Imposition of crossover interference through the nonrandom distribution of synapsis initiation complexes

Meiotic crossovers (COs) are nonrandomly distributed along chromosomes such that two COs seldom occur close together, a phenomenon known as CO interference. We have used genetic and cytological methods to investigate interference mechanisms in budding yeast. Assembly of the synaptonemal complex (SC) initiates at a few sites along each chromosome, triggered by a complex of proteins (including Zip2 and Zip3) called the synapsis initiation complex (SIC). We found that SICs, like COs, display interference, supporting the hypothesis that COs occur at synapsis initiation sites. Unexpectedly, we found that SICs show interference in mutants in which CO interference is abolished; one explanation is that these same mutations eliminate the subset of COs that normally occur at SICs. Since SICs are assembled in advance of SC and they are properly positioned even in the absence of SC formation, these data clearly demonstrate an aspect of interference that is independent of synapsis.     

Structure-activity relationships of piperazinebenzylamines as potent and selective agonists of the human melanocortin-4 receptor

SAR studies on a series of piperazinebenzenes directed toward the human melanocortin-4 receptor resulted in potent MC4R agonists. Replacement of the triazole moiety of an initial lead 4 by a basic nitrogen baring a lipophilic side-chain increased the binding affinities of these compounds. Analogs bearing an additional hetero-atom in the side-chain possessed good agonist potency. Thus, 11h had a Ki of 11 nM, and 13g exhibited an EC50 of 3.8 nM and a Ki of 6.4 nM.     

Recommendations for accelerating global action to prevent folic acid-preventable birth defects and other folate-deficiency diseases: meeting of experts on preventing folic acid-preventable neural tube defects

BACKGROUND: In April of 2003, The Micronutrient Initiative, in collaboration with several other organizations, convened a group of knowledgeable scientists and policy experts to discuss ways to accelerate the global pace at which countries implement effective and sustainable programs to prevent folic acid-preventable birth defects and other folate-deficiency diseases. Programs implemented to date by fewer than 40 countries have prevented only 10% of the estimated 240,000 annual cases of folic acid-preventable spina bifida and anencephaly. METHODS: Participants in this meeting summarized and presented scientific evidence showing that increased consumption of synthetic folic acid prevents a large proportion of spina bifida and anencephaly cases. They also reviewed related guidance and endorsement issued by national professional societies and advisory bodies as well as policies and programs implemented by some countries that have already demonstrated successful results in terms of reduced rates of neural tube defects and improved folate nutrition. CONCLUSIONS: The group formulated and discussed recommendations and strategies for increasing the pace of neural tube defect prevention globally. The recommendations and strategies are published here.     

Development by self-digestion: molecular mechanisms and biological functions of autophagy

Autophagy is the major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles. It involves the rearrangement of subcellular membranes to sequester cargo for delivery to the lysosome where the sequestered material is degraded and recycled. For many decades, it has been known that autophagy occurs in a wide range of eukaryotic organisms and in multiple different cell types during starvation, cellular and tissue remodeling, and cell death. However, until recently, the functions of autophagy in normal development were largely unknown. The identification of a set of evolutionarily conserved genes that are essential for autophagy has opened up new frontiers for deciphering the role of autophagy in diverse biological processes. In this review, we summarize our current knowledge about the molecular machinery of autophagy and the role of the autophagic machinery in eukaryotic development.     

Model parameterization, prior distributions, and the general time-reversible model in Bayesian phylogenetics

Bayesian phylogenetic methods require the selection of prior probability distributions for all parameters of the model of evolution. These distributions allow one to incorporate prior information into a Bayesian analysis, but even in the absence of meaningful prior information, a prior distribution must be chosen. In such situations, researchers typically seek to choose a prior that will have little effect on the posterior estimates produced by an analysis, allowing the data to dominate. Sometimes a prior that is uniform (assigning equal prior probability density to all points within some range) is chosen for this purpose. In reality, the appropriate prior depends on the parameterization chosen for the model of evolution, a choice that is largely arbitrary. There is an extensive Bayesian literature on appropriate prior choice, and it has long been appreciated that there are parameterizations for which uniform priors can have a strong influence on posterior estimates. We here discuss the relationship between model parameterization and prior specification, using the general time-reversible model of nucleotide evolution as an example. We present Bayesian analyses of 10 simulated data sets obtained using a variety of prior distributions and parameterizations of the general time-reversible model. Uniform priors can produce biased parameter estimates under realistic conditions, and a variety of alternative priors avoid this bias.     

Human rhinovirus type 2 is internalized by clathrin-mediated endocytosis

Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K(+) depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-beta-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.     

Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation

G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.     

Regulation of p70 S6 kinase by complex formation between the Rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin

Tiam1 is a ubiquitous guanine nucleotide exchange factor (GEF) that activates the Rac GTPase. We have shown previously that the N terminus of Tiam1 contributes to the signaling specificity of its downstream target Rac via association with IB2, a scaffold that promotes Rac activation of a p38 kinase cascade. Here we show that the N terminus of Tiam1 can influence Rac signaling specificity in a different way by interaction with spinophilin, a scaffold that binds to p70 S6 kinase, another protein regulated by Rac. In particular, spinophilin binding promotes the plasma membrane localization of Tiam1 and enhances the ability of Tiam1 to activate p70 S6 kinase. In contrast, spinophilin binding suppresses the ability of Tiam to activate Pak1, a different Rac effector. Finally, a mutant spinophilin that cannot bind to Tiam1 suppresses serum-induced p70 S6 kinase activation in cells, suggesting that a Tiam1/spinophilin complex contributes to p70 S6 kinase regulation by extracellular signals. These findings add to a growing body of evidence supporting the concept that some Rac-GEFs not only activate Rac GTPases but also participate in the selection of Rac effector by binding to particular scaffolds that complex with components of specific Rac effector pathways.