Neurotoxicological Examination of the Piglet Brain after Prenatal and Postnatal Exposure to Trichlorfon

Piglets were given 10 repeated doses of the orga-nophosphorus compound trichlorfon during the postnatal period in order to examine the effect on the brain development (Experiment 1). Following prenatal exposure to trichlorfon, the ability of a presumptive hypoplastic cerebellum and cerebrum at birth, to regenerate postnatally was investigated (Experiment 2). Administration of repeated doses of trichlorfon postnatally was accompanied by only small changes in brain weights, morphology and transmitter enzyme activity (choline acetyltransferase, glutamate decarboxylase, aromatic amino acid decarboxylase) in 35 days old piglets. Animals exposed prenatally, and sacrificed at the age of 35 days, showed a significant increase in brain weights and enzyme activities. The animals did, however, not reach control values in cerebral weight, cerebellar weight or total enzyme activities. Morphological changes still showed regional loss of Purkinje cells in the cerebellum. The study clearly indicated that the pig brain was less vulnerable to trichlorfon in the postnatal period of development than when exposed to the compound prenatally.     

The ontogeny of the uptake systems for glutamate,GABA, and glycine in synaptic vesicles isolated from rat brain

The ontogeny of the uptake of glutamate, GABA and glycine into synaptic vesicles isolated from rat brain has been investigated. The vesicular uptake of the three amino acids increased with developmental age in parallel with synaptogenesis, indicating a functional role of uptake of the amino acids by synaptic vesicles in the nerve terminals. Uptake of the amino acids by plasma membrane particles (synaptosomes) in brain homogenate showed a somewhat different developmental profile. The uptake of glutamate increased markedly with developmental time, while the uptake of GABA showed only a slight increase. Uptake of glycine by plasma membrane particles was very low and therefore not registered. The observed developmental increase in uptake of glycine by synaptic vesicles isolated from brain, supports previous reports indicating that glycine can be taken up by vesicles from non-glycine terminals.Special issue dedicated to Dr. Morris H. Aprison.     

Population Structure of a Hybrid Clonal Group of Methicillin-Resistant Staphylococcus aureus,ST239-MRSA-III

The methicillin-resistant Staphylococcus aureus (MRSA) clonal group known as ST239-MRSA-III is notable for its hybrid origin and for causing sustained hospital epidemics worldwide since the late 1970s. We studied the population structure of this MRSA clonal group using a sample of 111 isolates that were collected over 34 years from 29 countries. Genetic variation was assessed using typing methods and novel ascertainment methods, resulting in approximately 15 kb of sequence from 32 loci for all isolates. A single most parsimonious tree, free of homoplasy, partitioned 28 haplotypes into geographically-associated clades, including prominent European, Asian, and South American clades. The rate of evolution was estimated to be approximately 100× faster than standard estimates for bacteria, and dated the most recent common ancestor of these isolates to the mid-20th century. Associations were discovered between the ST239 phylogeny and the ccrB and dru loci of the methicillin resistance genetic element, SCCmec type III, but not with the accessory components of the element that are targeted by multiplex PCR subtyping tools. In summary, the evolutionary history of ST239 can be characterized by rapid clonal diversification that has left strong evidence of geographic and temporal population structure. SCCmec type III has remained linked to the ST239 chromosome during clonal diversification, but it has undergone homoplasious losses of accessory components. These results provide a population genetics framework for the precise identification of emerging ST239 variants, and invite a re-evaluation of the markers used for subtyping SCCmec.     

cAMP effector mechanisms. Novel twists for an 'old' signaling system

Cyclic AMP (cAMP) has traditionally been thought to act exclusively through cAMP-dependent protein kinase (cAPK, PKA), but a growing number of cAMP effects are not attributable to general activation of cAPK. At present, cAMP is known also to directly regulate ion channels and the ubiquitous Rap guanine exchange factors Epac 1 and 2. Adding to the sophistication of cAMP signaling is the fact that (1) the cAPK holoenzyme is incompletely dissociated even at saturating cAMP, the level of free R subunit of cAPK being able to regulate the maximal activity of cAPK, (2) cAPK activity can be modulated by oxidative glutathionylation, and (3) cAPK is anchored close to relevant substrates, other signaling enzymes, and local compartments of cAMP. Finally, we will demonstrate an example of fine-tuning of cAMP signaling through synergistic induction of neurite extensions by cAPK and Epac.     

cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension

Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAPKII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N6-benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2'-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2'-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10-100-fold. Phenylthio substituents in position 8, particularly with MeO- or Cl- in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT- and 2'-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2'-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.     

The surface-associated and secreted MopE protein of Methylococcus capsulatus (Bath) responds to changes in the concentration of copper in the growth medium

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress.     

Characterization of a prokaryotic haemerythrin from the methanotrophic bacterium Methylococcus capsulatus (Bath)

For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing differential protein expression in the methane-oxidizing bacterium, Methylococcus capsulatus (Bath), grown at a high and low copper-to-biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high-copper cultures. Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms. The diiron site is located in a hydrophobic pocket and is capable of binding O(2). We cloned the M. capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA. The haemerythrin protein was purified to homogeneity cleaved from its fusion partner. Recombinant M. capsulatus haemerythrin (McHr) was found to fold into a stable protein. Sequence similarity analysis identified all the candidate residues involved in the binding of diiron (His22, His58, Glu62, His77, His81, His117, Asp122) and the amino acids forming the hydrophobic pocket in which O(2) may bind (Ile25, Phe59, Trp113, Leu114, Ile118). We were also able to model a three-dimensional structure of McHr maintaining the correct positioning of these residues. Furthermore, UV/vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr. A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily.     

The potential for cleaner fish‐driven evolution in the salmon louse Lepeophtheirus salmonis: Genetic or environmental control of pigmentation?

The parasitic salmon louse represents one of the biggest challenges to environmentally sustainable salmonid aquaculture across the globe. This species also displays a high evolutionary potential, as demonstrated by its rapid development of resistance to delousing chemicals. In response, farms now use a range of non‐chemical delousing methods, including cleaner fish that eat lice from salmon. Anecdotal reports suggest that in regions where cleaner fish are extensively used on farms, lice have begun to appear less pigmented and therefore putatively less visible to cleaner fish. However, it remains an open question whether these observations reflect a plastic (environmental) or adaptive (genetic) response. To investigate this, we developed a pigment scoring system and conducted complimentary experiments which collectively demonstrate that, a) louse pigmentation is strongly influenced by environmental conditions, most likely light, and b) the presence of modest but significant differences in pigmentation between two strains of lice reared under identical conditions. Based on these data, we conclude that pigmentation in the salmon louse is strongly influenced by environmental conditions, yet there are also indications of underlying genetic control. Therefore, lice could display both plastic and adaptive responses to extensive cleaner fish usage where visual appearance is likely to influence survival of lice.     

GeneTools – application for functional annotation and statistical hypothesis testing

Background  

Modern biology has shifted from "one gene" approaches to methods for genomic-scale analysis like microarray technology, which allow simultaneous measurement of thousands of genes. This has created a need for tools facilitating interpretation of biological data in "batch" mode. However, such tools often leave the investigator with large volumes of apparently unorganized information. To meet this interpretation challenge, gene-set, or cluster testing has become a popular analytical tool. Many gene-set testing methods and software packages are now available, most of which use a variety of statistical tests to assess the genes in a set for biological information. However, the field is still evolving, and there is a great need for "integrated" solutions.     

Torularhodin and torulene are the major contributors to the carotenoid pool of marine Rhodosporidium babjevae (Golubev)

A carotenoid-producing yeast strain, isolated from the sub-arctic, marine copepod Calanus finmarchicus, was identified as Rhodosporidium babjevae (Golubev) according to morphological and biochemical characteristics and phylogenetic inference from the small-subunit ribosomal RNA gene sequence. The total carotenoids content varied with cultivation conditions in the range 66–117 μg per g dry weight. The carotenoid pool, here determined for the first time, was dominated by torularhodin and torulene, which collectively constituted 75–91% of total carotenoids under various regimes of growth. β-Carotene varied in the range 5–23%. A high-peptone/low-yeast extract (weight ratio 38:1) marine growth medium favoured the production of torularhodin, the carotenoid at highest oxidation level, with an average of 63% of total carotenoids. In standard yeast medium (YM; ratio 1.7:1), torularhodin averaged 44%, with increased proportions of the carotenes, torulene and β-carotene. The anticipated metabolic precursor γ-carotene (β,ψ-carotene) constituted a minor fraction (≤8%) under all conditions of growth.