Characterization of heterotrophic growth and sesquiterpene production by Rhodobacter sphaeroides on a defined medium

Rhodobacter sphaeroides is a metabolically versatile bacterium capable of producing terpenes natively. Surprisingly, terpene biosynthesis in this species has always been investigated in complex media, with unknown compounds possibly acting as carbon and nitrogen sources. Here, a defined medium was adapted for R. sphaeroides dark heterotrophic growth, and was used to investigate the conversion of different organic substrates into the reporter terpene amorphadiene. The amorphadiene synthase was cloned in R. sphaeroides, allowing its biosynthesis via the native 2-methyl-d-erythritol-4-phosphate (MEP) pathway and, additionally, via a heterologous mevalonate one. The latter condition increased titers up to eightfold. Consequently, better yields and productivities to previously reported complex media cultivations were achieved. Productivity was further investigated under different cultivation conditions, including nitrogen and oxygen availability. This novel cultivation setup provided useful insight into the understanding of terpene biosynthesis in R. sphaeroides, allowing to better comprehend its dynamics and regulation during chemoheterotrophic cultivation.

     

Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea

Plant Molecular Biology - Overexpression of BoMYB29 gene up-regulates the aliphatic glucosinolate pathway in Brassica oleracea plants increasing the production of the anti-cancer metabolite...     

Outer membrane protein gene expression traced by immunogold labelling in Rhizobium Leguminosarum bv. viciae strain 248 in Vicia sativa ssp. nigra.

During differentiation from bacterium into nitrogen fixing bacteroid, the outer membrane protein RopA decreases in density. Comparison between size increase of the rhizobia and density decrease of a RopA protein epitope after immuno-gold labelling, indicates that although ropA gene expression stops at the onset of nitrogen fixation, the amount of RopA protein probably remains the same.     

Ecological compensation of the impacts of a road. Preliminary method for the A50 road link (Eindhoven-Oss, The Netherlands)

After years of efforts to avoid or reduce the impact of infrastructure projects on nature, the principle of ecological compensation has been incorporated in Dutch governmental policy. Ecological compensation aims to recover those ecological functions and natural values that still remain affected after maximum effort has been made to reduce the impact of the intervention (mitigation). The accepted aim of current policy is thus no-net-loss of area and quality by means of mitigative and compensatory measures. As part of the planning process for construction of a stretch of road in The Netherlands, viz., the A50 road link in the province of North Brabant, a Nature Compensation Plan (NCP) was required to be drawn up. This work has recently been completed by the Regional Directorate of Public Works and Water Management, the initiator of the intervention. The NCP, initially presented as a Draft Plan, was drawn up by the Regional Directorate using a preliminary method designed by the Centre of Environmental Science of Leiden University for deriving compensatory measures. After an opportunity for public comment, the Draft Plan was revised to form a Final NCP. This article describes, firstly, the preliminary method for deriving ecological compensatory measures. The method starts by quantifying the effects of habitat loss, habitat disturbance (by changes in noise emissions, in the water table and in outdoor recreational patterns), barrier action and fauna casualties. Following mitigation of impacts on nature, compensation for non-mitigable effects focuses successively on area size, derived from the impacts on breeding birds, and on area quality, derived from the habitat requirements of the vegetation and fauna groups affected by the road. Guidelines for identifying appropriate locations for compensation are also formulated. Secondly, the compensation method is applied to calculate the mitigative and compensatory measures for the A50 trajectory between Eindhoven and Oss. Thirdly, two comparisons are made: the Draft NCP is compared with the results of the preliminary method, and the Draft and the Final NCP are compared with one another in order to identify the role of the interest groups that played a major role in commenting on the Draft Plan. Finally, realization of the compensatory measures and development of the preliminary method itself are discussed. On the basis of the experience with the A50 case study, a more robust compensation method for road projects is to be developed.     

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.     

Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa2

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.Resistance (R) proteins play a central role in the recognition-based immune system of plants. Unlike vertebrates, plants lack an adaptive immune system with highly specialized immune cells. Instead, they rely on an innate immune system in which each cell is autonomous. Two types of immune receptors can be distinguished in plants, pathogen-associated molecular patterns recognition receptors that detect conserved molecular patterns in plant pathogens and intracellular R proteins that recognize specific effectors employed by pathogens as modifiers of host metabolism or defense mechanisms (Jones and Dangl, 2006). Effector-triggered activation of R proteins leads to an array of protective responses, often culminating in programmed cell death at the site of infection (Greenberg and Yao, 2004), thereby preventing further ingress of the pathogen. Pathogens have evolved mechanisms to evade recognition by R proteins and to regain their virulence (Dodds and Rathjen, 2010). This continuous coevolutionary process between host and pathogen has resulted in a reservoir of highly diverse R proteins in plants, enabling them to counteract a wide range of pathogens and pests.The most common class of R proteins consists of nucleotide-binding (NB)-leucine-rich repeat (LRR) proteins with a tripartite domain architecture, which roughly corresponds to an N-terminal response domain (a coiled coil [CC] or Toll/Interleukin-1 receptor [TIR] domain) involved in downstream signaling, a central molecular switch domain (the NB domain present in the mammalian apoptosis regulator Apaf1, plant R proteins, and the Caenorhabditis elegans apoptosis regulator CED4 [NB-ARC]), and a C-terminal sensor domain (the LRR domain). The NB-ARC domain is an extended nucleotide-binding domain that plant immune receptors share with metazoan apoptosis regulators and immune receptors such as Apaf1, CED4, and nucleotide-binding oligomerization domain (NOD-like) receptors (NLRs) and belongs to the STAND (signal transduction ATPases with numerous domains) family of nucleoside triphosphatase domains (van der Biezen and Jones, 1998; Leipe et al., 2004; Albrecht and Takken, 2006; Maekawa et al., 2011b). The overall modular architecture of metazoan STAND nucleoside triphosphatase is similar to that of NB-LRR plant immune receptors, but the domains flanking the NB-ARC domain often differ. In NLRs, for example, several N-terminal domains can be found, including caspase-recruiting domains and Pyrin domains (Proell et al., 2008). In the mammalian protein Apaf1, the sensor involved in cytochrome c detection consists of C-terminal WD40 repeats (Zou et al., 1997).In plant NB-LRR resistance proteins, the recognition of a pathogen effector via the LRR domain is thought to switch the conformation of the protein from a closed, autoinhibited “off” state into an open, active “on” state (Lukasik and Takken, 2009). The activation of NB-LRR proteins is most likely a multistep process in which the NB-ARC domain plays a central role. The three subdomains of the NB-ARC, the NB, ARC1, and ARC2, collectively form a nucleotide-binding pocket that adopts different conformations depending on the bound nucleotide. This mechanism seems to be conserved between proteins from organisms as distant as bacteria, metazoans, and plants (Rairdan and Moffett, 2007; Danot et al., 2009; Takken and Tameling, 2009). The conformational change coincides with the exchange of bound ADP for ATP in the NB-ARC, probably stabilizing the active conformation (Tameling et al., 2006; Ade et al., 2007). Hydrolysis of the bound ATP is hypothesized to return the domains to their inactive state. The exact mechanism by which elicitor recognition via the LRR leads to a conformational change of the NB-ARC and the subsequent activation of immune signaling pathways is not clear.Previous studies have shown that the CC/TIR, NB-ARC, and LRR domains in plant immune receptors interact and cooperate with each other in an interdependent manner (Moffett et al., 2002; Leister et al., 2005; Ade et al., 2007; Rairdan et al., 2008). From these data, a picture emerges in which the LRR domain is not only involved in pathogen recognition, but also plays a role in maintaining an autoinhibited resting state in the absence of pathogens via its interactions with the other domains (Bendahmane et al., 2002; Hwang and Williamson, 2003; Ade et al., 2007; Qi et al., 2012). A similar role as regulatory domain has been found for the sensor domains of other NLRs, such as the mammalian Apaf1 (Hu et al., 1998). For the potato (Solanum tuberosum) immune receptor Rx1, a model plant NB-LRR protein, it has been shown that the LRR cooperates with the ARC subdomains in retaining the inactive state of the protein. The deletion of the ARC and LRR domains leads to a constitutive activity of the NB (Bendahmane et al., 2002; Rairdan et al., 2008). In addition, it was demonstrated that the elicitor, the Potato virus X (PVX) coat protein, modifies the interdomain interactions in Rx1 (Moffett et al., 2002; Rairdan et al., 2008). Sequence exchanges between Rx1 and the highly homologous nematode resistance protein Gpa2 (88% amino acid identity) resulted in incompatibilities between the domains that give rise to inappropriate activation of cell death responses (Rairdan and Moffett, 2006), indicating that the cooperation between the sensor and switch domains depends on an interaction fine tuned by intramolecular coevolution. In this light, it is interesting to note that a functional ortholog of Rx1, Rx2 from Solanum acaule, is almost identical to Rx1 in its LRR region but displays a higher similarity to Gpa2 in stretches of its CC-NB-ARC sequence (Bendahmane et al., 2000).The aim of our study was to pinpoint the molecular determinants controlling the switch between the resting and activation state of NB-LRR proteins. The incompatibility between the ARC and LRR domains of Rx1 and Gpa2 was used as a guideline to dissect the molecular and structural determinants involved in the cooperation between the switch (NB-ARC) and sensor (LRR) domain. An extensive exchange of polymorphic residues between these two homologous NB-LRR proteins resulted in the identification of a minimal fragment of 68 amino acid residues in the ARC2 domain and the first LRR repeats as being crucial for proper activation of Gpa2 and Rx1. Within this minimal region, we identified two amino acids that, despite their proximity in the amino acid sequence, differentiate between elicitor-dependent (position 401) and independent activation (position 403). However, structural modeling of the domains shows that the residue at position 403 operates at the interface of the ARC2 and N-terminal part of the LRR domain, while residue 401 mapped at the interface between the ARC2 and NB domain. Furthermore, an acidic loop region in the ARC2 domain and complementary-charged basic patches in the N-terminal half of the LRR domain are shown to be required for the physical interaction between these domains. We demonstrate that the binding between the CC- NB-ARC and LRR domains is disrupted upon elicitor-dependent activation and that the complementary-charged residues are predicted to facilitate reassociation. Two independent docking simulations of the NB-ARC and LRR domain indicate that the LRR domain binds to the NB-ARC domain at the surface formed by the interaction of the ARC2 and NB subdomains. We present a mechanistic model in which the first repeats of the LRR, the ARC2 subdomain, and the NB form a clamp, which governs the shuttling between a closed, autoinhibited “off” state and an open, active “on” state of the resistance protein. Finally, we discuss the consequences of the functional constraints imposed by the interface of the NB, ARC2, and LRR domain for the generation of novel resistance specificities via evolutionary processes and genetic engineering.     

Analysis of tissue flow patterns during primitive streak formation in the chick embryo

We have investigated the patterns of tissue flow underlying the formation of the primitive streak in the chick embryo. Analysis of time-lapse sequences of brightfield images to extract the tissue velocity field and of fluorescence images of small groups of DiI-labelled cells have shown that epiblast cells move in two large-scale counter-rotating streams, which merge at the site of streak formation. Despite the large-scale tissue flows, individual cells appear to move little relative to their neighbours. As the streak forms, it elongates in both the anterior and posterior directions. Inhibition of actin polymerisation via local application of the inhibitor latrunculin A immediately terminates anterior extension of the streak tip, but does not prevent posterior elongation. Inhibition of actin polymerisation at the base of the streak completely inhibits streak formation, implying that continuous movement of cells into the base of the forming streak is crucial for extension. Analysis of cycling cells in the early embryo shows that cell-cycle progression in the epiblast is quite uniform before the primitive streak forms then decreases in the central epiblast and incipient streak and increases at the boundary between the area pellucida and area opaca during elongation. The cell-cycle inhibitor aphidicolin, at concentrations that completely block cell-cycle progression, permits initial streak formation but arrests development during extension. Our analysis suggests that cell division maintains the cell-flow pattern that supplies the streak with cells from the lateral epiblast, which is critical for epiblast expansion in peripheral areas, but that division does not drive streak formation or the observed tissue flow.     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.     

Consensus statement understanding health and malnutrition through a systems approach: the ENOUGH program for early life

Nutrition research, like most biomedical disciplines, adopted and often uses experimental approaches based on Beadle and Tatum’s one gene—one polypeptide hypothesis, thereby reducing biological processes to single reactions or pathways. Systems thinking is needed to understand the complexity of health and disease processes requiring measurements of physiological processes, as well as environmental and social factors, which may alter the expression of genetic information. Analysis of physiological processes with omics technologies to assess systems’ responses has only become available over the past decade and remains costly. Studies of environmental and social conditions known to alter health are often not connected to biomedical research. While these facts are widely accepted, developing and conducting comprehensive research programs for health are often beyond financial and human resources of single research groups. We propose a new research program on essential nutrients for optimal underpinning of growth and health (ENOUGH) that will use systems approaches with more comprehensive measurements and biostatistical analysis of the many biological and environmental factors that influence undernutrition. Creating a knowledge base for nutrition and health is a necessary first step toward developing solutions targeted to different populations in diverse social and physical environments for the two billion undernourished people in developed and developing economies.