The isoprenoid end product N6-isopentenyladenosine reduces inflammatory response through the inhibition of the NFκB and STAT3 pathways in cystic fibrosis cells.

OBJECTIVE: N6-isopentenyladenosine (iPA) is an intermediate of the mevalonate pathway that exhibits various anti-cancer effects. However, studies on its anti-inflammatory activity are scarce and underlying molecular mechanisms are unknown. Therefore, we aimed to investigate the ability of iPA to exert anti-inflammatory effects in the human cystic fibrosis (CF) cell model of exacerbated inflammation. MATERIALS AND METHODS: TNFα-stimulated CF cells CuFi-1 and its normal counterpart NuLi-1 were pre-treated with increasing concentrations of iPA and cell viability and proliferation were assessed by MTT and BrdU assays. The effect of iPA on IL-8 and RANTES secretion was determined by ELISA, and the activation and expression of signaling molecules and selenoproteins were studied by Western blot. To assess the direct effect of iPA on NFκB activity, luciferase assay was performed on TNFα-stimulated HEK293/T cells transfected with a NFκB reporter plasmid. RESULTS: We demonstrated for the first time that iPA prevents IL-8 and RANTES release in TNFα-stimulated CF cells and this effect is mediated by increasing the expression of the direct NFκB inhibitor IκBα and decreasing the levels of STAT3. Consistent with this, we showed that iPA inhibited TNFα-mediated NFκB activation in HEK/293T cells. Finally, we also found that iPA improved the levels of glutathione peroxidase 1 and thioredoxin reductase 1 only in CF cells suggesting its ability to maintain sufficient expression of these anti-oxidant selenoproteins. CONCLUSIONS: Our findings indicate that iPA can exert anti-inflammatory activity especially in the cases of excessive inflammatory response as in CF.

BAG3 is involved in neuronal differentiation and migration.

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

Morphological and bioenergetic demands underlying the mitophagy in post-mitotic neurons: the pink-parkin pathway.

Evidence suggests a striking causal relationship between changes in quality control of neuronal mitochondria and numerous devastating human neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Contrary to replicating mammalian cells with a metabolism essentially glycolytic, post-mitotic neurons are distinctive owing to (i) their exclusive energetic dependence from mitochondrial metabolism and (ii) their polarized shape, which entails compartmentalized and distinct energetic needs. Here, we review the recent findings on mitochondrial dynamics and mitophagy in differentiated neurons focusing on how the exceptional characteristics of neuronal populations in their morphology and bioenergetics needs make them quite different to other cells in controlling the intracellular turnover of these organelles.

Tissue distribution of P2X4 receptors studied with an ectodomain antibody.

A monoclonal antibody was developed to the extracellular domain of the rat P2X4 receptor. The antibody was highly selective among all rat P2X receptor subunits, and recognised only the oligomeric, non-denatured form of the P2X4 receptor. Immunohistochemistry showed an extensive pattern of distribution throughout the central and peripheral nervous systems, the epithelia of ducted glands and airways, smooth muscle of bladder, gastrointestinal tract, uterus, and arteries, uterine endometrium and fat cells. The protein was identified by Western blotting in membrane extracts of these tissues, and the ectodomain antibody immunoprecipitated a protein that was recognised with a P2X4 receptor C terminus antibody. The findings indicate that the P2X4 receptor subunit has a very extensive distribution among mammalian tissues, and this suggests possible new functional roles.

Biochemical and immunohistochemical evidence for a non-muscle myosin at the neuromuscular junction in bovine skeletal muscle.

We identified 220-kD protein in bovine skeletal muscle homogenate by affinity chromatography on an agarose column and subsequent SDS-PAGE. Peptide mass fingerprinting (MALDI mass spectrometry) and internal sequence analysis revealed that this protein has homology with several members of the myosin superfamily, particularly with human cardiac beta-myosin heavy chain (beta-MHC). A rabbit polyclonal antibody against the 220-kD protein specifically stained a 220-kD band in Western blots of skeletal muscle homogenate. Immunohistochemical experiments on cryostat sections demonstrated that in skeletal muscle this protein is exclusively localized at the neuromuscular junctions, no immunoreactivity being present at the myofibril level. Because of its relative homology with cardiac beta-MHC, we also investigated the distribution of the 220-kD protein in bovine heart. In cardiac fibers, 220-kD protein-related immunoreactivity was restricted to the intercalated disks, whereas myofibrils were completely devoid of specific immunoreactivity. This distribution pattern was completely different from that of cardiac beta-MHC, which involved myofibrils. Because of the above biochemical and immunohistochemical features, the 220-kD protein we have identified is suggested to be a novel member of the non-muscle (non-sarcomeric) myosin family.

Underlying anatomy for CTV contouring and lymphatic drainage in rectal cancer radiation therapy.

Despite the low local recurrence rate that can be achieved by adequate surgery (total mesorectal excision--TME), radiation therapy was shown to play a significant role in reducing this risk. The widespread use of TME in many European Centers has introduced a new terminology and the need to identify the area at major risk for local failure using this surgical procedure. In the surgical series where extended extra-mesorectal surgery was performed, the role of lymphatic spread was evidenced, especially for low rectal cancer, through the pelvic parietal fascia and lateral pelvic spaces. The aim of this study was to better define some anatomic concepts and the main risk factors which impact on CTV contouring and field conformation in rectal cancer treatment. This information helps formulating guidelines for CTV contouring in daily radiotherapy practice, in order to define the best therapy, according to the tumor stage and location.