The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide

The major contribution of this paper is the finding of a glycolytic source of ATP in the isolated postsynaptic density (PSD). The enzymes involved in the generation of ATP are glyceraldehyde-3-phosphate dehydrogenase (G3PD) and phosphoglycerate kinase (PGK). Lactate dehydrogenase (LDH) is available for the regeneration of NAD+, as well as aldolase for the regeneration of glyceraldehyde-3-phosphate (G3P). The ATP was shown to be used by the PSD Ca2+/calmodulin-dependent protein kinase and can probably be used by two other PSD kinases, protein kinase A and protein kinase C. We confirmed by immunocytochemistry the presence of G3PD in the PSD and its binding to actin. Also present in the PSD is NO synthase, the source of NO. NO increases the binding of NAD, a G3PD cofactor, to G3PD and inhibits its activity as also found by others. The increased NAD binding resulted in an increase in G3PD binding to actin. We confirmed the autophosphorylation of G3PD by ATP, and further found that this procedure also increased the binding of G3PD to actin. ATP and NO are connected in that the formation of NO from NOS at the PSD resulted, in the presence of NAD, in a decrease of ATP formation in the PSD. In the discussion, we raise the possible roles of G3PD and of ATP in protein synthesis at the PSD, the regulation by NO, as well as the overall regulatory role of the PSD complex in synaptic transmission.

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of ≈20-kDa C-terminal fragments (CTF) and ≈30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181–190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the ≈20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181–190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.

A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5′-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.

Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: Clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant

A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52/2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription–PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.

Involvement of the Ubiquitin/Proteasome System in Sorting of the Interleukin 2 Receptor β Chain to Late Endocytic Compartments

Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor β chain (IL2Rβ) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rβ was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rβ, with little effect on its internalization. In addition, we show that a fraction of IL2Rβ can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rβ targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rβ were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rβ toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing.

Intermediate hosts of Toxoplasma gondii and food safety:epidemiology, genetics and parasite survival in meat-producing animals in Italy

Toxoplasma gondii is a coccidian parasite with a global distribution. The definitive host is the cat (and other felids). All warm-blooded animals can act as intermediate hosts, including humans. Sexual reproduction (gametogony) takes place in the final host and oocysts are released in the environment, where they then sporulate to become infective. In intermediate hosts the cycle is extra-intestinal and results in the formation of tachyzoites and bradyzoites. Tachyzoites represent the invasive and proliferative stage and on entering a cell it multiplies asexually by endodyogeny. Bradyzoites within tissue cysts are the latent form. T. gondii is a food-borne parasite causing toxoplasmosis, which can occur in both animals and humans. Infection in humans is asymptomatic in more than 80% of cases in Europe and North-America. In the remaining cases patients present fever, cervical lymphadenopathy and other non-specific clinical signs. Nevertheless, toxoplasmosis is life threatening if it occurs in immunocompromised subjects. The main organs involved are brain (toxoplasmic encephalitis), heart (myocarditis), lungs (pulmonary toxoplasmosis), eyes, pancreas and parasite can be isolated from these tissues. Another aspect is congenital toxoplasmosis that may occur in pregnant women and the severity of the consequences depends on the stage of pregnancy when maternal infection occurs. Acute toxoplasmosis in developing foetuses may result in blindness, deformation, mental retardation or even death. The European Food Safety Authority (EFSA), in recent reports on zoonoses, highlighted that an increasing numbers of animals resulted infected with T. gondii in EU (reported by the European Member States for pigs, sheep, goats, hunted wild boar and hunted deer, in 2011 and 2012). In addition, high prevalence values have been detected in cats, cattle and dogs, as well as several other animal species, indicating the wide distribution of the parasite among different animal and wildlife species. The main route of transmission is consumption of food and water contaminated with sporulated oocysts. However, infection through the ingestion of meat contaminated with tissue cysts is frequent. Finally, although less frequent, other food products contaminated with tachyzoites such as milk, may also pose a risk. The importance of this parasite as a risk for human health was recently highlighted by EFSA’s opinion on modernization of meat inspection, where Toxoplasma gondii was identified as a relevant hazard to be addressed in revised meat inspection systems for pigs, sheep, goats, farmed wild boar and farmed deer (Call for proposals -GP/EFSA/BIOHAZ/2013/01). The risk of infection is more highly associated to animals reared outside, also in free-range or organic farms, where biohazard measure are less strict than in large scale, industrial farms. Here, animals are kept under strict biosecurity measures, including barriers, which inhibit access by cats, thus making soil contamination by oocysts nearly impossible. A growing demand by the consumer for organic products, coming from free-range livestock, in respect of animal-welfare, and the desire for the best quality of derived products, have all led to an increase in the farming of free-range animals. The risk of Toxoplasma gondii infection increases when animals have access to environment and the absence of data in Italy, together with need for in depth study of both the prevalence and genotypes of Toxoplasma gondii present in our country were the main reasons for the development of this thesis project. A total of 152 animals have been analyzed, including 21 free-range pigs (Suino Nero race), 24 transhumant Cornigliese sheep, 77 free-range chickens and 21 wild animals. Serology (on meat juice) and identification of T. gondii DNA through PCR was performed on all samples, except for wild animals (no serology). An in-vitro test was also applied with the aim to find an alternative and valid method to bioassay, actually the gold standard. Meat samples were digested and seeded onto Vero cells, checked every day and a RT-PCR protocol was used to determine an eventual increase in the amount of DNA, demonstrating the viability of the parasite. Several samples were alos genetically characterized using a PCR-RFLP protocol to define the major genotypes diffused in the geographical area studied. Within the context of a project promoted by Istituto Zooprofilattico of Pavia and Brescia (Italy), experimentally infected pigs were also analyzed. One of the aims was to verify if the production process of cured “Prosciutto di Parma” is able to kill the parasite. Our contribution included the digestion and seeding of homogenates on Vero cells and applying the Elisa test on meat juice. This thesis project has highlighted widespread diffusion of T. gondii in the geographical area taken into account. Pigs, sheep, chickens and wild animals showed high prevalence of infection. The data obtained with serology were 95.2%, 70.8%, 36.4%, respectively, indicating the spread of the parasite among numerous animal species. For wild animals, the average value of parasite infection determined through PCR was 44.8%. Meat juice serology appears to be a very useful, rapid and sensitive method for screening carcasses at slaughterhouse and for marketing “Toxo-free” meat. The results obtained on fresh pork meat (derived from experimentally infected pigs) before (on serum) and after (on meat juice) slaughter showed a good concordance. The free-range farming put in evidence a marked risk for meat-producing animals and as a consequence also for the consumer. Genotyping revealed the diffusion of Type-II and in a lower percentage of Type-III. In pigs is predominant the Type-II profile, while in wildlife is more diffused a Type-III and mixed profiles (mainly Type-II/III). The mixed genotypes (Type-II/III) could be explained by the presence of mixed infections. Free-range farming and the contact with wildlife could facilitate the spread of the parasite and the generation of new and atypical strains, with unknown consequences on human health. The curing process employed in this study appears to produce hams that do not pose a serious concern to human health and therefore could be marketed and consumed without significant health risk. Little is known about the diffusion and genotypes of T. gondii in wild animals; further studies on the way in which new and mixed genotypes may be introduced into the domestic cycle should be very interesting, also with the use of NGS techniques, more rapid and sensitive than PCR-RFLP. Furthermore wildlife can become a valuable indicator of environmental contamination with T. gondii oocysts. Other future perspectives regarding pigs include the expansion of the number of free-range animals and farms and for Cornigliese sheep the evaluation of other food products as raw milk and cheeses. It should be interesting to proceed with the validation of an ELISA test for infection in chickens, using both serum and meat juice on a larger number of animals and the same should be done also for wildlife (at the moment no ELISA tests are available and MAT is the reference method for them). Results related to Parma ham do not suggest a concerning risk for consumers. However, further studies are needed to complete the risk assessment and the analysis of other products cured using technological processes other than those investigated in the present study. For example, it could be interesting to analyze products such as salami, produced with pig meat all over the Italian country, with very different recipes, also in domestic and rural contexts, characterized by a very short period of curing (1 to 6 months). Toxoplasma gondii is one of the most diffuse food-borne parasites globally. Public health safety, improved animal production and protection of endangered livestock species are all important goals of research into reliable diagnostic tools for this infection. Future studies into the epidemiology, parasite survival and genotypes of T. gondii in meat producing animals should continue to be a research priority.

Noise and PSRR improvement technique for TPC readout front-end in CMOS. technology.

ALICE is one of four major experiments of particle accelerator LHC installed in the European laboratory CERN. The management committee of the LHC accelerator has just approved a program update for this experiment. Among the upgrades planned for the coming years of the ALICE experiment is to improve the resolution and tracking efficiency maintaining the excellent particles identification ability, and to increase the read-out event rate to 100 KHz. In order to achieve this, it is necessary to update the Time Projection Chamber detector (TPC) and Muon tracking (MCH) detector modifying the read-out electronics, which is not suitable for this migration. To overcome this limitation the design, fabrication and experimental test of new ASIC named SAMPA has been proposed . This ASIC will support both positive and negative polarities, with 32 channels per chip and continuous data readout with smaller power consumption than the previous versions. This work aims to design, fabrication and experimental test of a readout front-end in 130nm CMOS technology with configurable polarity (positive/negative), peaking time and sensitivity. The new SAMPA ASIC can be used in both chambers (TPC and MCH). The proposed front-end is composed of a Charge Sensitive Amplifier (CSA) and a Semi-Gaussian shaper. In order to obtain an ASIC integrating 32 channels per chip, the design of the proposed front-end requires small area and low power consumption, but at the same time requires low noise. In this sense, a new Noise and PSRR (Power Supply Rejection Ratio) improvement technique for the CSA design without power and area impact is proposed in this work. The analysis and equations of the proposed circuit are presented which were verified by electrical simulations and experimental test of a produced chip with 5 channels of the designed front-end. The measured equivalent noise charge was <550e for 30mV/fC of sensitivity at a input capacitance of 18.5pF. The total core area of the front-end was 2300?m × 150?m, and the measured total power consumption was 9.1mW per channel.

Academie to atelier : the artistic education of American women in nineteenth-century Paris

This paper puts forth an alternate reading of the artistic climate in late nineteenth-century Paris than that which has traditionally been suggested. I propose that the expansion of creative opportunity during this time reveals a climate of communal support, consent, and progressive reform for women artists, rather than a struggle to undermine central (masculine) control, as many scholars have claimed. Specifically, I explore the work of American expatriates living in Paris, including but not limited to Cecilia Beaux, Anna Klumpke, Alice Kellogg, and Ellen Day Hale. The birth of the private academy in Paris offered women the chance to develop their artistic ability and assert their independence. The Académie Julian in particular provided a comparatively accepting and progressive environment where American women studying abroad could study from the nude model, receive proper training, and explore their full creative potential. Through an examination of a) these women’s self-portraits, and b) depictions of them painted by their contemporaries – both male and female – I further investigate the artistic education of American women in the highly-gendered cultural milieu of late nineteenth-century France.

Cyclically-arranged, storm-controlled, prograding lithosomes in Messinian terrigenous shelves (Bajo Segura Basin, western Mediterranean)

This work focuses on a Messinian shallow-marine terrigenous unit, termed the La Virgen Formation, which forms part of the sedimentary infill of the Bajo Segura Basin (Betic margin of the western Mediterranean). This formation was deposited during a high sea level phase prior to the onset of the Messinian Salinity Crisis. Stratigraphically, it comprises a prograding stack of sandstone lithosomes alternating with marly intervals (1st-order cyclicity). These lithosomes are characterized by a homoclinal geometry that tapers distally, and interfinger with pelagic sediments rich in planktonic and benthic microfauna (Torremendo Formation). An analysis of sedimentary facies of each lithosome reveals a repetitive succession of sandy storm beds (tempestites), occasionally amalgamated, which are separated by thin marly layers (2nd-order cyclicity). Each storm bed contains internal erosional surfaces (3rd-order cyclicity) that delimit sets of laminae. Two categories of storm beds have been differentiated. The first one includes layers formed below storm wave base (SWB), characterized by traction structures associated to unidirectional flows (scoured base, planar lamination, and parting lineation). The second category consists of layers deposited above the SWB which display typical high regime oscillatory flow structures (swaley and hummocky cross lamination). In both cases, the ichnological record is characterized by an oligotypic association of Ophiomorpha nodosa, which can be interpreted as the result of allochthonous tracemakers (crustaceans) transported during storm events together with the sediment. The benthic microfauna in the marly intervals that separate the sandstone lithosomes (1st-order cyclicity) indicates that the storm ebb surges were deposited at depths ranging from those of inner shelf settings (with Elphidium spp. and Cibicides lobatulus) to those of outer shelf (with Valvulineria complanata and Uvigerina cylindrica). At the distal end of the sandstone lithosomes, the planktonic microfauna is characterized by a high content of taxa indicative of warm-oligotrophic waters (Globigerinoides obliquus and Globigerinoides bulloideus). In contrast, in the marly intervals, the microfauna is dominated by species typical of cold-eutrophic waters (Globigerina and Neogloboquadrina). This alternation of planktic foraminiferal assemblages is interpreted as being the expression of climatic cycles, in which every episode of progradation of tempestite-dominated lithosomes corresponds to maximum insolation and warm waters, whereas episodes of marly deposition correspond to minimal insolation and cold waters. The 1st-order cyclicity recorded in the La Virgen Formation, in a context of terrigenous storm-dominated shelf, corresponds to sapropel/homogeneous marl cycles formed in a pelagic basin (Torremendo Fm). These cycles in pelagic sediments are commonplace throughout the Mediterranean during the Messinian and reflect precession orbital changes: repeated periods of maximum insolation – minimum precession (sapropels) and minimal insolation – maximum precession (homogeneous marls). The fact that the example of terrigenous unit studied herein is coetaneous with the well-developed reef complexes in the Mediterranean basins points out the importance of sediment supply in the formation of large-scale sandy lithosomes. This is a crucial aspect to understanding reservoir genesis as well as lateral stratigraphic relationships with potential seal and/or source rocks.

Evidence of Intracrystalline Mesostructured Porosity in Zeolites by Advanced Gas Sorption, Electron Tomography and Rotation Electron Diffraction

The small size of micropores (typically <1 nm) in zeolites causes slow diffusion of reactant and product molecules in and out of the pores and negatively impacts the product selectivity of zeolite based catalysts, for example, fluid catalytic cracking (FCC) catalysts. Size-tailored mesoporosity was introduced into commercial zeolite Y crystals by a simple surfactant-templating post-synthetic mesostructuring process. The resulting mesoporous zeolite Y showed significantly improved product selectivity in both laboratory testing and refinery trials. Advanced characterization techniques such as electron tomography, three-dimensional rotation electron diffraction, and high resolution gas adsorption coupled with hysteresis scanning and density functional theory, unambiguously revealed the intracystalline nature and connectivity of the introduced mesopores. They can be considered as molecular highways that help reactant and product molecules diffuse quickly to and away from the catalytically active sites within the zeolite crystals and, thus, shift the selectivity to favor the production of more of the valuable liquid fuels at reduced yields of coke and unconverted feed.

Integrated taphonomy in an open-marine platform: The Lower Cretaceous of Sierra Helada (Betic Cordillera, SE Spain)

A detailed taphonomic analysis was carried out on the lower Albian deposits of the Sierra Helada section (Alicante, Betic Cordillera, southeastern Spain). Ten taphonomic characters were studied and ten skeletal concentrations were defined on the basis of taphonomic features and the dominant taxa. Cluster analysis was performed on the dataset represented by the abundance of the taphonomic characters in each skeletal concentration. This enabled the definition of four different taphonomic categories: 1) skeletal concentrations characterized by the presence of fossils preserved in life position, 2) skeletal concentrations showing very little physical reworking, 3) skeletal concentrations related to high-energy background conditions, and 4) skeletal concentrations produced by medium- to high-energy events. Four taphofacies were defined on the basis of the main sedimentological features and the most representative skeletal concentrations. Taphofacies A represents the low energy outer platform, rich in skeletal concentrations with echinoids in life position and only slightly reworked. The second taphofacies (taphofacies B) is very rich in reworked echinoid tests and calcarenitic beds and records the transition to shallower areas, while taphofacies C shows abundant thick-bedded calcarenites and skeletal concentrations produced by sediment transport and rapid deposition. Finally, cross-bedded grainstone beds, which are rich in fine-grained fragmented, locally reoriented bioclasts (taphofacies D), record the existence of shifting sandy dunes in the shallow inner part of the platform.

Evaluating Taphonomic Bias in a Storm-Disturbed Carbonate Platform: Effects of Compositional and Environmental Factors in Lower Jurassic Brachiopod Accumulations (Eastern Subbetic Basin, Spain)

In order to evaluate taxonomic and environmental control on the preservation pattern of brachiopod accumulations, sedimentologic and taphonomic data have been integrated with those inferred from the structure of brachiopod accumulations from the easternmost Lower Jurassic Subbetic deposits in Spain. Two brachiopod communities (Praesphaeroidothyris and Securina communities) were distinguished showing a mainly free-lying way of life in soft-bottom habitats. Three taphofacies are discriminated based on proportion of disarticulation, fragmentation, packing, and shell filling. Taphofacies 1 is represented by thinly fragmented, dispersed brachiopod shells in wackestone beds. Taphofacies 2 is spatially restricted to small lenses where shells are poorly fragmented, rarely disarticulated, usually void filled, and highly packed. Taphofacies 3 is represented by mud or cement filled, loosely packed, articulated brachiopods forming large pocket-like structures. Temporal and spatial averaging were minimally involved in taphofacies 2 and 3. It is interpreted that patchy preservation implies preservation of primary original patchiness of brachiopod communities on the seafloor. The origin of shell-rich taphofacies (2 and 3) is related to rapid burial due to episodic storm activity, while shell-poor taphofacies 1 records background conditions. The nature and comparative diversity of these taphofacies underscores the importance of rapid burial for shell beds preservation. Differences in preservation between taphofacies 2 and 3 are mainly related to environmental criteria, most importantly storm energy and water depth. In contrast, the taxonomic-specific pattern of the communities is a subordinate element of control, controlling only minor within-taphofacies differences in preservation.