PrP(106-126) activates neuronal intracellular kinases and Egr1 synthesis through activation of NADPH-oxidase independently of PrPc

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106-126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106-126) in cortical neurons and the participation of PrPc. We show that PrP(106-126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.     

Phosphorylation of the RGS protein Sst2 by the MAP kinase Fus3 and use of Sst2 as a model to analyze determinants of substrate sequence specificity

Previously, we used mass spectrometry to demonstrate pheromone-stimulated phosphorylation of Ser-539 in Sst2, a regulator of G protein signaling in yeast Saccharomyces cerevisiae [Garrison, T. R., et al. (1999) J. Biol. Chem. 274, 36387-36391]. Here, we show that Sst2 phosphorylation is mediated by the mitogen-activated protein (MAP) kinase Fus3. Phosphorylation occurs within a canonical MAP kinase phosphorylation site (Pro-X-Ser/Thr-Pro, where "X" at the -1 position can be any amino acid), a consensus sequence deduced earlier from analysis of synthetic peptide substrates. In a direct test of the model, we compared Sst2 phosphorylation following systematic substitution of the -1 residue His-538. Each of the substitution mutants was suitable as a MAP kinase substrate, as shown by phosphorylation-dependent mobility shifts in vivo and/or by direct phosphorylation in vitro followed by peptide mapping and mass spectrometry sequencing. This analysis documents phosphorylation of Sst2 by Fus3 and demonstrates that the prevailing model for MAP kinase recognition is valid for a native substrate protein in vivo as well as for small synthetic peptides tested in vitro.     

Genetic heterogeneity in Italian families with IgA nephropathy: suggestive linkage for two novel IgA nephropathy loci

IgA nephropathy (IgAN) is the most common glomerulonephritis worldwide, but its etiologic mechanisms are still poorly understood. Different prevalences among ethnic groups and familial aggregation, together with an increased familial risk, suggest important genetic influences on its pathogenesis. A locus for familial IgAN, called "IGAN1," on chromosome 6q22-23 has been described, without the identification of any responsible gene. The partners of the European IgAN Consortium organized a second genomewide scan in 22 new informative Italian multiplex families. A total of 186 subjects (59 affected and 127 unaffected) were genotyped and were included in a two-stage genomewide linkage analysis. The regions 4q26-31 and 17q12-22 exhibited the strongest evidence of linkage by nonparametric analysis (best P=.0025 and .0045, respectively). These localizations were also supported by multipoint parametric analysis, in which peak LOD scores of 1.83 ( alpha =0.50) and 2.56 ( alpha =0.65) were obtained using the affected-only dominant model, and by allowance for the presence of genetic heterogeneity. Our results provide further evidence for genetic heterogeneity among families with IgAN. Evidence of linkage to multiple chromosomal regions is consistent with both an oligo/polygenic and a multiple-susceptibility-gene model for familial IgAN, with small or moderate effects in determining the pathological phenotype. Although we identified new candidate regions, replication studies are required to confirm the genetic contribution to familial IgAN.     

Reactive extraction of acylglycerides using a column bioreactor containing Rhizopus oryzae resting-cells

The work evaluated the use of a bioreactor for the enzymatic transesterification of oilseed. Rhizopus oryzae resting-cells, isolated from Foeniculum vulgare Fennel, were used as a biocatalyst. The bioreactor consisted of two glass-jacketed flasks, interconnected by a circuit using isooctane as a solvent, which was recirculated with the help of a peristaltic pump. The system enabled the extraction and hydrolysis of triacylglycerides (TAG), and the subsequent esterification of the free fatty acids formed (FFA). Esterification was carried out by the addition of alcohol using a peristaltic pump. This reactive extraction of vegetable oil was optimized for sunflower seeds using 4-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (solketal) as alcohol. Subsequently, a variety of oil seeds and alcohols were tested. Final ester conversions ranged from 84 to 98% depending on the alcohol and the oilseed used.     

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate—one where methanogenesis was allowed and another in which it was completely inhibited with 2‐bromoethane sulfonate. We observed a three‐way syntrophy among ethanol fermenters, acetate‐oxidizing anode‐respiring bacteria (ARB), and a H₂ scavenger. When methanogenesis was allowed, H₂‐oxidizing methanogens were the H₂ scavengers, but when methanogenesis was inhibited, homo‐acetogens became a channel for electron flow from H₂ to current through acetate. We established the presence of homo‐acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo‐acetogens. Both methods documented the presence of the homo‐acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol‐fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H₂‐scavenging syntrophic partners that were either H₂‐oxidizing methanogens or homo‐acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron‐flow distribution and H₂‐scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69–78. © 2009 Wiley Periodicals, Inc.     

Proteasome inhibitors shorten replicative life span and induce a senescent-like phenotype of human fibroblasts

The proteasome constitutes the main non-lysosomal cellular protease activity, and plays a crucial role not only in the disposal of unwanted material, but also in the regulation of numerous cellular processes. Previously, we have reported that during the replicative senescence of WI-38 fibroblasts there is a significant impairment in proteasome activity, which probably has important implications in the control of MAPK signaling and cellular proliferation. In this study, we report the potential role of the proteasome in the generation of the senescent phenotype in WI-38 fibroblasts. Our results indicate that inhibition of proteasome activity leads to an impairment in cell proliferation, and a shortening of the life span. The results also indicate that inhibition of the proteasome in young cells induces a premature senescent-like phenotype, as indicated by the increase in senescence-associated beta-galactosidase (SA beta-gal) activity and the abundance of both p21 and collagenase mRNAs, as well as a decreased level of EPC-1 mRNA known markers of cellular senescence, not previously shown to depend on proteasome activity. Together, our results suggest a molecular mechanism for the lack of responsiveness of human cells to growth factors, and point towards a role for the proteasome in the control of the life span of both cells and organisms.     

Physiological and biochemical responses of fruit exocarp of tomato (Lycopersicon esculentum Mill.) mutants to natural photo-oxidative conditions

Photo-oxidative stress was imposed under natural solar radiation on exposed and shaded sections of detached fruit of immature green tomato (Lycopersicon esculentum Miller = Solanum lycopersicum L.) mutants (anthocyanin absent, beta-carotene, Delta, and high pigment-1) and their nearly isogenic parents ('Ailsa Craig' and 'Rutgers'). After 5 h exposure to high solar irradiance, either with or without ultraviolet (UV) radiation, surface colour changes, pigment composition, photosynthetic efficiency, antioxidant metabolites and enzyme activities, and selected flavonoids and antioxidant proteins in exocarp tissue were evaluated. The imposed photo-oxidative stress reproduced the symptoms observed on attached fruit. Both high temperature and solar irradiance caused fruit surface discoloration with faster degradation of chlorophyll (Chl) than carotenoids (Car), leading to an increase in the Car/Chl ratio. Surface bleaching was mostly caused by visible light, whereas elevated temperatures were mostly responsible for the inactivation of photosynthesis, measured as decreased F(v)/F(m). Ascorbate, glutathione, and total soluble protein concentrations decreased in the exocarp as the duration of exposure increased. Specific activities of superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), and catalase increased with exposure, suggesting that these proteins were conserved during the imposed stress. GR protein expression remained stable during the imposed stress, whereas, MDHAR protein expression increased. Quercetin and kaempferol concentrations increased rapidly upon exposure, but not to UV radiation, suggesting rapid photo-protection in response to visible light; however, naringenin synthesis was not induced. The apparent increased tolerance of hp-1 fruit is discussed.     

Functional cGMP-gated channels in cerebellar granule cells

Cyclic nucleotide-gated channels (CNGCs) are important transducers of external signals in sensory processes. These channels are ubiquitously expressed in a variety of neurons, and are necessary to transduce signals for growth cone guidance and plasticity. Here, we demonstrate that the CNGC subunits (CNGA1 and CNGB1, presumably the 1b isoform) are expressed in rat cerebellar granule cells and that they combine to form functional channels. The expression of the mRNAs that encode these proteins is maximal after 7 days in cell culture, when the channels are expressed at synapses and co-localize with the synaptic marker synapsin I. These ligand-gated channels are functional and can be blocked by Mg(2+) or L-cis-diltiazem. Moreover, channel opening in response to increases in intracellular cGMP results in Ca(2+) entry into the cell. Chronic blockade (96 h) of these channels with L-cis-diltiazem significantly decreases the number of functional boutons, as determined by their capacity to load and unload the styryl dye FM1-43 when stimulated. Moreover, the unloading kinetics is modified from a biphasic to a monophasic profile in a subset of synaptic boutons. These channels are also expressed in early developmental stages, both in the soma and in emerging processes, and CNGA1 can be detected in growth cones. Pharmacological blockade of these channels with L-cis-diltiazem causes an overall change in growth cone morphology, impairing the formation of lamellipodia between filopodia and increasing the number of filopodia. J     

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ～threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.