Genetic Variability in European Populations of an Invasive American Crayfish: Preliminary Results

Since 1973, the red swamp crayfish, Procambarus clarkii, native to south-central United States and northeastern Mexico, has spread throughout Europe. Here, we surveyed the genetic variability of five European populations of the species using RAPD markers. Genetic variation was found to be so high as to uniquely fingerprint most of the surveyed individuals. Analysis of molecular variance (AMOVA) of the RAPD markers showed that 1) a large part of the genetic variation can be attributed to the differentiation among localities, and 2) the differentiation was mainly due to the separation of the samples from Louisiana with respect to the European set. A hypothesis emerged in which subsequent introductions of crayfish from different sources were performed. This hypothesis might explain the high genetic diversity observed within each population and the genetic differentiation among populations, as the result, respectively, of the introduction of different sets of crayfish and the casual bias of introductions. Although preliminary, our results suggest that RAPDs could be helpful in providing information about human-mediated introduced populations.     

Temperature-, SDS-, and pH-induced conformational changes in protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a dynamic simulation and fourier transform infrared spectroscopic study

The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites.     

Patterns of Rodent Species Diversity and Abundance in a Kenyan Relict Tropical Rainforest

Patterns of rodent species abundance and diversity were examined over a 5 months period in two areas of a Kenyan relict tropical rainforest. The two areas are subjected to different administrations which lead to various levels of anthropogenic disturbance: one can be considered relatively disturbed and one relatively undisturbed. Anthropogenic disturbance causes a reduction in woody stem density between 0 and 1.5 m and reduced understory tree canopy cover. Rodent abundance was estimated using the program CAPTURE and compared with the number of individuals actually captured. Density was estimated with three different methods, two of these utilised a boundary strip to estimate effective size of the area trapped. Density resulted in being relatively high in both areas, so population might have been at a peak. Species richness was higher in the disturbed forest, while species diversity and evenness was higher in the undisturbed forest. We suggest that in the disturbed forest the increase in number of species might be due to sporadical entrance in the forest by non-forest species, while the decrease in diversity might be due to the decrease of lower strata vegetation that occurs in the disturbed forest, hence this factor might affect species equitability. Bibliographic data supports this hypothesis as rodent species diversity and ground vegetation cover have been found to be correlated.     

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.     

PPARγ and RXRγ ligands act synergistically as potent antineoplastic agents in vitro and in vivo glioma models

Glioblastoma represent the most common primary brain tumor in adults and are currently considered incurable. We investigated antiproliferative and anti-invasive mechanisms of 6-OH-11- O -hydroxyfenantrene (IIF), a retinoid X receptor ligand, and pioglitazone (PGZ), a peroxisome proliferator-activated receptor γ activator, in three different glioblastoma cell lines. A dose-dependent reduction of tumor invasion and strong decrease of matrix metalloproteinases 2 and 9 expression was observed, especially when a combination therapy of IIF and PGZ was administered. Combined treatment also markedly reduced proliferation and induced apoptosis in all glioma cell lines tested. This was in particular accompanied by decrease of antiapoptotic proteins Bcl2 and p53, while simultaneously pro-apoptotic cytochrome c , cleaved caspase 3, Bax and Bad levels increased. These in vitro findings were further substantiated in a murine glioma model in vivo , where oral administration of PGZ and IIF resulted in significantly reduced tumor volume and proliferation. Of note, treatment with nuclear receptor ligands was not only effective when the treatment was initiated shortly after the intraparenchymal seeding of the glioma cells, but even when initiated in the last third of the observation period. Collectively, our results demonstrate the effectiveness of a combined treatment of ligands of proliferator-activated receptor and retinoid X receptor against glioblastoma.     

Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD⁺ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD⁺ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD⁺-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD⁺ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.     

Nuclear shield: a multi-enzyme task-force for nucleus protection

Background

In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions.

Methodology/Principal Findings

Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization.

Conclusions/Significance

The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and accumulation of protection enzymes near sensitive targets represents a new safeguard modality which deeply differs from the adaptive response which is based on expression of specific enzymes.     

Nanoscale enzyme inhibitors: Fullerenes inhibit carbonic anhydrase by occluding the active site entrance

We investigated a series of derivatized fullerenes possessing alcohol, amine, and amino acid pendant groups as inhibitors of the zinc enzymes carbonic anhydrases (CAs, EC 4.2.1.1). We discovered that fullerenes bind CAs with submicromolar—low micromolar affinity, despite the fact that these compounds do not possess moieties normally associated with CA inhibitors such as the sulfonamides and their isosteres, or the coumarins. The 13 different mammalian CA isoforms showed a diverse inhibition profile with these compounds. By means of computational methods we assessed the inhibition mechanism as being due to occlusion of the active site entrance by means of the fullerene cage (possessing dimension of the same order of magnitude as the opening of the enzyme cavity, of 1 nm). The pendant moieties to the fullerene cage make interactions with amino acid residues from the active site, among which His64, His94, His96, Val121, and Thr200. Fullerenes thus represent a totally new class of nanoscale CA inhibitors which may show applications for targeting physiologically relevant isoforms, such as the dominant CA II and the tumor-associated CA IX.     

A continuous kinetic assay for RNA-cleaving deoxyribozymes,exploiting ethidium bromide as an extrinsic fluorescent probe

We describe a rapid and inexpensive method to monitor the kinetics of small RNA-cleaving deoxyribozymes, based on the exogenous fluorophore ethidium bromide. Ethidium binds preferentially to double-stranded nucleic acids, and its fluorescence emission increases dramatically upon intercalation. Thus, ethidium can be used in single-turnover experiments to measure both annealing of the deoxyribozyme to its substrate and release of the products. Under conditions in which dissociation of the product is fast compared with cleavage, the apparent rate of product release reflects the cleavage step. The method was developed for characterizing the so-called 8-17 catalytic DNA, but its general applicability in the deoxyribozyme field was verified using the 10-23 RNA-cleaving construct. Catalysis by both deoxyribozymes was not inhibited in the presence of substoichiometric amounts of ethidium, and the rates obtained through the ethidium assay were virtually identical to the rates determined using radiolabeled substrates. In contrast, the assay cannot be applied to the large, structured ribozymes, and its use to study the kinetics of the small hammerhead ribozyme was hampered by the presence on the catalyst of at least one high-affinity ethidium binding site.