Synthesis and structural characterization of two donor-functionalized terphenyl magnesium compounds

The synthesis and structural characterization of two novel donor-functionalized terphenyl magnesium compounds, namely (Danip)₂Mg (1) [Danip = 2,6-di(o-anisyl)phenyl] and [DinapOMg(THF)]₂ (Dinap = 2,6-di(o-2-methoxynaphthyl)phenyl; DinapO = 2-(o-2-methoxynaphthyl)-6-(o-2-naphthoxide)phenyl) is reported. Compound 1 represents a first structurally authenticated bis terphenyl magnesium compound, while the molecular structure of dimeric tetrahydrofuran solvated 2 shows a five-coordinate magnesium atom stabilized by a partially demethylated dianionic mixed arylether/aryloxide terphenyl ligand (= DinapO).     

Cell–cell communication in Arabidopsis early embryogenesis

The basic body plan of the adult plant is established during embryogenesis, resulting in the juvenile form of the seedling. Arabidopsis embryogenesis is distinguished by a highly regular pattern of cell divisions. Some of these divisions are asymmetric, generating daughter cells with different fates. However, their subsequent differentiation might still depend on cell–cell communication to be fully accomplished or maintained. In some cases, cell fate specification solely depends on cell–cell communication that in general plays an important role in the generation of positional information within the embryo. Although auxin-dependent signalling has received much attention, other ways of cell–cell communication have also been demonstrated or suggested. This review focuses on aspects of pattern formation and cell–cell communication during Arabidopsis embryogenesis up to the mid-globular stage of development.     

Long-term subarachnoid catheter placement in the middle cranial fossa of the rat

Research using rats sometimes requires long-term placement of catheters in the subarachnoid space, the cavity between the arachnoid mater and the pia mater in the brain. These catheters can be used to experimentally induce subarachnoid bleeding by injecting blood or to locally administer drugs or other substances. To date, published techniques for penetrating the subarachnoid space of small experimental animals require the use of inflexible or relatively inflexible catheters. These catheters typically consist of metal or stiff plastic and are used to access the occipital or frontal cranial cavity or to directly access the cisterna magna via the atlantooccipital membrane. However, inflexible catheters are not ideal for long-term placement in the subarachnoid space. In this paper, the authors describe a reliable procedure for long-term catheterization of the subarachnoid cavity of the rat. For this method, personnel insert the catheter and keep it in place in the rat's middle cranial cavity, in the vicinity of the cerebral arterial circle. This new approach allows personnel to repeatedly use the catheter for a period of at least 2 weeks. The catheter, which is well-tolerated by rats, can be used for administering saline solutions and for injecting blood that has not been treated with heparin into the subarachnoid space.     

Primary cilia regulate mTORC1 activity and cell size through Lkb1

The mTOR pathway is the central regulator of cell size. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD), where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins. We therefore investigated if cilia have a role in mTOR regulation. Here, we show that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. Our results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling.     

The Mechanism of nucleotide-assisted molybdenum insertion into molybdopterin. A novel route toward metal cofactor assembly

The molybdenum cofactor (Moco) is synthesized by an ancient and conserved biosynthetic pathway. In plants, the two-domain protein Cnx1 catalyzes the insertion of molybdenum into molybdopterin (MPT), a metal-free phosphorylated pyranopterin carrying an ene-dithiolate. Recently, we identified a novel biosynthetic intermediate, adenylated molybdopterin (MPT-AMP), which is synthesized by the C-terminal G domain of Cnx1. Here, we show that MPT-AMP and molybdate bind in an equimolar and cooperative way to the other N-terminal E domain (Cnx1E). Tungstate and sulfate compete for molybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn(2+)-/Mg(2+)-dependent hydrolysis of MPT-AMP but only when molybdate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release of Moco that was quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is release as second product of the reaction. When comparing MPT-AMP hydrolysis with the formation of Moco and AMP a 1.5-fold difference in reaction rates were observed. Together with the strict dependence of the reaction on molybdate the formation of adenylated molybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to molybdopterin is proposed.     

Use of laccase as a novel, versatile reporter system in filamentous fungi

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.     

Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.