Cloning and expression of pkaC and pkaR,the genes encoding the cAMP-dependent protein kinase of Aspergillus fumigatus

This report describes the cloning and expression of both subunits of PKA in the opportunistic fungal pathogen Aspergillus fumigatus. The predicted translation product of the regulatory subunit, pkaR, is defined as a type II regulatory subunit. The gene encoding the A. fumigatus catalytic subunit, pkaC, contains the conserved kinase and activation domains that are characteristic of PkaC proteins. Both subunit mRNAs are expressed throughout the asexual life cycle of A. fumigatus. Message levels of pkaR and pkaC are higher during co-cultivation with alveolar epithelial cells than during culture alone.This revised version was published online in October 2005 with corrections to the Cover Date.     

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Role of menaquinone in the reduction of fumarate, nitrate, iron(III) and manganese(IV) by Shewanella putrefaciens MR-1

Abstract Mutants of Shewanella putrefaciens MR-1 deficient in menaquinone and methylmenaquinone, but which have wild-type levels of ubiquinone, retain the ability to use trimethylamine N -oxide as an electron acceptor, but they lose the ability to use nitrate, iron(III), and fumarate as electron acceptors. These mutants also show a reduced rate of manganese(IV) reduction. One of these mutants could be restored to essentially wild-type phenotype by supplementing the medium with 1,4-dihydroxy-2-naphthoic acid. A requirement for naphthoquinones in iron(III) reduction and a preference for naphthoquinones in manganese(IV) reduction provide further support that the metal reducing systems in MR-1 are linked to anaerobic respiration.     

Transgenerational effects of parent and grandparent gender on offspring development in a biparental beetle species

Parental effects on offspring life-history traits are common and increasingly well-studied. However, the extent to which these effects persist into offspring in subsequent generations has received less attention. In this experiment, maternal and paternal effects on offspring and grand-offspring were investigated in the biparental burying beetle Nicrophorus vespilloides, using a split-family design. This allowed the separation of prenatal and postnatal transgenerational effects. Grandparent and parent gender were found to have a cumulative effect on offspring development and may provide a selection pressure on the division of parental investment in biparental species.     

Functional Organization of the Action Observation Network in Autism: A Graph Theory Approach

Background

The ability to recognize, understand and interpret other’s actions and emotions has been linked to the mirror system or action-observation-network (AON). Although variations in these abilities are prevalent in the neuro-typical population, persons diagnosed with autism spectrum disorders (ASD) have deficits in the social domain and exhibit alterations in this neural network.

Method

Here, we examined functional network properties of the AON using graph theory measures and region-to-region functional connectivity analyses of resting-state fMRI-data from adolescents and young adults with ASD and typical controls (TC).

Results

Overall, our graph theory analyses provided convergent evidence that the network integrity of the AON is altered in ASD, and that reductions in network efficiency relate to reductions in overall network density (i.e., decreased overall connection strength). Compared to TC, individuals with ASD showed significant reductions in network efficiency and increased shortest path lengths and centrality. Importantly, when adjusting for overall differences in network density between ASD and TC groups, participants with ASD continued to display reductions in network integrity, suggesting that also network-level organizational properties of the AON are altered in ASD.

Conclusion

While differences in empirical connectivity contributed to reductions in network integrity, graph theoretical analyses provided indications that also changes in the high-level network organization reduced integrity of the AON.     

A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites

Apicomplexan parasites constitute one of the most significant groups of pathogens infecting humans and animals. The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasma gondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This amoeboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actin-myosin motor, to the inner membrane complex. The Plasmodium blood stage merozoite, however, does not exhibit gliding motility. Here we show that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins 45 and 50 (GAP40 and GAP50), are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we identify a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.