Unusual intraindividual variation of the nuclear 18S rRNA gene is widespread within the Acipenseridae

Significant intraindividual variation in the sequence of the 18S rRNA gene is unusual in animal genomes. In a previous study, multiple 18S rRNA gene sequences were observed within individuals of eight species of sturgeon from North America but not in the North American paddlefish, Polyodon spathula, in two species of Polypterus (Polypterus delhezi and Polypterus senegalus), in other primitive fishes (Erpetoichthys calabaricus, Lepisosteus osseus, Amia calva) or in a lungfish (Protopterus sp.). These observations led to the hypothesis that this unusual genetic characteristic arose within the Acipenseriformes after the presumed divergence of the sturgeon and paddlefish families. In the present study, a survey of nearly all Eurasian acipenseriform species was conducted to examine 18S rDNA variation. Intraindividual variation was not found in the polyodontid species, the Chinese paddlefish, Psephurus gladius, but variation was detected in all Eurasian acipenserid species. The comparison of sequences from two major segments of the 18S rRNA gene and identification of sites where insertion/deletion events have occurred are placed in the context of evolutionary relationships within the Acipenseriformes and the evolution of rDNA variation in this group.     

Alzheimer-like plaque formation by human macrophages is reduced by fibrillation inhibitors and lovastatin

The cerebral deposition of Abeta-peptide as amyloid fibrils and plaques represents a hallmark characteristic of Alzheimer's disease (AD). AD plaques are defined by their green birefringence after Congo red staining, their spherulite-like superstructure and their association with specific secondary components. Here we show that primary human macrophages promote the formation of amyloid plaques that correspond in all aforementioned characteristics to typical amyloid plaques from diseased tissues: they consist of aggregated Abeta-peptide, they reveal the typical 'Maltese cross" structure and they are associated with the secondary components glycosaminoglycanes, apolipoprotein E (apoE) and the raft lipids cholesterol and sphingomyelin. Plaque formation can be impaired in this cell system by addition of small molecules, such as Congo red, melantonine and lovastatin, suggesting potential applications for the study of cellular amyloid formation and for the identification or validation of drug candidates.     

Proteomic profiling of Cronobacter turicensis 3032, a food‐borne opportunistic pathogen

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface‐associated and whole‐cell proteins by two complementary proteomics approaches, 1D‐SDS‐PAGE combined with LC‐ESI‐MS/MS and 2D‐LC‐MALDI‐TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin‐receptor protein for the uptake of siderophore‐bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.     

First evidence of a membrane‐bound,tyramine and β‐phenylethylamine producing,tyrosine decarboxylase in Enterococcus faecalis: A two‐dimensional electrophoresis proteomic study

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of β‐phenylethylamine. Kinetics of tyramine and β‐phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine‐enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to β‐phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2‐DE and MALDI‐TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and β‐phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down‐regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy‐supplying routes. The most interesting finding is a membrane‐bound TDC highly over‐expressed during amine production. This is the first evidence of a true membrane‐bound TDC, longly suspected in bacteria on the basis of the gene sequence.     

Quantitative detection of changes in the leaf‐mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

Although the vacuole is the most important final store for toxic heavy metals like cadmium (Cd²⁺), our knowledge on how they are transported into the vacuole is still insufficient. It has been suggested that Cd²⁺ can be transported as phytochelatin‐Cd²⁺ by an unknown ABC transporter or in exchange with protons by cation/proton exchanger (CAX) transporters. To unravel the contribution of vacuolar transporters to Cd²⁺ detoxification, a quantitative proteomics approach was performed. Highly purified vacuoles were isolated from barley plants grown under minus, low (20 μM), and high (200 μM) Cd²⁺ conditions and protein levels of the obtained tonoplast samples were analyzed using isobaric tag for relative and absolute quantitation (iTRAQ?). Although 56 vacuolar transporter proteins were identified, only a few were differentially expressed. Under low‐Cd²⁺ conditions, an inorganic pyrophosphatase and a γ‐tonoplast intrinsic protein (γ‐TIP) were up‐regulated, indicating changes in energization and water fluxes. In addition, the protein ratio of a CAX1a and a natural resistance‐associated macrophage protein (NRAMP), responsible for vacuolar Fe²⁺ export was increased. CAX1a might play a role in vacuolar Cd²⁺ transport. An increase in NRAMP activity leads to a higher cytosolic Fe²⁺ concentration, which may prevent the exchange of Fe²⁺ by toxic Cd²⁺. Additionally, an ABC transporter homolog to AtMRP3 showed up‐regulation. Under high Cd²⁺ conditions, the plant response was more specific. Only a protein homologous to AtMRP3 that showed already a response under low Cd²⁺ conditions, was up‐regulated. Interestingly, AtMRP3 is able to partially rescue a Cd²⁺‐sensitive yeast mutant. The identified transporters are good candidates for further investigation of their roles in Cd²⁺ detoxification.     

Mapping Loci Associated With Tail Color and Sex Determination in the Short-Lived Fish Nothobranchius furzeri

The African fish Nothobranchius furzeri is the shortest-lived vertebrate species that can reproduce in captivity, with a median life span of 9–11 weeks for the shortest-lived strain. Natural populations of N. furzeri display differences in life span, aging biomarkers, behavior, and color, which make N. furzeri a unique vertebrate system for studying the genetic basis of these traits. We mapped regions of the genome involved in sex determination and tail color by genotyping microsatellite markers in the F₂ progeny of a cross between a short-lived, yellow-tailed strain and a long-lived, red-tailed strain of N. furzeri. We identified one region linked with the yellow/red tail color that maps close to melanocortin 1 receptor (mc1r), a gene involved in pigmentation in several vertebrate species. Analysis of the segregation of sex-linked markers revealed that N. furzeri has a genetic sex determination system with males as the heterogametic sex and markedly reduced recombination in the male sex-determining region. Our results demonstrate that both naturally-evolved pigmentation differences and sex determination in N. furzeri are controlled by simple genetic mechanisms and set the stage for the molecular genetic dissection of factors underlying such traits. The microsatellite-based linkage map we developed for N. furzeri will also facilitate analysis of the genetic architecture of traits that characterize this group of vertebrates, including short life span and adaptation to extreme environmental conditions.THE Nothobranchius fish species are present in eastern and southeastern Africa, where they populate ephemeral water pools that often undergo complete desiccation during the dry season (Terzibasi et al. 2008; Reichard et al. 2009; Wildekamp 2009). Nothobranchius species tend to live in extreme habitats and have evolved unique adaptations to harsh environmental conditions, including extremely short life cycles, resistance to a wide range of temperatures and water salinity, embryonic development that does not require the presence of water, and a developmental diapause that allows embryos to survive for months in dry conditions (Wourms 1972; Inglima et al. 1981; Genade et al. 2005).Nothobranchius furzeri is the shortest-lived species of the Nothobranchius genus, with an intergeneration time of 40 days, a median life span of 9–11 weeks, and a maximum life span of 12–15 weeks for the shortest-lived strain GRZ (Valdesalici and Cellerino 2003; Genade et al. 2005; Valenzano et al. 2006; Terzibasi et al. 2008, 2009; Hartmann et al. 2009). Natural populations of N. furzeri can vary substantially in phenotypic traits. For example, N. furzeri strains derived from Zimbabwe and northern Mozambique (e.g., GRZ) exhibit a shorter life span than strains derived from more humid areas in southern Mozambique (e.g., MZM-0403) under controlled conditions (Terzibasi et al. 2008). The extremely short life cycle of N. furzeri and the presence of natural populations with phenotypic variations make this species a promising model system for studying aging and adult-specific traits, including color and behavior.The color pattern of the adult male tail differs among N. furzeri strains. GRZ males show a yellow submarginal band and a black marginal band (yellow morph) whereas MZM-0403 males display a broad red band (red morph) in the caudal fin (Figure 1A). This dichromatism is present in natural populations of N. furzeri (Terzibasi et al. 2008; Reichard et al. 2009). Similar color polymorphism among males is also observed in other species of Nothobranchius (Wildekamp 2009) and in other fish species, including guppies and cichlids (Hughes et al. 1999; Brooks and Endler 2001; Maan et al. 2004). Differences in male color morphs within the same species are associated with sexual preference by females, different recognition by predators depending on the habitat, and differential susceptibility to pathogens (Price et al. 2008), which could all influence the evolution of this trait. Despite the widespread variation in Nothobranchius coloration, the genetic basis of this trait is unknown.Open in a separate windowFigure 1.—Cross between two strains of N. furzeri that differ in color and life span (A) Color phenotypes of GRZ and MZM-0403. (B) A yellow-tailed, short-lived male GRZ and a red-tailed, long-lived female MZM-0403 were the founders of cross 1.Genetic information on N. furzeri is still limited. The N. furzeri genome is 1.6–1.9 Gb in size and is characterized by a high repeat content (45%) (Reichwald et al. 2009). N. furzeri has 19 chromosomes, but no morphologically discernible sex chromosomes (Reichwald et al. 2009). The sex determination system in N. furzeri has not been characterized yet. Sex can be determined either genetically or environmentally in fish (Volff 2005; Marshall Graves 2008). For example, medaka, platyfish, guppy, and sticklebacks all have recently evolved genetic sex-determining systems (Volff and Schartl 2002; Peichel et al. 2004; Shapiro et al. 2009; Tripathi et al. 2009a), whereas zebrafish do not have a clear genetic basis of sex determination (von Hofsten and Olsson 2005).Genetic studies in N. furzeri would greatly benefit from the building of a linkage map in this species of fish. However, to date there is no linkage map available for N. furzeri or for any Nothobranchius species, although linkage maps have been generated for fish of the same order, e.g., Poecilia reticulata (guppy) and Xiphophorus maculatus (platyfish) (Khoo et al. 2003; Walter et al. 2004; Tripathi et al. 2009b), and of the same superorder, e.g., Oryzias latipes (medaka) (Wada et al. 1995).Here, we report a microsatellite-based linkage map for N. furzeri using a genetic cross between the short-lived yellow-tailed GRZ strain and the long-lived red-tailed MZM-0403 strain. This N. furzeri linkage map allowed us to map the male-specific tail color trait on linkage group (LG) V. Synteny analysis revealed that LG V has homology to a region of the medaka genome that contains the melanocortin 1 receptor (mc1r) gene, which is known to play a key role in vertebrate pigmentation. We identified a sequence polymorphism in mc1r between the two strains of N. furzeri, allowing us to map mc1r on LG V. This analysis revealed that mc1r is located in close proximity to the color locus, but that the sequence polymorphism is probably not causative for the color difference. We also found that sex is genetically determined in N. furzeri, with males as the heterogametic sex. The sex-determining region is located on LG XIII and is characterized by male-specific suppression of recombination. Our findings will be pivotal for the identification of the genetic determinants of color in N. furzeri and for expanding our knowledge about sex-determination mechanisms in vertebrates. Due to the array of intraspecific phenotypic differences displayed by the various populations of N. furzeri, this linkage map will also be a key tool for mapping phenotypic variation in this short-lived vertebrate species, including differences in life span.     

Src-protein tyrosine kinases are required for cocaine-induced increase in the expression and function of the NMDA receptor in the ventral tegmental area

Cocaine-induced long-term potentiation of glutamatergic synapses in the ventral tegmental area (VTA) has been proposed as a key process that contributes to the development of addictive behaviors. In particular, the activation of ionotrophic glutamate NMDA receptor (NMDAR) in the VTA is critical for the initiation of cocaine sensitization. Here we show that application of cocaine both in slices and in vivo induced an increase in tyrosine phosphorylation of the NR2A, but not the NR2B subunit of the NMDAR in juvenile rats. Cocaine induced an increase in the activity of both Fyn and Src kinases, and the Src-protein tyrosine kinase (Src-PTKs) inhibitor, 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4-d]pyrimidine (PP2), abolished both cocaine-induced increase in tyrosine phosphorylation of the NR2A subunit and the increase in the expression of NR1, NR2A, and NR2B in the VTA. Moreover, cocaine-induced enhancement in NMDAR-mediated excitatory post-synaptic currents was completely abolished by PP2. Taken together, these results suggest that acute cocaine induced an increase in the expression of NMDAR subunits and enhanced tyrosine phosphorylation of NR2A-containing NMDAR through members of the Src-PTKs. This in turn, increased NMDAR-mediated currents in VTA dopamine neurons. These results provide a potential cellular mechanism by which cocaine triggers NMDAR-dependent synaptic plasticity of VTA neurons that may underlie the development of behavioral sensitization.     

De novo biosynthesis of glycosaminoglycans in the extracellular matrix of skin studied by matrix-assisted laser desorption/ionization mass spectrometry

The self-healing capacity of skin is limited, and medical intervention is often unavoidable. Skin may be generated ex vivo from cultured fibroblasts. Because the molecular composition of de novo formed skin (mostly collagen and glycosaminoglycans [GAGs]) is crucial, analytical methods are required for the quality control of tissue-engineered products. Here, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of fibroblast cultures subsequent to digestion with chondroitinase ABC is a reliable and fast method to monitor the GAG content of native and bioengineered skin. Furthermore, the supplementation of the fibroblast medium with 13C-labeled glucose provides insights into the biosynthesis of GAGs.     

Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition

In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.     

HIF-1α protein is upregulated in HIF-2α depleted cells via enhanced translation

The hypoxia-inducible factors HIF-1 and HIF-2 are primarily regulated via stabilization of their respective α-subunits under hypoxic conditions. Previously, compensatory upregulation of one HIF-α-subunit upon depletion of the other α-subunit was described, yet the underlying mechanism remained elusive. Here we provide evidence that enhanced HIF-1α protein expression in HIF-2α knockdown (k/d) cells neither results from elevated HIF-1α mRNA expression, nor from increased HIF-1α protein stability. Instead, we identify enhanced HIF-1α translation as molecular mechanism. Moreover, we found elevated levels of the RNA-binding protein HuR and provide evidence that HuR is critical for the compensatory HIF-1α regulation in HIF-2α k/d cells.