The wheat Lr34 multipathogen resistance gene confers resistance to anthracnose and rust in sorghum

The ability of the wheat Lr34 multipathogen resistance gene (Lr34res) to function across a wide taxonomic boundary was investigated in transgenic Sorghum bicolor. Increased resistance to sorghum rust and anthracnose disease symptoms following infection with the biotrophic pathogen Puccinia purpurea and the hemibiotroph Colletotrichum sublineolum, respectively, occurred in transgenic plants expressing the Lr34res ABC transporter. Transgenic sorghum lines that highly expressed the wheat Lr34res gene exhibited immunity to sorghum rust compared to the low‐expressing single copy Lr34res genotype that conferred partial resistance. Pathogen‐induced pigmentation mediated by flavonoid phytoalexins was evident on transgenic sorghum leaves following P. purpurea infection within 24–72 h, which paralleled Lr34res gene expression. Elevated expression of flavone synthase II, flavanone 4‐reductase and dihydroflavonol reductase genes which control the biosynthesis of flavonoid phytoalexins characterized the highly expressing Lr34res transgenic lines 24‐h post‐inoculation with P. purpurea. Metabolite analysis of mesocotyls infected with C. sublineolum showed increased levels of 3‐deoxyanthocyanidin metabolites were associated with Lr34res expression, concomitant with reduced symptoms of anthracnose.     

Contrasting microcystin production and cyanobacterial population dynamics in two Planktothrix-dominated freshwater lakes

Microcystin concentrations in two Dutch lakes with an important Planktothrix component were related to the dynamics of cyanobacterial genotypes and biovolumes. Genotype composition was analysed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rrn operon (rRNA-ITS), and biovolumes were measured by using microscopy. In Lake Tjeukemeer, microcystins were present throughout summer (maximum concentration 30 microg l(-1)) while cyanobacterial diversity was low and very constant. The dominant phototroph was Planktothrix agardhii. In contrast, Lake Klinckenberg showed a high microcystin peak (up to 140 microg l(-1)) of short duration. In this lake, cyanobacterial diversity was higher and very dynamic with apparent genotype successions. Several genotypes derived from DGGE field profiles matched with genotypes from cultures isolated from field samples. The microcystin peak measured in Lake Klinckenberg could be confidently linked to a bloom of Planktothrix rubescens, as microscopic and genotypic analysis showed identity of bloom samples and a toxin-producing P. rubescens culture. Toxin-producing genotypes were detected in the microbial community before they reached densities at which they were detected by using microscopy. Cyanobacterial biovolumes provided additional insights in bloom dynamics. In both lakes, the microcystin content per cell was highest at the onset of the blooms. Our results suggest that while genotypic characterization of a lake can be valuable for detection of toxic organisms, for some lakes a monitoring of algal biomass has sufficient predictive value for an assessment of toxin production.     

The extraneuronal monoamine transporter Slc22a3/Orct3 co-localizes with the Maoa metabolizing enzyme in mouse placenta

Monoamine clearance is a combined function of uptake mechanisms in the plasma membrane with intracellular metabolizing enzymes. Two different uptake mechanisms have been described. Uptake(1) is located in presynaptic neurones, whereas uptake(2) is extraneuronal. Recently, the Slc22a3/Orct3 gene was identified as the extraneuronal monoamine transporter. In mouse embryonic development Orct3 expression is restricted to the placenta, which is also a site of expression of neuronal transporters. We have used RNA blots and in situ hybridization to examine the expression of Orct3 and other members of the monoamine uptake and metabolizing pathways in mouse placenta. The results show that Orct3 expression overlaps that of the monoamine metabolizing enzyme Maoa in the labyrinth layer of the placenta with an expression pattern distinct from that of the neuronal transporters Slc6a2/Net and Slc6a4/Sert.     

Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakes

We characterized the bacterioplankton community and its seasonal dynamics in two neighbouring hypertrophic lakes by denaturing gradient gel electrophoresis (DGGE) analysis of short (193 bp) 16S ribosomal DNA polymerase chain reaction (PCR) products obtained with primers specific for the domain Bacteria. Lake Blankaart is turbid and has a high phytoplankton biomass and episodic cyanobacterial blooms, whereas biomanipulated Lake Visvijver is characterized by clearwater conditions and the establishment of a dense charophyte vegetation. Both lakes were dominated by bacterial groups commonly found in freshwater habitats (e.g. ACK4 cluster of Actinomycetes; ACK stands for clones isolated from the Adirondack mountain lakes) . Yet, cluster analysis and principal components analysis (PCA) revealed that taxon composition of the bacterioplankton community of the two lakes differs substantially and consistently throughout the season. During the study year (1998), the bacterioplankton community of both lakes showed a distinct seasonal pattern. Lake Blankaart showed a clear differentiation between winter, spring, summer and autumn. In Lake Visvijver, summer samples differed greatly from spring, autumn and winter samples. We hypothesize that the contrasting bacterioplankton in the two neighbouring shallow lakes is determined largely by the presence or absence of macrophytes.     

Impaired activity of the extraneuronal monoamine transporter system known as uptake-2 in Orct3/Slc22a3-deficient mice

Two uptake systems that control the extracellular concentrations of released monoamine neurotransmitters such as noradrenaline and adrenaline have been described. Uptake-1 is present at presynaptic nerve endings, whereas uptake-2 is extraneuronal and has been identified in myocardium and vascular and nonvascular smooth muscle cells. The gene encoding the uptake-2 transporter has recently been identified in humans (EMT), rats (OCT3), and mice (Orct3/Slc22a3). To generate an in vivo model for uptake-2, we have inactivated the mouse Orct3 gene. Homozygous mutant mice are viable and fertile with no obvious physiological defect and also show no significant imbalance of noradrenaline or dopamine. However, Orct3-null mice show an impaired uptake-2 activity as measured by accumulation of intravenously administered [(3)H]MPP(+) (1-methyl-4-phenylpyridinium). A 72% reduction in MPP(+) levels was measured in hearts of both male and female Orct3 mutant mice. No significant differences between wild-type and mutant mice were found in any other adult organ or in plasma. When [(3)H]MPP(+) was injected into pregnant females, a threefold-reduced MPP(+) accumulation was observed in homozygous mutant embryos but not in their placentas or amniotic fluid. These data show that Orct3 is the principal component for uptake-2 function in the adult heart and identify the placenta as a novel site of action of uptake-2 that acts at the fetoplacental interface.     

Counting stem cells: methodological constraints

The number of stem cells contributing to hematopoiesis has been a matter of debate. Many studies use retroviral tagging of stem cells to measure clonal contribution. Here we argue that methodological factors can impact such clonal analyses. Whereas early studies had low resolution, leading to underestimation, recent methods may result in an overestimation of stem-cell counts. We discuss how restriction enzyme choice, PCR bias, high-throughput sequencing depth and tagging method could affect the conclusions of clonal studies.     

Phenotypic and functional heterogeneity of human bone marrow- and amnion-derived MSC subsets

Bone marrow-derived mesenchymal stromal/stem cells (MSCs) are nonhematopoietic cells that are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In addition, they are known to participate in niche formation for hematopoietic stem cells and to display immunomodulatory properties. Conventionally, these cells are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks. This isolation procedure is hampered by the unpredictable influence of secreted molecules, the interactions between cocultured hematopoietic and other unrelated cells, and by the arbitrarily selected removal time of nonadherent cells before the expansion of MSCs. Finally, functionally isolated cells do not provide biological information about the starting population. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression, or absence, of surface markers. In this report, we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow- and amnion-derived MSCs.