Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp

Four filamentous ascomycetes, Leptosphaeria maculans, L. biglobosa, Oculimacula yallundae and O. acuformis, were transformed via Agrobacterium tumefaciens-mediated transformation with the genes encoding DsRed and GFP. Using vectors pCAMDsRed and pCAMBgfp, either germinated conidia of Leptosphaeria spp. and O. yallundae or physically fragmented cultures of Oculimacula spp. were transformed. In vitro, the expression of the two reporter proteins in mycelium of both Oculimacula and both Leptosphaeria species was sufficient to distinguish each species in co-inoculated cultures. In planta, transformants of L. maculans or L. biglobosa expressing DsRed or GFP could be observed together in leaves of Brassica napus. Either reporter protein could be used to view the colonization of leaf petioles by both Leptosphaeria spp. and growth in the xylem vessels could be clearly observed. With the generation of these transformants, further studies on interactions between pathogen species involved in disease complexes on various host species and between opposite mating types of the same species are now possible.     

Correlation of probiotic Lactobacillus salivarius growth phase with its cell wall-associated proteome

Lactobacillus salivarius subsp. salivarius UCC118 is a probiotic bacterium that was originally isolated from human intestinal tissues and was subsequently shown in a pilot study to alleviate symptoms associated with mild-moderate Crohn's disease. Strain UCC118 can adhere to animal and human intestinal tissue, and to both healthy and inflamed ulcerative colitis mucosa, irrespective of location in the gut. In this study, an enzymatic technique has been combined with proteomic analysis to correlate bacterial growth phase with the presence of factors present in the cell wall of the bacterium. Using PAGE electrophoresis, it was determined that progression from lag to log to stationary growth phases in vitro correlated with increasing prominence of an 84kD protein associated with in vitro adherence ability. Isolated proteins from the 84kD band region were further separated by two-dimensional electrophoresis, resolving this band into 20 individual protein spots at differing isoelectric points. The protein moieties were excised, trypsin digested and subjected to tandem mass spectrometry. The observed proteins are analogous to those reported to be associated with the Listeria monocytogenes cell-wall proteome, and include DnaK, Ef-Ts and pyruvate kinase. These data suggest that at least some of the beneficial attributes of probiotic lactobacilli, and in particular this strain, may be due to nonpathogenic mimicry of pathogens and potentially be mediated through a form of attenuated virulence.     

The metal permease ZupT from Escherichia coli is a transporter with a broad substrate spectrum

The Escherichia coli zupT (formerly ygiE) gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An E. coli strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if zupT was expressed. Heterologous expression of Arabidopsis thaliana ZIP1 could also alleviate iron deficiency in this E. coli strain, as could expression of indigenous mntH or feoABC. Transport studies with intact cells showed that ZupT facilitates uptake of 55Fe2+ similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using 57Co2+. Expression of zupT rendered E. coli cells hypersensitive to Co2+ and sensitive to Mn2+. ZupT did not appear to be metal regulated: expression of a Phi(zupT-lacZ) operon fusion indicated that zupT is expressed constitutively at a low level.     

Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes

Exposure of unirradiated human keratinocytes to irradiated cell conditioned medium (ICCM) is known to cause a cascade of events that leads to reproductive death and apoptosis. This study investigates the effect of ICCM on clonogenic survival, mitochondrial mass and BCL2 expression in unirradiated keratinocytes. Exposure to 5 mGy, 0.5 Gy and 5 Gy ICCM resulted in a significant decrease in clonogenic survival. Human keratinocytes incubated with ICCM containing an antioxidant, N-acetylcysteine, showed no significant decrease in clonogenic survival. HPV-G cells incubated with ICCM containing a caspase 9 inhibitor showed no significant decrease in clonogenic survival when the ICCM dose was < or =0.5 Gy. A significant increase in mitochondrial mass per cell was observed after exposure to 5 mGy and 0.5 Gy ICCM. A change in the distribution of the mitochondria from a diffuse cytoplasmic distribution to a more densely concentrated perinuclear distribution was also observed at these doses. No significant increase in mitochondrial mass or change in distribution of the mitochondria was found for 5 Gy ICCM. Low BCL2 expression was observed in HPV-G cells exposed to 5 mGy or 0.5 Gy ICCM, whereas a large significant increase in BCL2 expression was observed in cells exposed to 5 Gy ICCM. This study has shown that low-dose irradiation can cause cells to produce medium-borne signals that can cause mitochondrial changes and the induction of BCL2 expression in unirradiated HPV-G cells. The dose dependence of the mitochondrial changes and BCL2 expression suggests that the mechanisms may be aimed at control of response to radiation at the population level through signaling pathways.     

Effects of cobalt hexammine on folding and self-cleavage of the Neurospora VS ribozyme

We have investigated the effects of Co(NH3)6(3+), an analog of hexahydrated Mg2+, on folding and catalysis of the Neurospora VS ribozyme. Most of the metal ion-induced changes detected by chemical modification structure probing in either metal ion are similar, but occur at approximately 33-fold lower concentrations of Co(NH3)6(3+) than Mg2+. However, Co(NH3)6(3+) is not as effective at inducing two functionally important structural changes: stabilizing the pseudoknot interaction between loops I and V, and rearranging the secondary structure of helix Ib. Comparison of the folding of the precursor and the downstream cleavage product, which lacks helix Ia, shows that helix Ia inhibits stable pseudoknot formation and rearrangement of helix Ib. The VS ribozyme does not self-cleave with Co(NH3)6(3+) as the sole polyvalent cation; however, mixed-metal kinetic experiments show that Co(NH3)6(3+) does not inhibit Mg2+-induced self-cleavage. In contrast, at sub-saturating concentrations of Mg2+, Co(NH3)6(3+) increases the rate of Mg2+-induced self-cleavage, indicating that Co(NH3)6(3+) contributes to the functionally relevant folding of the VS ribozyme.     

Neuroimaging studies of autobiographical event memory

Commonalities and differences in findings across neuroimaging studies of autobiographical event memory are reviewed. In general terms, the overall pattern across studies is of medial and left-lateralized activations associated with retrieval of autobiographical event memories. It seems that the medial frontal cortex and left hippocampus in particular are responsive to such memories. However, there are also inconsistencies across studies, for example in the activation of the hippocampus and dorsolateral prefrontal cortex. It is likely that methodological differences between studies contribute to the disparate findings. Quantifying and assessing autobiographical event memories presents a challenge in many domains, including neuroimaging. Methodological factors that may be pertinent to the interpretation of the neuroimaging data and the design of future experiments are discussed. Consideration is also given to aspects of memory that functional neuroimaging might be uniquely disposed to examine. These include assessing the functionality of damaged tissue in patients and the estimation of inter-regional communication (effective connectivity) between relevant brain regions.     

Characterization of cytochrome P450 2D6 alleles using the Invader system

The cytochrome p450 (CYP) superfamily comprises enzymes that play an essential role in the transformation of medically relevant compounds. Accurate genotyping of polymorphisms in members of this family is drawing increasing interest because certain allelic variants may result in either loss of efficacy or toxic accumulation of therapeutic agents. Debrisoquine 4-hydroxylase, or CYP2D6, is among the most widely studied of the CYPs. The complexity of the CYP2D6 genomic region, including pseudogenes, gene deletions, and gene duplications, has offered numerous challenges to developing a genotyping strategy. We describe a comprehensive CYP2D6 genotyping strategy that employs both a PCR/Invader genotyping assay system and an Invader genomic copy number assay The Invader system is a homogeneous, isothermal, highly specific, and robust signal amplification system. Resultsfrom II CYP2D6 assays in an alle frequency study compare well to published allele frequency values for Caucasians. Further, Invader assays provided unambiguous genotyping determinations for 100% of the 171 samples that yielded a visible PCR product on an agarose gel. A copy number assay yielded only one equivocal result in 205 samples. We identified 17 single-copy individuals and 17 three-copy (or more) individuals.