Neuroprotective role of nerve growth factor in hypoxicischemic injury. From brain to skin

Hypoxic-ischemic injuries (HII) of the brain, optic pathways, and skin are frequently associated with poor neurological and clinical outcome. Unfortunately, no new therapeutic approaches have been proposed for these conditions. Recently, experimental and clinical studies showed that nerve growth factor (NGF) can improve neurological deficits, visual loss and skin damage after HII. Based on these studies, we report the effects of NGF administration in different lesions of the brain, optic pathways and skin. 2.5S NGF purified and lyophilized from male mouse submaxillary glands was utilized for the treatment. NGF administration was started in absence of recovery after conventional and standardized treatment. One mg NGF was administered via the external catheter into the brain, by drop administration in the eye, and by subcutaneous administration in the skin. We treated 4 patients: 2 children with hypoxic-ischemic brain damage, an adult patient with an optic glioma-induced visual loss and a child with a severe crush syndrome of the lower left limb. After NGF treatment, we observed an amelioration of both neurological and electrophysiological function of the brain, a subjective and objective improvement of visual function, and a gradual improvement of ischemic skin lesion. No side effects were related to NGF treatment in all patients studied. Our observation shows that NGF administration may be an effective and safe adjunct therapy in patients with severe HII. The beneficial and prolonged effect on nerve function suggests a neuroprotective mechanism exerted by NGF on the residual viable neurological pathways of these patients.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Influence of temperature on the rheological behavior of a new fucose-containing bacterial exopolysaccharide

The effect of temperature on the rheology of a new fucose-containing extracellular polysaccharide (EPS) was evaluated. The steady state data revealed a shear-thinning behavior, with the viscosity being immediately recovered when the shear rate was decreased. The mechanical spectra indicated viscous solutions with entangled polymer molecules in the range of temperatures studied (from 15 °C to 65 °C). In addition, the Time-Temperature Superposition principle was successfully applied and the Cox-Merz rule was valid, reinforcing the idea of a thermorheologically simple behavior for the EPS in aqueous solution. Furthermore, the viscous and viscoelastic properties at 25 °C were maintained after consecutive heating and cooling cycles, indicating a good thermal stability under temperature fluctuations.     

Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing

SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K(m) values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k(cat)/K(m) = 5.5 × 10(3) M(-1)·s(-1)), but SsoPox can also degrade N-butyryl homoserine lactone (C(4)-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C(12)-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C(4)-HSL- and oxo-C(12)-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.     

Identification of a Novel Gene Signature of ES Cells Self-Renewal Fluctuation through System-Wide Analysis

Embryonic Stem cells (ESCs) can be differentiated into ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. In regular culture conditions, ESCs'' self-renewal is maintained through molecules that inhibit spontaneous differentiation enabling long-term cellular expansion. This undifferentiating condition is characterized by multiple metastable states that fluctuate between self-renewal and differentiation balance. Here, we aim to characterize the high-pluripotent ESC metastate marked by the expression of Zscan4 through a supervised machine learning framework based on an ensemble of support vector machine (SVM) classifiers. Our study revealed a leukaemia inhibitor factor (Lif) dependent not-canonical pluripotency signature ({"type":"entrez-nucleotide","attrs":{"text":"AF067063","term_id":"3171143"}}AF067063, {"type":"entrez-nucleotide","attrs":{"text":"BC061212","term_id":"38173992"}}BC061212, Dub1, Eif1a, Gm12794, Gm13871, Gm4340, Gm4850, Tcstv1/3, and Zfp352), that specifically marks Zscan4 ESCs'' fluctuation. This novel ESC metastate is enhanced by high-pluripotency culture conditions obtained through Extracellular signal Regulated-Kinase (ERK) and Glycogen synthase kinase-3 (Gsk-3) signaling inhibition (2i). Significantly, we reported that the conditional ablation of the novel ESC metastate marked by the expression of Gm12794 is required for ESCs self-renewal maintenance. In conclusion, we extend the comprehension of ESCs biology through the identification of a novel molecular signature associated to pluripotency programming.     

Evaluation of AMCase and CHIT-1 expression in monocyte macrophages lineage

Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT-1) are two active chitinases expressed in humans. The chitinase activity of AMCase was found to be causative in allergic inflammation and its expression was found to be induced by interleukin-13. CHIT1-1 is expressed by phagocytic cells and extremely high levels are seen in lysosomal storage diseases. Despite that AMCase expression in the inflammation is under investigation, little is known regarding its regulation during macrophages' full maturation and polarization. In this study, we compared AMCase and CHIT-1 modulation during monocyte to macrophage transition and polarization. Gene expression analysis was investigated by real-time PCR from mRNA of human monocytes obtained from buffy coat of healthy volunteers, from mRNA of polarized to classically activated macrophages (or M1), obtained by interferon (IFN)-γ and lipopolysaccharide (LPS) treatment, and from mRNA of alternatively activated macrophages (or M2) obtained by interleukin (IL)-4 exposure. Our results showed that the expression of AMCase and CHIT-1 were differently modulated in HMMs at different stage of maturation. The behavior of these two active chitinase suggests that in the immune response their role is complementary.     

8-Oxoguanine DNA-glycosylase repair activity and expression: a comparison between cryopreserved isolated lymphocytes and EBV-derived lymphoblastoid cell lines

Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.     

Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability

A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge–charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme.