Kif14 Mutation Causes Severe Brain Malformation and Hypomyelination

We describe a novel spontaneous mouse mutant, laggard (lag), characterized by a flat head, motor impairment and growth retardation. The mutation is inherited as an autosomal recessive trait, and lag/lag mice suffer from cerebellar ataxia and die before weaning. lag/lag mice exhibit a dramatic reduction in brain size and slender optic nerves. By positional cloning, we identify a splice site mutation in Kif14. Transgenic complementation with wild-type Kif14-cDNA alleviates ataxic phenotype in lag/lag mice. To further confirm that the causative gene is Kif14, we generate Kif14 knockout mice and find that all of the phenotypes of Kif14 knockout mice are similar to those of lag/lag mice. The main morphological abnormality of lag/lag mouse is severe hypomyelination in central nervous system. The lag/lag mice express an array of myelin-related genes at significantly reduced levels. The disrupted cytoarchitecture of the cerebellar and cerebral cortices appears to result from apoptotic cell death. Thus, we conclude that Kif14 is essential for the generation and maturation of late-developing structures such as the myelin sheath, cerebellar and cerebral cortices. So far, no Kif14-deficient mice or mutation in Kif14 has ever been reported and we firstly define the biological function of Kif14 in vivo. The discovery of mammalian models, laggard, has opened up horizons for researchers to add more knowledge regarding the etiology and pathology of brain malformation.     

Interaction between Meloidogyne incognita and Hoplolaimus columbus on Davis Soybean

Greenhouse and laboratory experiments were performed to determine if an interaction exists between Meloidogyne incognita and Hoplolaimus columbus on Davis soybean. Greenhouse tests were performed with three population levels of M. incognita and H. columbus (0, 1,500, 6,000/1.5-liter pot) separately and in all combinations. Dry root weight (DRT) declined nonlinearly and dry shoot weight (DST) declined linearly with respect to increasing initial populations of M. incognita and H. columbus. When the two nematode species were added to the soil together, the amount of DRT and DST suppression by one species was dependent on the initial level of the concomitant species. The final root population of M. incognita or H. columbus declined linearly with increasing initial population density of the concomitant species. H. columbus suppressed M. incognita populations in the soil nonlinearly, but M. incognita had no effect on H. columbus.     

Kif3a Controls Murine Nephron Number Via GLI3 Repressor,Cell Survival,and Gene Expression in a Lineage-Specific Manner

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3^Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms.     

Interaction between Autonomous and Microtubule Guidance Systems Controls Cellulose Synthase Trajectories

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Oxidizable Residues Mediating Protein Stability and Cytoprotective Interaction of DJ-1 with Apoptosis Signal-regulating Kinase 1

Parkinson disease (PD)-associated genomic deletions and the destabilizing L166P point mutation lead to loss of the cytoprotective DJ-1 protein. The effects of other PD-associated point mutations are less clear. Here we demonstrate that the M26I mutation reduces DJ-1 expression, particularly in a null background (knockout mouse embryonic fibroblasts). Thus, homozygous M26I mutation causes loss of DJ-1 protein. To determine the cellular consequences, we measured suppression of apoptosis signal-regulating kinase 1 (ASK1) and cytotoxicity for [M26I]DJ-1, and systematically all other DJ-1 methionine and cysteine mutants. C106A mutation of the central redox site specifically abolished binding to ASK1 and the cytoprotective activity of DJ-1. DJ-1 was apparently recruited into the ASK1 signalosome via Cys-106-linked mixed disulfides. The designed higher order oxidation mimicking [C106DD]DJ-1 non-covalently bound to ASK1 even in the absence of hydrogen peroxide and conferred partial cytoprotection. Interestingly, mutations of peripheral redox sites (C46A and C53A) and M26I also led to constitutive ASK1 binding. Cytoprotective [wt]DJ-1 bound to the ASK1 N terminus (which is known to bind another negative regulator, thioredoxin 1), whereas [M26I]DJ-1 bound to aberrant C-terminal site(s). Consequently, the peripheral cysteine mutants retained cytoprotective activity, whereas the PD-associated mutant [M26I]DJ-1 failed to suppress ASK1 activity and nuclear export of the death domain-associated protein Daxx and did not promote cytoprotection. Thus, cytoprotective binding of DJ-1 to ASK1 depends on the central redox-sensitive Cys-106 and may be modulated by peripheral cysteine residues. We suggest that impairments in oxidative conformation changes of DJ-1 might contribute to PD neurodegeneration.Loss-of-function mutations in the DJ-1 gene (PARK7) cause autosomal-recessive hereditary Parkinson disease (PD)² (1). The most dramatic PD-associated mutation L166P impairs DJ-1 dimer formation and dramatically destabilizes the protein (2–7). Other mutations such as M26I (8) and E64D (9) have more subtle defects with unclear cellular consequences (4, 7, 10, 11). In addition to this genetic association, DJ-1 is neuropathologically linked to PD. DJ-1 is up-regulated in reactive astrocytes, and it is oxidatively modified in brains of sporadic PD patients (12–14).DJ-1 protects against oxidative stress and mitochondrial toxins in cell culture (15–17) as well as in diverse animal models (18–21). The cytoprotective effects of DJ-1 may be stimulated by oxidation and mediated by molecular chaperoning (22, 23), and/or facilitation of the pro-survival Akt and suppression of apoptosis signal-regulating kinase 1 (ASK1) pathways (6, 24, 25). The cytoprotective activity of DJ-1 against oxidative stress depends on its cysteine residues (15, 17, 26). Among the three cysteine residues of DJ-1, the most prominent one is the easiest oxidizable Cys-106 (27) that is in a constrained conformation (28), but the other cysteine residues Cys-46 and Cys-53 have been implicated with DJ-1 activity as well (22). However, the molecular basis of oxidation-mediated cytoprotective activity of DJ-1 is not clear. Moreover, the roles of PD-mutated and in vivo oxidized methionines are not known.Here we have mutagenized all oxidizable residues within DJ-1 and studied the effects on protein stability and function. The PD-associated mutation M26I within the DJ-1 dimer interface selectively reduced protein expression as well as ASK1 suppression and cytoprotective activity in oxidatively stressed cells. These cell culture results support a pathogenic effect of the clinical M26I mutation (8). Furthermore, oxidation-defective C106A mutation abolished binding to ASK1 and cytoprotective activity of DJ-1, whereas the designed higher order oxidation mimicking mutant [C106DD]DJ-1 bound to ASK1 even in the absence of H₂O₂ and conferred partial cytoprotection. The peripheral cysteine mutants [C46A]DJ-1 and [C53A]DJ-1 were also cytoprotective and were incorporated into the ASK1 signalosome even in the basal state. Thus, DJ-1 may be activated by a complex mechanism, which depends on the redox center Cys-106 and is modulated by the peripheral cysteine residues. Impairments of oxidative DJ-1 activation might contribute to the pathogenesis of PD.     

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen

The infectious process starts with an initial contact between pathogenand host. We have previously demonstrated that Paracoccidioidesbrasiliensis conidia interact with plasma proteins includingfibrinogen, which is considered the major component of the coagulation system.In this study, we evaluated the in vitro capacity of P.brasiliensis conidia to aggregate with plasma proteins andcompounds involved in the coagulation system. We assessed the aggregation ofP. brasiliensis conidia after incubation with human serumor plasma in the presence or absence of anticoagulants, extracellular matrix(ECM) proteins, metabolic and protein inhibitors, monosaccharides and othercompounds. Additionally, prothrombin and partial thromboplastin times weredetermined after the interaction of P. brasiliensis conidiawith human plasma. ECM proteins, monosaccharides and human plasma significantlyinduced P. brasiliensis conidial aggregation; however,anticoagulants and metabolic and protein inhibitors diminished the aggregationprocess. The extrinsic coagulation pathway was not affected by the interactionbetween P. brasiliensis conidia and plasma proteins, while theintrinsic pathway was markedly altered. These results indicate that P.brasiliensis conidia interact with proteins involved in thecoagulation system. This interaction may play an important role in the initialinflammatory response, as well as fungal disease progression caused byP. brasiliensis dissemination.     

Interaction between rhizobia and potato tissues10

Several cultivars of Solanum tuberosum L., the potato, weregrown on tissue culture media and their roots inoculated withstrains of rhizobia known to infect legumes at root junctionsor between epidermal cells. Infection incidence and severityshowed considerable cultivar/bacterial strain interaction. Bacteriaspread through intercellular spaces and invaded cells in a non-structuredway: some infections penetrated to the root xylem. There wasno evidence that potato root cells produced nod-inducing factorsand nitrogenase activity was not detected. In some host/rhizobialcombinations outgrowths were formed on roots. These varied fromloose infected callus tissue at the junctions of lateral roots,to modified lateral roots of limited growth which showed somecolonization by rhizobia. Key words: Solarium tuberosum L, potato, glycoprotein, immunogold labelling, rhizobia, flavonoids     

How Food Controls Aggression in Drosophila

How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. For instance, it is unclear whether food increases aggression directly, or as a secondary consequence of increased social interactions caused by attraction to food. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner by which food influences aggression. We show that food promotes aggression in flies, and that it does so independently of any effect on frequency of contact between males, increase in locomotor activity or general enhancement of social interactions. Importantly, the level of aggression depends on the absolute amount of food, rather than on its surface area or concentration. When food resources exceed a certain level, aggression is diminished, suggestive of reduced competition. Finally, we show that detection of sugar via Gr5a⁺ gustatory receptor neurons (GRNs) is necessary for food-promoted aggression. These data demonstrate that food exerts a specific effect to promote aggression in male flies, and that this effect is mediated, at least in part, by sweet-sensing GRNs.     

Soluble Adenylyl Cyclase Controls Mitochondria-dependent Apoptosis in Coronary Endothelial Cells

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.Increasing evidence suggests that apoptosis of endothelial cells (EC)³ may be responsible for acute and chronic vascular diseases, e.g. through atherogenesis (1), endothelial dysfunction (2), or thrombosis (3). Within several signaling mechanisms, a cAMP-dependent signaling pathway plays a substantial role in mediating apoptotic cell death induced by various stress factors. Elevation of the cellular cAMP either by forskolin-induced stimulation of the G-protein-responsive transmembrane adenylyl cyclase (tmAC) or by treatment with cAMP analogs has been shown to lead to both induction and suppression of apoptosis in different cell types (4–7). This discrepancy may be due to differences in cell types and experimental models. Alternatively, a lack of specificity of tmAC-induced signals, especially directed to distant intracellular targets like mitochondria, may be a cause of the discrepancy. Indeed, the classical model of cAMP signaling requires the diffusion of cAMP from plasma membrane-localized tmAC to targets localized throughout the cell. Diffusion of cAMP throughout the cytosol makes it difficult to selectively activate distally localized targets without also activating more proximal targets. Therefore, such diffusion of cAMP would likely diminish specificity, selectivity, and signal strength. This model is further complicated by the presence of phosphodiesterases, which degrade cAMP, thus preventing its diffusion.In addition to tmAC, a second source of cAMP, soluble adenylyl cyclase (sAC), was demonstrated for mammalian cells (8, 9). Cytosolic localization of sAC provides both specificity and selectivity by permitting generation of cAMP proximal to intracellular targets. Furthermore, this model for cAMP action incorporates phosphodiesterases, which would act to limit diffusion and prevent nonspecific effector activation.Whether sAC participates in apoptosis was unknown. A previous report demonstrated that sAC is co-localized with mitochondria (10). Because mitochondria play a fundamental role in apoptosis (11), we hypothesized that sAC may influence the development of apoptosis by modulating the mitochondrial pathway of apoptosis. Therefore, we aimed to examine the role of sAC in apoptotic cell death, especially its role in the modulation of the mitochondria-dependent pathway of apoptosis. For this purpose, apoptosis was induced in rat coronary EC by simulated in vitro ischemia or by acidosis. By applying pharmacological inhibition of sAC or small interfering RNA (siRNA)-mediated sAC knockdown, we found that sAC activity is required for the induction of apoptosis by ischemia or acidosis. Additionally, translocation of sAC to mitochondria and the sAC-dependent release of cytochrome c suggest that this cyclase specifically regulates the mitochondrial pathway of apoptosis.     

Interaction between Fiscal and Monetary Policy in a Dynamic Nonlinear Model

The objective of this study is to verify the dynamics between fiscal policy, measured by public debt, and monetary policy, measured by a reaction function of a central bank. Changes in monetary policies due to deviations from their targets always generate fiscal impacts. We examine two policy reaction functions: the first related to inflation targets and the second related to economic growth targets. We find that the condition for stable equilibrium is more restrictive in the first case than in the second. We then apply our simulation model to Brazil and United Kingdom and find that the equilibrium is unstable in the Brazilian case but stable in the UK case.     

A Negative Regulatory Loop between MicroRNA and Hox Gene Controls Posterior Identities in Caenorhabditis elegans

MicroRNAs (miRNAs) have been found to regulate gene expression across eukaryotic species, but the function of most miRNA genes remains unknown. Here we describe how the analysis of the expression patterns of a well-conserved miRNA gene, mir-57, at cellular resolution for every minute during early development of Caenorhabditis elegans provided key insights in understanding its function. Remarkably, mir-57 expression shows strong positional bias but little tissue specificity, a pattern reminiscent of Hox gene function. Despite the minor defects produced by a loss of function mutation, overexpression of mir-57 causes dramatic posterior defects, which also mimic the phenotypes of mutant alleles of a posterior Hox gene, nob-1, an Abd homolog. More importantly, nob-1 expression is found in the same two posterior AB sublineages as those expressing mir-57 but with an earlier onset. Intriguingly, nob-1 functions as an activator for mir-57 expression; it is also a direct target of mir-57. In agreement with this, loss of mir-57 function partially rescues the nob-1 allele defects, indicating a negative feedback regulatory loop between the miRNA and Hox gene to provide positional cues. Given the conservation of the miRNA and Hox gene, the regulatory mechanism might be broadly used across species. The strategy used here to explore mir-57 function provides a path to dissect the regulatory relationship between genes.     

The Interaction between Thermodynamic Stability and Buried Free Cysteines in Regulating the Functional Half-Life of Fibroblast Growth Factor-1

Protein biopharmaceuticals are an important and growing area of human therapeutics; however, the intrinsic property of proteins to adopt alternative conformations (such as during protein unfolding and aggregation) presents numerous challenges, limiting their effective application as biopharmaceuticals. Using fibroblast growth factor-1 as model system, we describe a cooperative interaction between the intrinsic property of thermostability and the reactivity of buried free-cysteine residues that can substantially modulate protein functional half-life. A mutational strategy that combines elimination of buried free cysteines and secondary mutations that enhance thermostability to achieve a substantial gain in functional half-life is described. Furthermore, the implementation of this design strategy utilizing stabilizing mutations within the core region resulted in a mutant protein that is essentially indistinguishable from wild type as regard protein surface and solvent structure, thus minimizing the immunogenic potential of the mutations. This design strategy should be generally applicable to soluble globular proteins containing buried free-cysteine residues.     

Functional Overlap between eIF4G Isoforms in Saccharomyces cerevisiae

Initiation factor eIF4G is a key regulator of eukaryotic protein synthesis, recognizing proteins bound at both ends of an mRNA to help recruit messages to the small (40S) ribosomal subunit. Notably, the genomes of a wide variety of eukaryotes encode multiple distinct variants of eIF4G. We found that deletion of eIF4G1, but not eIF4G2, impairs growth and global translation initiation rates in budding yeast under standard laboratory conditions. Not all mRNAs are equally sensitive to loss of eIF4G1; genes that encode messages with longer poly(A) tails are preferentially affected. However, eIF4G1-deletion strains contain significantly lower levels of total eIF4G, relative to eIF4G2-delete or wild type strains. Homogenic strains, which encode two copies of either eIF4G1 or eIF4G2 under native promoter control, express a single isoform at levels similar to the total amount of eIF4G in a wild type cell and have a similar capacity to support normal translation initiation rates. Polysome microarray analysis of these strains and the wild type parent showed that translationally active mRNAs are similar. These results suggest that total eIF4G levels, but not isoform-specific functions, determine mRNA-specific translational efficiency.     

Human Urinary Composition Controls Antibacterial Activity of Siderocalin

During Escherichia coli urinary tract infections, cells in the human urinary tract release the antimicrobial protein siderocalin (SCN; also known as lipocalin 2, neutrophil gelatinase-associated lipocalin/NGAL, or 24p3). SCN can interfere with E. coli iron acquisition by sequestering ferric iron complexes with enterobactin, the conserved E. coli siderophore. Here, we find that human urinary constituents can reverse this relationship, instead making enterobactin critical for overcoming SCN-mediated growth restriction. Urinary control of SCN activity exhibits wide ranging individual differences. We used these differences to identify elevated urinary pH and aryl metabolites as key biochemical host factors controlling urinary SCN activity. These aryl metabolites are well known products of intestinal microbial metabolism. Together, these results identify an innate antibacterial immune interaction that is critically dependent upon individualistic chemical features of human urine.