Three new hybrids of Ophioglossum (Ophioglossaceae) from Monte Pisano,Tuscany (Central Italy)

Based on general morphology, spore measurements and ornamentation (scanning electron microscope), genome size estimation, and molecular systematics (trnL-trnF IGS), we show the extreme systematic complexity within the European representatives of the genus Ophioglossum. In particular, three hybrids from Tuscany are described: the tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane, seen as the hybrid between diploid O. lusitanicum L. and hexaploid O. azoricum C.Presl; the tetraploid O. × giovanninii Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the homoploid hybrid between tetraploid O. vulgatum L. and tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane; the pentaploid O. × pseudoazoricum Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the hybrid between hexaploid O. azoricum C.Presl and tetraploid O. vulgatum L. All the three new taxa grow in different localities in the Monte Pisano mountain range.     

Complex of Rutin with β-Cyclodextrin as Potential Delivery System

This study aimed to obtain and characterize an RU-β-CD complex in the context of investigating the possibility of changes in the solubility, stability, antioxidative and microbiological activity as well as permeability of complexated rutin as against its free form. The formation of the RU-β-CD complex via a co-grinding technique was confirmed by using DSC, SEM, FT-IR and Raman spectroscopy, and its geometry was assessed through molecular modeling. It was found that the stability and solubility of the so-obtained complex were greater compared to the free form; however, a slight decrease was observed inits antibacterial potency. An examination of changes in the EPR spectra of thecomplex excluded any reducing effect of complexation on the antioxidative activity of rutin. Considering the prospect of preformulation studies involving RU-β-CD complexes, of significance is also the observed possibility of prolongedly releasing rutin from the complex at a constant level over along period of 20 h, and the fact that twice as much complexated rutin was able topermeate compared to its free form.     

Effects of Detergent β-Octylglucoside and Phosphate Salt Solutions on Phase Behavior of Monoolein Mesophases

Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent β-octylglucoside (βOG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures of MO with salt solutions above the hydration boundary; and 2), quaternary mixtures of MO with βOG and salt solutions over a wide range of hydration conditions. All quaternary mixtures showed highly regular lyotropic phase behavior with the same sequence of phases (Lα, Ia3d, and Pn3m) as MO/water mixtures at similar temperatures. The effects of additives in quaternary systems agreed qualitatively with those found in ternary mixtures in which only one additive is present. However, quantitative differences in the effects of additives on the lattice parameters of fully hydrated mesophases were found between ternary and quaternary mixtures. We discuss the implications of these findings for mechanistic investigations of membrane protein crystallization in lipidic mesophases and for studies of the suitability of precipitants for mesophase-based crystallization methods.     

The caspase 6 derived N-terminal fragment of DJ-1 promotes apoptosis via increased ROS production

In pathological conditions, the amount of DJ-1 determines whether a cell can survive or engage a cell death program. This is exemplified in epithelial cancers, in which DJ-1 expression is increased, while autosomal recessive early onset Parkinson''s disease mutations of DJ-1 generally lead to decreased stability and expression of the protein. We have shown previously that DJ-1 is cleaved by caspase-6 during induction of apoptosis. We demonstrate here that the N-terminal cleaved fragment of DJ-1 (DJ-1 Nt) is specifically expressed in the nucleus and promotes apoptosis in SH-SY5Y neuroblastoma cell lines. In addition, overexpression of DJ-1 Nt in different cell lines leads to a loss of clonogenic potential and sensitizes to staurosporin and 1-methyl-4-phenylpyridinium (MPP+)-mediated caspase activation and apoptosis. Importantly, inhibition of endogenous DJ-1 expression with sh-RNA or DJ-1 deficiency mimics the effect of DJ-1 Nt on cell growth and apoptosis. Moreover, overexpression of DJ-1 Nt increases reactive oxygen species (ROS) production, and sensitizes to MPP+-mediated apoptosis and DJ-1 oxidation. Finally, specific exclusion of DJ-1 Nt from the nucleus abrogates its pro-apoptotic effect. Taken together, our findings identify an original pathway by which generation of a nuclear fragment of DJ-1 through caspase 6-mediated cleavage induces ROS-dependent amplification of apoptosis.     

Identification,purification, and characterization of an eukaryotic-like phosphopantetheine adenylyltransferase (coenzyme A biosynthetic pathway) in the hyperthermophilic archaeon Pyrococcus abyssi

Although coenzymeA (CoA) is essential in numerous metabolic pathways in all living cells, molecular characterization of the CoA biosynthetic pathway in Archaea remains undocumented. Archaeal genomes contain detectable homologues for only three of the five steps of the CoA biosynthetic pathway characterized in Eukarya and Bacteria. In case of phosphopantetheine adenylyltransferase (PPAT) (EC 2.7.7.3), the putative archaeal enzyme exhibits significant sequence similarity only with its eukaryotic homologs, an unusual situation for a protein involved in a central metabolic pathway. We have overexpressed in Escherichia coli, purified, and characterized this putative PPAT from the hyperthermophilic archaeon Pyrococcus abyssi (PAB0944). Matrix-assisted laser desorption ionization-time of flight mass spectrometry and high performance liquid chromatography measurements are consistent with the presence of a dephospho-CoA (dPCoA) molecule tightly bound to the polypeptide. The protein indeed catalyzes the synthesis of dPCoA from 4'-phosphopantetheine and ATP, as well as the reverse reaction. The presence of dPCoA stabilizes PAB0944, as it induces a shift from 76 to 82 degrees C of the apparent Tm measured by differential scanning microcalorimetry. Potassium glutamate was found to stabilize the protein at 400 mm. The enzyme behaves as a monomeric protein. Although only distantly related, secondary structure prediction indicates that archaeal and eukaryal PPAT belong to the same nucleotidyltransferase superfamily of bacterial PPAT. The existence of operational proteins highly conserved between Archaea and Eukarya involved in a central metabolic pathway challenge evolutionary scenarios in which eukaryal operational proteins are strictly of bacterial origin.     

Effect of lowered incubation temperatures on nucleic acid and protein synthesis by a mesophilic and a psychrophilic bacterium

The authors studied changes in the synthesis of nucleic acids (RNA, DNA) and protein by a mesophilic strain ofEscherichia coli B and a psychrophilic strain ofPseudomonas fluorescens at a low incubation temperature giving tenfold prolongation of the generation time. It was found that lowering the incubation temperature was followed by an increase in the intracellular nucleic acid content during the lag phase and the phase of accelerated growth, in which maximum nucleic acid (NA) values were reached. As a result, the total NA level in the cell also remained relatively high during further proliferation, when the increase in NA (particularly RNA) slows down at low incubation temperatures. Proteosynthesis, however, fell in the mesophilic culture. The smaller effect of a lowered temperature on DNA biosynthesis was manifested specifically in the lag phase ofEscherichia coli, in which disproportion developed between the amount of DNA (which was synthesized at a relatively higher rate) and RNA; this was afterwards equalized by a temporary break in DNA production. Pronounced differences in the given types of biosynthesis were found only in the mesophilic culture, while at suboptimal temperatures the metabolism of the psychrophilic strain slowed down but no marked changes occurred.     

Integracides: tetracyclic triterpenoid inhibitors of HIV-1 integrase produced by Fusarium sp

HIV-1 integrase is a critical enzyme in the replication of HIV-1. It is absent in the host cells and therefore is a good target for treatment of HIV-1 infections. Integracides are members of the tetracyclic triterpenoids family that were isolated from the fermentation broth of a Fusarium sp. Integracide A, a sulfated ester, exhibited significant inhibitory activity against strand transfer reaction of HIV-1 integrase. The discovery, structure elucidation including single crystal X-ray structure and HIV-1 inhibitory activity of these compounds are described.     

Involvement of p53 in phthalate effects on mouse and rat osteoblasts

The role of two estrogen‐mimicking compounds in regulating osteoblast activities were examined. Previously, our attention was focused on benzyl butyl phthalate (BBP) and di‐n‐butyl phthalate (DBP) since previous works showed that they enter the cytoplasm, bioaccumulate, modify actin cytoarchitecture and exert mitogenic effects involving microfilament disruption, and nuclear actin and lamin A regulation in Py1a rat osteoblasts. In this study we showed that BBP and DBP cause DNA base lesions both in MT3T3‐E1 osteoblasts and in mouse primary calvarial osteoblasts (COBs). In addition, treatment with the above effectors caused an increase of p53 and phospho‐p53 (ser‐15 and ser‐20) as well as an increase of apoptotic proteins with consequent decrease of cell viability. Moreover, treatment with phthalates did not modified p53 and phospho‐p53 expression in Py1a rat osteoblasts. It is of relevance that in p53 knockdown mouse osteoblasts a proliferative effect of phthalates, similar to that observed in rat Py1a osteoblasts, was found. In conclusion, our data demonstrated that phthalates induce osteoblast apoptosis, which is, at least in part, mediated by p53 activation, suggesting that the proliferative effects could be due to p53 missing activation or p53 mutation. J. Cell. Biochem. 107: 316–327, 2009. © 2009 Wiley‐Liss, Inc.     

Dynamic formation of optically trapped microstructure arrays for biosensor applications

We explore the dynamic properties of multiple-beam optical traps to manipulate arrays of microstructures for biosensor applications. Multiple optical traps are generated by a virtually loss-less transformation of input phase patterns into high-intensity trapping-beams. A direct image projection of the phase patterns enables an adjustable number of optical traps in addition to instantaneous control of the position, size, shape and intensity of each trapping-beam. We present experimental results showing various colloidal formations through dynamic optical manipulation of polystyrene microspheres and yeast cells in aqueous media. The experimental configurations are geared towards the use of multiple-beam optical traps for biosensor applications.