PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β₂-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β₂-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.     

Multiscale mechanisms of cell migration during development: theory and experiment

Long-distance cell migration is an important feature of embryonic development, adult morphogenesis and cancer, yet the mechanisms that drive subpopulations of cells to distinct targets are poorly understood. Here, we use the embryonic neural crest (NC) in tandem with theoretical studies to evaluate model mechanisms of long-distance cell migration. We find that a simple chemotaxis model is insufficient to explain our experimental data. Instead, model simulations predict that NC cell migration requires leading cells to respond to long-range guidance signals and trailing cells to short-range cues in order to maintain a directed, multicellular stream. Experiments confirm differences in leading versus trailing NC cell subpopulations, manifested in unique cell orientation and gene expression patterns that respond to non-linear tissue growth of the migratory domain. Ablation experiments that delete the trailing NC cell subpopulation reveal that leading NC cells distribute all along the migratory pathway and develop a leading/trailing cellular orientation and gene expression profile that is predicted by model simulations. Transplantation experiments and model predictions that move trailing NC cells to the migratory front, or vice versa, reveal that cells adopt a gene expression profile and cell behaviors corresponding to the new position within the migratory stream. These results offer a mechanistic model in which leading cells create and respond to a cell-induced chemotactic gradient and transmit guidance information to trailing cells that use short-range signals to move in a directional manner.     

Cancer gene discovery in the mouse

Developments in high-throughput genome analysis and in computational tools have made it possible to rapidly profile entire cancer genomes with basepair resolution. In parallel with these advances, mouse models of cancer have evolved into powerful tools for cancer gene discovery. Here we discuss some of the approaches that may be used for cancer gene identification in the mouse and discuss how a cross-species 'oncogenomics' approach to cancer gene discovery represents a powerful strategy for finding genes that drive tumorigenesis.     

Cleavable linkers in chemical biology

Interest in cleavable linkers is growing due to the rapid development and expansion of chemical biology. The chemical constrains imposed by the biological conditions cause significant challenges for organic chemists. In this review we will present an overview of the cleavable linkers used in chemical biology classified according to their cleavage conditions by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents.     

Interactions between age,sex, and hormones in experimental ischemic stroke

Age, sex, and gonadal hormones have profound effects on ischemic stroke outcomes, although how these factors impact basic stroke pathophysiology remains unclear. There is a plethora of inconsistent data reported throughout the literature, primarily due to differences in the species examined, the timing and methods used to evaluate injury, the models used, and confusion regarding differences in stroke incidence as seen in clinical populations vs. effects on acute neuroprotection or neurorepair in experimental stroke models. Sex and gonadal hormone exposure have considerable independent impact on stroke outcome, but these factors also interact with each other, and the contribution of each differs throughout the lifespan. The contribution of sex and hormones to experimental stroke will be the focus of this review. Recent advances and our current understanding of age, sex, and hormone interactions in ischemic stroke with a focus on inflammation will be discussed.     

Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation

Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression.     

Alternatively folded proteins with unexpected beneficial functions

HAMLET (human α-lactalbumin made lethal to tumour cells) and its related partially unfolded protein-fatty acid complexes are novel biomolecular nanoparticles that possess relatively selective cytotoxic activities towards tumour cells. One of the key characteristics is the requirement for the protein to be partially unfolded, hence endowing native proteins with additional functions in the alternatively folded states. Beginning with the history of its discovery and development, the cellular targets that appear to be strongly correlated with tumour cell death are introduced in the present article.     

Analysis of the microbial community structure in a membrane bioreactor during initial stages of filtration

Membrane biofouling was investigated during the early stages of filtration in a laboratory-scale membrane bioreactor operated on molasses wastewater. The bacterial diversity and composition of the membrane biofilm and activated sludge were analyzed using terminal restriction fragment length polymorphism coupled with 16S rRNA clone library construction and sequencing. The amount of extracellular polymeric substances produced by bacteria was investigated using spectroscopic methods. The results reveal that the bacterial community of activated sludge differs significantly from that of the membrane biofilm, especially at the initial phase. Phylogenetic analysis based on 16S rRNA gene sequences identified 25 pioneer OTUs responsible for membrane surface colonization. Also, the relationship between the identified bacterial strains and the system specifications was explored.