Modelling the impact of geometric parameters on the redox potential of blue copper proteins

The synthesis and structure of a homologous series of cationic N(2)S(2) copper(I) Schiff base complexes constructed using o-tert-butylthiobenzaldehyde and a series of terminal diamines (ethane, propane, butane) are reported. The complexes differ only in the length of the methylene chain between the imine groups. This simple modification forces the copper centre to shift geometry from a planar (1,2-diaminoethane) to a more distorted tetrahedral motif (1,4-diaminobutane). The redox potentials of the three cations were measured using cyclic voltammetry in donor (acetonitrile) and non-donor solvents (dichloromethane). The S-Cu-N angles for each complex are correlated against the respective redox potential allowing an analysis of the geometric impact on the redox potential in soft copper centres. The redox potential is observed to increase as the metal centre moves from a planar towards a tetrahedral motif. Comparing this data with the reported structures of the blue copper proteins (rusticyanin and plastocyanin) allows an assessment of the contribution of the geometry of the metal binding site to the operating potential of these proteins to be made.     

Extensive carbon isotopic heterogeneity among methane seep microbiota

To assess and study the heterogeneity of δ¹³C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the δ¹³C compositions of methane seep-associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal δ¹³C values (> 50‰) in this microbial community. The δ¹³C of ANME-1 rods ranged from −24‰ to −87‰. The δ¹³C of ANME-2 sarcina ranged from −18‰ to −75‰. Initial measurements of shell aggregates were as heavy as −19.5‰ with none observed to be lighter than −57‰. Subsequent measurements on shell aggregates trended lighter reaching values as ¹³C-depleted as −73‰. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more ¹³C-depleted (with values as light as −56‰). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME-1 and ANME-2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean δ¹³C = −38 ± 3‰), suggesting that this bacterial morphotype was capable of rapid colonization and growth.     

Identification of adult mineralized tissue zebrafish mutants

Zebrafish craniofacial, skeletal, and tooth development closely resembles that of higher vertebrates. Our goal is to identify viable adult zebrafish mutants that can be used as models for human mineralized craniofacial, dental, and skeletal system disorders. We used a large-scale forward-genetic chemical N-ethyl-nitroso-urea mutagenesis screen to identify 17 early lethal homozygous recessive mutants with defects in craniofacial cartilage elements, and 7 adult homozygous recessive mutants with mineralized tissue phenotypes including craniofacial shape defects, fused sutures, dysmorphic or missing skeletal elements, scoliosis, and neural arch defects. One mutant displayed both an early lethal homozygous phenotype and an adult heterozygous phenotype. These results extend the utility of the zebrafish model beyond the embryo to study human bone and cartilage disorders.     

Alterations in the mitochondrial proteome of neuroblastoma cells in response to complex 1 inhibition

Increasing evidence points to mitochondrial dysfunction in Parkinson's disease (PD) associated with complex I dysfunction, but the exact pathways which lead to cell death have not been resolved. 2D-gel electrophoresis profiles of isolated mitochondria from neuroblastoma cells treated with subcytotoxic concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well-characterized complex I inhibitor, were assessed to identify associated targets. Up to 27 differentially expressed proteins were observed, of which 16 were identified using peptide mass fingerprinting. Changes in protein levels were validated by immunoprobing 1D blots, confirming increases in heat shock cognate 71 kDa (Hsc70), 60 kDa heat shock protein (Hsp60), fumarase, glutamate oxaloacetate transaminase 2, ATP synthase subunit d, and voltage-dependent anion-channel 1 (VDAC1). Immunoprobing of 2D blots revealed isoform changes in Hsc70, Hsp60, and VDAC1. Subcytotoxic concentrations of MPTP modulated a host of mitochondrial proteins including chaperones, metabolic enzymes, oxidative phosphorylation-related proteins, an inner mitochondrial protein (mitofilin), and an outer mitochondrial membrane protein (VDAC1). Early changes in chaperones suggest a regulated link between complex 1 inhibition and protein folding. VDAC1, a multifunctional protein, may have a key role in signaling between mitochondria and the rest of the cell prior to cell death. Our work provides new important information of relevance to PD.     

Isolation and identification of a 92-kDa stress induced protein from Candida albicans

Mycopathologia - It was previously shown that the presence of estrogen enhances survival of Candida albicans under heat and oxidative stresses. A 92-kDa protein is inducible by heat shock and...     

HPLC-PAD-APCI/MS assay of phenylpropanoids in cereals

A new, rapid HPLC-PAD-APCI/MS assay has been developed in order to measure accurately the amount of p-coumaric, E- and Z-ferulic acid and the dehydrodimers of ferulic acid in cereal grain. In the positive ionisation mode, MS patterns gave additional information for the identification of the dimers. The time required and the quantities of solvents employed in the developed analytical method are much lower than those involved in previously available assays of these compounds, thus making the method suitable for the screening of cereal genotypes. Application of the method to accessions of maize, wheat and sorghum showed that E-ferulic was the most abundant phenylpropanoid, whilst the major dimer was 8-O-4' dehydrodimer of ferulic acid followed by the 5-5' and then the 8-5' forms. Maize grains, especially of the Mexican landraces, contained the highest levels of these dimers.     

Electron and light microscopic study of various glands and the secretions released into the environment by the turbellarian Urastoma cyprinae

The turbellarian Urastoma cyprinae (Graff) occurs on the gills of various bivalve species including the mussel Mytilus galloprovinciallis, where it is known to cause serious damage. More recently, it has been shown that the worms are strongly attracted to the gill of the American oyster (Crassostrea virginica) and are capable of inducing changes to the composition of proteolytic enzymes of the host mucus. Such changes may be attributable to secretory products released by the worms. Mucous glands (11-18 mum in diameter) produce minute spherules (0.7-0.9 mum in diameter) tightly bound together. The glands occupy approximately 20% of the body volume and are the most voluminous secretory organs in the worm. The smaller rhabdoid glands are unevenly distributed throughout the peripheral parenchyma and contain secretory granules of 0.35-1.2 mum in diameter. The latter occur most prominently along the distal margins of the epidermis. The frontal pole of U. cyprinae consists of a complex assembly of mucous and rhabdoid gland cells as well as other glandular structures. Collectively, these bodies release their contents to the outside via narrow gland necks. The overall organization is consistent with the frontal gland previously described for other free-living turbellarians, including other rhabdocoels. A variety of secretory products, displaying variations in staining properties, have likewise been identified in association with the body wall from other regions of the worm. This work attempts to gain a better appreciation of the secretory structures associated with the worm tegument, focusing primarily on the widespread mucous and rhabdoid glands. The secretions play a role in host-parasite interactions.     

Adaptation for horizontal transfer in a homing endonuclease

Selfish genes of no function other than self-propagation are susceptible to degeneration if they become fixed in a population, and regular transfer to new species may be the only means for their long-term persistence. To test this idea we surveyed 24 species of yeast for VDE, a nuclear, intein-associated homing endonuclease gene (HEG) originally discovered in Saccharomyces cerevisiae. Phylogenetic analyses show that horizontal transmission has been a regular occurrence in its evolutionary history. Moreover, VDE appears to be specifically adapted for horizontal transmission. Its 31-bp recognition sequence is an unusually well-conserved region in an unusually well-conserved gene. In addition, the nine nucleotide sites most critical for homing are also unusually well conserved. Such adaptation for horizontal transmission presumably arose as a consequence of selection, both among HEGs at different locations in the genome and among variants at the same location. The frequency of horizontal transmission must therefore be a key feature constraining the distribution and abundance of these genes.     

Meningeal worm is a long-lived parasitic nematode in white-tailed deer

A natural infection of the meningeal worm, Parelaphostrongylus tenuis, persisted for at least 3.7 yr in a white-tailed deer (Odocoileus virginianus). The deer was 5-7 yr old and was shedding dorsal-spined nematode larvae at the time of quarantine. Larvae were extracted from all fecal samples collected up to 730 days post-quarantine (dpq) and thereafter only at 862 dpq and at necropsy (1,350 dpq). Live adults of P. tenuis, one male and one female, were recovered from the cranium at necropsy. Parelaphostrongylus tenuis infections are long lived and latent periods may be extended. Our findings reaffirm the need for reliable antemortem diagnosis to identify non-patent P. tenuis infections to prevent inadvertent introduction of infected animals to non-endemic areas.     

Comparative mapping of Z-orthologous genes in vertebrates: implications for the evolution of avian sex chromosomes

Sex chromosomes of birds and mammals are highly differentiated and share several cytological features. However, comparative gene mapping reveals extensive conserved synteny between the chicken Z sex chromosome and human chromosome 9 but not the human X sex chromosome, implying an independent origin of avian and mammalian sex chromosomes. To better understand the evolution of the avian Z chromosome we analysed the synteny of chicken Z-linked genes in zebrafish, which is the best-mapped teleost genome so far. Existing zebrafish maps do not support the existence of an ancestral Z linkage group in the zebrafish genome, whereas mammalian X-linked genes show at least some degree of synteny conservation. This is consistent with in situ hybridisation mapping data in the freshwater pufferfish, Tetraodon nigroviridis where mammalian X-linked genes show a much higher degree of conserved synteny than human chromosome 9 or the avian Z chromosome. Collectively, these data argue in favour of a more recent evolution of the avian Z chromosome, compared with the mammalian X.