Peptidomics of Cpefat/fat mouse brain regions: implications for neuropeptide processing

Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpe^fat/fat mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpe^fat/fat mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpe^fat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpe^fat/fat mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpe^fat/fat mice; these peptide processing intermediates with C‐terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.     

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.     

Evidence for the involvement of endothelial cell products in adrenal CITED2 expression

The adrenal gland contains a well-organized network of blood vessels, and adrenocortical cells are situated in close proximity to endothelial cells. Recently, several new mechanisms have been characterized that control the release of aldosterone by adrenocortical cells, including the involvement of endothelial-cell-derived factors. Interestingly, a CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), which is necessary for adrenal development, has been linked to aldosterone synthesis. We have therefore examined the effects of endothelial-cell-conditioned medium (ECCM), as produced during the incubation of human umbilical vein endothelial cells for 24 h, on the promoter activity and mRNA and protein expression of CITED2 in adrenocortical cells as represented by the NCI-H295R cell line. We have found a dose-dependent effect of ECCM on CITED2 promoter activity; this peaks at 480%. Activation of the CITED2 promoter occurs in parallel to an increase in CITED2 messenger RNA (as quantified by real-time polymerase chain reaction) and protein. The stimulatory effect of ECCM can be reversed by blocking mitogen-activated protein kinase activity with the MEK1-inhibitor PD98059. We conclude that products secreted by endothelial cells control not only steroidogenesis, but also factors that are important for adrenocortical development, thereby highlighting the role of cellular interactions within adrenocortical development and physiology. This work was supported by a grant from the Doktor Robert Pfleger-Stiftung, Bamberg, Germany, to H.S.W.     

Manufacture of stable palladium and gold nanoparticles on native and genetically engineered flagella scaffolds

The use of bacterial flagella as templates for the immobilization of Pd and Au nanoparticles is described. Complete coverage of D. desulfuricans flagellar filaments by Pd(0) nanoparticles was obtained via the H(2)-mediated reduction of Pd(NH3)4]Cl2 but similar results were not obtained using HAuCl4. The introduction of additional cysteine-derived thiol residues in the E. coli FliC protein increased Au(III) sorption and reduction onto the surface of the flagellar filament and resulted in the production of stabilized Au(0) nanoparticles of approximately 20-50 nm diameter. We demonstrate the application of molecular engineering techniques to manufacture biologically passivated Au(0) nanoparticles of a size suitable for catalytic applications.     

The Ca2+ Sensor Protein Swiprosin-1/EFhd2 Is Present in Neurites and Involved in Kinesin-Mediated Transport in Neurons

Swiprosin-1/EFhd2 (EFhd2) is a cytoskeletal Ca²⁺ sensor protein strongly expressed in the brain. It has been shown to interact with mutant tau, which can promote neurodegeneration, but nothing is known about the physiological function of EFhd2 in the nervous system. To elucidate this question, we analyzed EFhd2^−/−/lacZ reporter mice and showed that lacZ was strongly expressed in the cortex, the dentate gyrus, the CA1 and CA2 regions of the hippocampus, the thalamus, and the olfactory bulb. Immunohistochemistry and western blotting confirmed this pattern and revealed expression of EFhd2 during neuronal maturation. In cortical neurons, EFhd2 was detected in neurites marked by MAP2 and co-localized with pre- and post-synaptic markers. Approximately one third of EFhd2 associated with a biochemically isolated synaptosome preparation. There, EFhd2 was mostly confined to the cytosolic and plasma membrane fractions. Both synaptic endocytosis and exocytosis in primary hippocampal EFhd2^−/− neurons were unaltered but transport of synaptophysin-GFP containing vesicles was enhanced in EFhd2^−/− primary hippocampal neurons, and notably, EFhd2 inhibited kinesin mediated microtubule gliding. Therefore, we found that EFhd2 is a neuronal protein that interferes with kinesin-mediated transport.     

Sequential Utilization of Hosts from Different Fly Families by Genetically Distinct,Sympatric Populations within the Entomophthora muscae Species Complex

The fungus Entomophthora muscae (Entomophthoromycota, Entomophthorales, Entomophthoraceae) is a widespread insect pathogen responsible for fatal epizootic events in many dipteran fly hosts. During epizootics in 2011 and 2012 in Durham, North Carolina, we observed a transition of fungal infections from one host, the plant-feeding fly Delia radicum, to a second host, the predatory fly Coenosia tigrina. Infections first appeared on Delia in the middle of March, but by the end of May, Coenosia comprised 100% of infected hosts. Multilocus sequence typing revealed that E. muscae in Durham comprises two distinct subpopulations (clades) with several haplotypes in each. Fungi from either clade are able to infect both fly species, but vary in their infection phenologies and host-specificities. Individuals of the more phylogenetically diverse clade I predominated during the beginning of the spring epizootic, infecting mostly phytophagous Delia flies. Clade II dominated in late April and May and affected mostly predatory Coenosia flies. Analysis of population structure revealed two subpopulations within E. muscae with limited gene exchange. This study provides the first evidence of recombination and population structure within the E. muscae species complex, and illustrates the complexity of insect-fungus relationships that should be considered for development of biological control methods.     

Establishment of transgenic lettuce plants producing potentially antihypertensive ShRNA

Development of RNAi-based therapeutics is a fast growing field of the pharmaceutical industry. Using plants for production of pharmaceutically valuable siRNAs may have significant advantages of costeffectiveness, scalability, and low risk of contamination with human pathogens. If edible plant species are genetically engineered to synthesize siRNAs, the costly stage of target product purification may be omitted. We describe the establishment of transgenic lettuce plants producing shRNA targeting delta isoform of protein kinase C (PKC-delta), an effective target for RNAi-based treatment of arterial hypertension. Transgenic lettuce plants were obtained by Agrobacterium-mediated transformation with genetic constructs harboring antiPKC and scrambled (control) shRNA genes. The presence of transgenes was proven by PCR analysis, and the accumulation of antiPKC shRNA was estimated using the RT-qPCR technique. Six transgenic lettuce lines showed varying levels of antiPKC shRNA expression with the highest value reaching 14 ± 9% of highly abundant endogenous lettuce micro RNA (miR156a), or 12.7 fmol/g dry weight. Plants carrying either antiPKC or scrambled shRNA genes flowered normally but did not produce seeds. The described transgenic lettuce plants accumulating antiPKC siRNA are the subject for animal testing and can be considered as raw material for the development of novel antihypertensive drugs.     

Neurotoxicity of Alzheimer's disease A�� peptides is induced by small changes in the A��42 to A��40 ratio

The amyloid peptides Aβ₄₀ and Aβ₄₂ of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ₄₂ seems more pathogenic than Aβ₄₀, the reason for this is not well understood. We show here that small alterations in the Aβ₄₂:Aβ₄₀ ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ₄₂:Aβ₄₀ ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates.