Alterations in capsaicin-evoked electrolyte transport during the evolution of guinea pig TNBS ileitis

The efferent secretomotor activity of capsaicin-sensitive nerves was monitored during the evolution of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis in the guinea pig by recording changes in short-circuit current (DeltaI(sc)) in response to capsaicin, substance P (SP), and carbachol. Submucosal-mucosal preparations mounted in standard Ussing chambers were studied at time 0, at 8 h, and 1, 3, 5, 7, 14, and 30 days following the intraluminal instillation of TNBS or saline. Maximal DeltaI(sc) responses to capsaicin were dramatically attenuated (54%) by 24 h. By day 7, SP- and TTX-insensitive carbachol-stimulated DeltaI(sc) were also significantly reduced. Similar attenuation in capsaicin and carbachol responses was observed in jejunal tissue 20 cm proximal to the inflamed site at day 7. These studies demonstrate that efferent secretomotor function of capsaicin-sensitive nerves is maintained early in TNBS ileitis but significantly reduced by 24 h. By day 7, defects in enterocyte secretory function at inflamed and noninflamed sites also occurred, an effect that may be mediated by circulating cytokines.     

Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases

Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn’s disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account.     

Differentially expressed cDNAs in Alternaria alternata treated with 2-propenyl isothiocyanate

The molecular mechanism of the fungal tolerance phenotype to fungicides is not completely understood. This knowledge would allow for the development of environmentally friendly strategies for the control of fungal infection. With the goal of determining genes induced by 2p-ITC, a forward suppressive subtractive hybridization was performed using cDNAs from ITC-treated Alternaria alternata as a “tester” and from untreated fungus as a “driver.” Using this approach, a library containing 102 ESTs was generated that resulted in 50 sequences after sequence assembly (17 contigs and 33 singletons). Blastx analysis revealed that 38% and 40% of the sequences showed significant similarity with known and hypothetical proteins, respectively, whereas 18% were not similar to known genes. These last sequences could represent novel genes that play an unknown role in the molecular responses of fungi during adaptation to 2p-ITC. Clones similar to opsins, ABC transporters, calmodulin, ATPases and SNOG proteins were identified. Using real-time RT-PCR analysis, significant inductions of an ABC transporter and a Ca⁺⁺ ATPase during 2p-ITC treatment were discovered. These results suggest that the fungal resistance phenotype to 2p-ITC involves calcium ions and 2p-ITC efflux via an ABC transporter.     

Gene Expression Analysis of PTEN Positive Glioblastoma Stem Cells Identifies DUB3 and Wee1 Modulation in a Cell Differentiation Model

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.     

Two novel defensin-encoding genes of the Chagas disease vector Triatoma brasiliensis (Reduviidae, Triatominae): Gene expression and peptide-structure modeling

Defensins are cysteine-rich peptides involved in the innate immunity of insects and many other organisms. In the present study, two novel defensin-encoding cDNAs and the respective genomic DNAs (def3 and def4) of Triatoma brasiliensis were identified and their tissue-specific and temporal expression was characterized. Both of the deduced mature peptides consisted of 43 amino acid residues and were highly similar to previously identified triatomine defensins (81.4-100%). Semi-quantitative RT-PCR data showed that def3 was constitutively expressed in the fat body and was induced in salivary glands and the small intestine at 5 and 3 days after feeding (daf), respectively. The def4 mRNA level was highly up-regulated in the stomach and fat-body tissues at 5 and 3 daf, respectively. The three-dimensional structures of these defensins were predicted using a homology modeling approach with Def-AAA, the defensin from Anopheles gambiae, as template (62-74% identity). A map of the electrostatic potential of these models revealed that, despite their similar folding patterns, mature Def2 and Def4 have a more cationic structure than is the case for Def1 and Def3. Such differences may orient the antimicrobial profile of these defensins against distinct targets in different organs of the insect.     

Involvement of the mitochondrial compartment in human NCL fibroblasts

Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.     

Novel large-range mitochondrial DNA deletions and fatal multisystemic disorder with prominent hepatopathy

Hepatic involvement in mitochondrial cytopathies rarely manifests in adulthood, but is a common feature in children. Multiple OXPHOS enzyme defects in children with liver involvement are often associated with dramatically reduced amounts of mtDNA. We investigated two novel large scale deletions in two infants with a multisystem disorder and prominent hepatopathy. Amount of mtDNA deletions and protein content were measured in different post-mortem tissues. The highest levels of deleted mtDNA were in liver, kidney, pancreas of both patients. Moreover, mtDNA deletions were detected in cultured skin fibroblasts in both patients and in blood of one during life. Biochemical analysis showed impairment of mainly complex I enzyme activity. Patients manifesting multisystem disorders in childhood may harbour rare mtDNA deletions in multiple tissues. For these patients, less invasive blood specimens or cultured fibroblasts can be used for molecular diagnosis. Our data further expand the array of deletions in the mitochondrial genomes in association with liver failure. Thus analysis of mtDNA should be considered in the diagnosis of childhood-onset hepatopathies.