Tetanus, botulinum and snake presynaptic neurotoxins

Tetanus and botulinum neurotoxins, produced by anaerobic bacteria of the genus Clostridium, are the most toxic proteins known and are solely responsible for the pathogenesis of tetanus and botulism. They are metallo-proteases that enter nerve terminals and cleave proteins of the neuroexocytosis apparatus causing a persistent, but reversible, inhibition of neurotransmitter release. Botulinum neurotoxins are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals.

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Structural and mechanical properties of UV-photo-cross-linked poly(N-vinyl-2-pyrrolidone) hydrogels

Biocompatible poly( N-vinyl-2-pyrrolidone) (PVP) hydrogels have been produced by UV irradiation of aqueous polymer mixtures, using a high-pressure mercury lamp. The resulting materials have been characterized by a combination of experimental techniques, including rheology, small-angle neutron scattering (SANS), electron paramagnetic resonance (EPR), and pulsed gradient spin-echo nuclear magnetic resonance (PGSE-NMR), to put in evidence the relationship between the microstructural properties and the macrofunctional behavior of the gels. Viscoelastic measurements showed that UV photo-cross-linked PVP hydrogels present a strong gel mechanical behavior and viscoelastic moduli values similar to those of biological gels. The average distance between the cross-linking points of the polymer network was estimated from the hydrogels elastic modulus. However, SANS measurements showed that the network microstructure is highly inhomogeneous, presenting polymer-rich regions more densely cross-linked, surrounded by a water-rich environment. EPR and PGSE-NMR data further support the existence of these water-rich domains. Inclusion of a third component, such as glycerol, in the PVP aqueous mixture to be irradiated has been also investigated. A small amount of glycerol (<3% w/w) can be added keeping satisfactory properties of the hydrogel, while higher amounts significantly affect the cross-linking process.     

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.     

Microbial Deterioration of Artistic Tiles from the Façade of the Grande Albergo Ausonia &; Hungaria (Venice,Italy)

The Grande Albergo Ausonia &; Hungaria (Venice Lido, Italy) has an Art Nouveau polychrome ceramic coating on its façade, which was restored in 2007. Soon after the conservation treatment, many tiles of the façade decoration showed coloured alterations putatively attributed to the presence of microbial communities. To confirm the presence of the biological deposit and the stratigraphy of the Hungaria tiles, stereomicroscope, optical and environmental scanning electron microscope observations were made. The characterisation of the microbial community was performed using a PCR–DGGE approach. This study reported the first use of a culture-independent approach to identify the total community present in biodeteriorated artistic tiles. The case study examined here reveals that the coloured alterations on the tiles were mainly due to the presence of cryptoendolithic cyanobacteria. In addition, we proved that the microflora present on the tiles was generally greatly influenced by the environment of the Hungaria hotel. We found several microorganisms related to the alkaline environment, which is in the range of the tile pH, and related to the aquatic environment, the presence of the acrylic resin Paraloid B72® used during the 2007 treatment and the pollutants of the Venice lagoon.     

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2^Wt and DDB2^PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2^PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> sp. A59 GH10 and GH11 Extracellular Endoxylanases: Application in Biomass Bioconversion

The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45–50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass.     

CD4+ Th0 cell clones,isolated from a metastatic lymph node of a melanoma patient,possess cytolytic function

In the present study T lymphocytes isolated from a metastatic lymph node (T-LNL) of a melanoma patient have been cloned. In the attempt to verify whether T-LNL may acquire in vitro functional activities in the absence of tumour-associated antigens, they were cloned utilizing allogeneic lymphocytes as feeder cells. Nineteen clones generated from T-LNL proved to be CD4+ and, among these, five were able to kill autologous and allogeneic human melanoma cells in HLA-class-II-restricted way. On the basis of their cytokine production, these CD4+ cytolytic T-LNL clones were shown to belong to the Th0 subset and three of them expersseed the V17 chain of the T cell receptor. These results suggest the presence of melanomaspecific but functionally inactive lymphocytes with T cell receptor oligoclonality in the lymph node environment. These specific T cells may acquire in vitro the capacity to kill autologous and allogeneic tumours without any induction by autologous melanoma cells.