated in durum wheat seedlings of Ciccio and Svevo cultivars. Eight small RNA libraries from leaves and roots of chronically stressed plants were sequenced to detect conserved and novel miRNAs. A total of 294 miRNAs were identified, 7 of which were described here for the first time. The expression level of selected miRNAs and target genes was analyzed by qPCR in seedlings subjected to chronic (Ciccio and Svevo, leaves and roots) or short-term (Svevo roots) stress conditions. Some miRNAs showed an immediate stress response, and their level of expression was either maintained or returned to a basal level during a long-term stress. Other miRNAs showed a gradual up- or downregulation during the short-term stress. The newly identified miRNA ttu-novel-106 showed an immediate strongly downregulation after nitrogen stress, which was negatively correlated with the expression of MYB-A, its putative target gene. PHO2 gene was significantly upregulated after 24–48-h stress, corresponding to a downregulation of miR399b. Ttu-miR399b putative binding sites in the 5′ UTR region of the Svevo PHO2 gene were identified in the A and B genomes. Both MYB-A and PHO2 genes were validated for their cleavage site using 5′ RACE assay.