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In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. [ABSTRACT FROM AUTHOR]

The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons. [ABSTRACT FROM AUTHOR]

Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes in vitro. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig. [ABSTRACT FROM AUTHOR]

Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP’s) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp. [ABSTRACT FROM AUTHOR]

No universal approach has been reported for specific monitoring of the catalytic activity of a wide range of kinases in cells. The present study describes an original platform for detecting the autonomous activity of serine/threonine kinases in cells through the introduction of expression vectors encoding modified substrate kinase fusion proteins. The surrogate substrate used consists of the p53 tumor suppressor protein fused with individual kinase domains (Chk1, DYRK3, and Cdk5) at its carboxy-terminal through four tandem Gly-Gly-Gly-Gly-Ser repeats. After transfection into cells, phosphorylation of the p53 moiety could be specifically induced by the catalytic activity of kinases contained in the fusion protein. Moreover, p53 phosphorylation was significantly blocked when a kinase-inactive mutant was used as the fusion partner instead of the active kinase. Using this system, the cell-based evaluation of several Cdk5 inhibitors was demonstrated. Thus, this approach provides a novel platform for the specific, cell-based screening of inhibitors of a wide prospective range of protein kinases and is of tremendous potential for drug discovery efforts. [ABSTRACT FROM AUTHOR]

RsbW, an anti-sigma factor possessing kinase activity, is expressed by many Gram-positive bacteria including Staphylococcus aureus. To obtain clues about the domain structure and the folding-unfolding mechanism of RsbW, we have elaborately studied rRsbW, a recombinant S. aureus RsbW. Sequence analysis of the protein fragments, generated by the limited proteolysis of rRsbW, has proposed it to be a single-domain protein. The unfolding of rRsbW in the presence of GdnCl or urea was completely reversible in nature and occurred through the formation of at least two intermediates. The structure, shape, and the surface hydrophobicity of no intermediate completely matches with those of other intermediates or the native rRsbW. Interestingly, one of the intermediates, formed in the presence of less GdnCl concentrations, has a molten globule-like structure. Conversely, all of the intermediates, like native rRsbW, exist as dimers in aqueous solution. The putative molten globule and the urea-generated intermediates also have retained some kinase activity. Additionally, the putative ATP binding site/catalytic site of rRsbW shows higher denaturant sensitivity than the tentative dimerization region of this enzyme. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of MKK2 or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2. [ABSTRACT FROM AUTHOR]

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T. [ABSTRACT FROM AUTHOR]

Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3’-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3’-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3’-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst. [ABSTRACT FROM AUTHOR]

Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4), a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The experimental results showed that EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells. [ABSTRACT FROM AUTHOR]

Water currents, plants, and animals all disperse fungal spores, but the most commonly considered form of spore transport is wind, where astonishingly high fungal spore fluxes have been observed in terrestrial ecosystems (513 spores per m SP 2 sp s SP 1 sp ) [[6], [7]]. In the mucoralean fungus I Rhizopus oryzae i , the CotH proteins found on the surface of spores promote adhesion and invasion by acting as ligands for glucose-regulated protein 78 (GRP78) on the surface of endothelial cells, similar to the examples from I A i . I fumigatus i conidia is the Mep1p metalloprotease, which is released from spores in the mammalian lung to cleave host complement proteins and enhance infection, similar to the Alp1p serine protease released from mycelia for the same purpose [[44]]. We urge the fungal community to learn from other systems, like the prokaryotes, in which additional activities have already been ascribed to surface proteins, as with the kinase activity of the CotH proteins that phosphorylate extracellular proteins to aid in germination of the endospore [[47]]. [Extracted from the article]

Host plant resistance is the most practical approach to control the Southern root-knot nematode (Meloidogyne incognita; RKN), which has emerged as one of the most serious economic pests of Upland cotton (Gossypium hirsutum L.). Previous QTL analyses have identified a resistance locus on chromosome 11 (qMi-C11) affecting galling and another locus on chromosome-14 (qMi-C14) affecting egg production. Although these two QTL regions were fine mapped and candidate genes identified, expression profiling of genes would assist in further narrowing the list of candidate genes in the QTL regions. We applied the comparative transcriptomic approach to compare expression profiles of genes between RKN susceptible and resistance genotypes at an early stage of RKN development that coincides with the establishment of a feeding site and at the late stage of RKN development that coincides with RKN egg production. Sequencing of cDNA libraries produced over 315 million reads of which 240 million reads (76%) were mapped on to the Gossypium hirsutum genome. A total of 3,789 differentially expressed genes (DEGs) were identified which were further grouped into four clusters based on their expression profiles. A large number of DEGs were found to be down regulated in the susceptible genotype at the late stage of RKN development whereas several genes were up regulated in the resistant genotype. Key enriched categories included transcription factor activity, defense response, response to phyto-hormones, cell wall organization, and protein serine/threonine kinase activity. Our results also show that the DEGs in the resistant genotype at qMi-C11 and qMi-C14 loci displayed higher expression of defense response, detoxification and callose deposition genes, than the DEGs in the susceptible genotype. [ABSTRACT FROM AUTHOR]

The host limits adenovirus infections by mobilizing immune systems directed against infected cells that also represent major barriers to clinical use of adenoviral vectors. Adenovirus early transcription units encode a number of products capable of thwarting antiviral immune responses by co-opting host cell pathways. Although the EGF receptor (EGFR) was a known target for the early region 3 (E3) RIDα protein encoded by nonpathogenic group C adenoviruses, the functional role of this host-pathogen interaction was unknown. Here we report that incoming viral particles triggered a robust, stress-induced pathway of EGFR trafficking and signaling prior to viral gene expression in epithelial target cells. EGFRs activated by stress of adenoviral infection regulated signaling by the NFκB family of transcription factors, which is known to have a critical role in the host innate immune response to infectious adenoviruses and adenovirus vectors. We found that the NFκB p65 subunit was phosphorylated at Thr254, shown previously by other investigators to be associated with enhanced nuclear stability and gene transcription, by a mechanism that was attributable to ligand-independent EGFR tyrosine kinase activity. Our results indicated that the adenoviral RIDα protein terminated this pathway by co-opting the host adaptor protein Alix required for sorting stress-exposed EGFRs in multivesicular endosomes, and promoting endosome-lysosome fusion independent of the small GTPase Rab7, in infected cells. Furthermore RIDα expression was sufficient to down-regulate the same EGFR/NFκB signaling axis in a previously characterized stress-activated EGFR trafficking pathway induced by treatment with the pro-inflammatory cytokine TNF-α. We also found that cell stress activated additional EGFR signaling cascades through the Gab1 adaptor protein that may have unappreciated roles in the adenoviral life cycle. Similar to other E3 proteins, RIDα is not conserved in adenovirus serotypes associated with potentially severe disease, suggesting stress-activated EGFR signaling may contribute to adenovirus virulence. [ABSTRACT FROM AUTHOR]

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells. [ABSTRACT FROM AUTHOR]

Inhibition of human papillomavirus (HPV) replication is a promising therapeutic approach for intervening with HPV-related pathologies. Primary targets for interference are two viral proteins, E1 and E2, which are required for HPV replication. Both E1 and E2 are phosphoproteins; thus, the protein kinases that phosphorylate them might represent secondary targets to achieve inhibition of HPV replication. In the present study, we show that CX4945, an ATP-competitive small molecule inhibitor of casein kinase 2 (CK2) catalytic activity, suppresses replication of different HPV types, including novel HPV5NLuc, HPV11NLuc and HPV18NLuc marker genomes, but enhances the replication of HPV16 and HPV31. We further corroborate our findings using short interfering RNA (siRNA)-mediated knockdown of CK2 α and α’ subunits in U2OS and CIN612 cells; we show that while both subunits are expressed in these cell lines, CK2α is required for HPV replication, but CK2α’ is not. Furthermore, we demonstrate that CK2α acts in a kinase activity-dependent manner and regulates the stability and nuclear retention of endogenous E1 proteins of HPV11 and HPV18. This unique feature of CK2α makes it an attractive target for developing antiviral agents. [ABSTRACT FROM AUTHOR]

The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM_Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM_Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM_Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM_Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM_Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM_Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs produce by cell endogenously. They are 18–26 nucleotides in length and play important roles at the post-transcriptional stage of gene regulation. Evolutionarily, miRNAs are conserved and their conservation plays an important role in the prediction of new miRNAs in different plants. Wheat (Triticum aestivum L.) is an important diet and consumed as second major crop in the world. This significant cereal crop was focused here through comparative genomics-based approach to identify new conserved miRNAs and their targeted genes. This resulted into a total of 212 new conserved precursor miRNAs (pre-miRNAs) belonging to 185 miRNA families. These newly profiled wheat’s miRNAs are also annotated for stem-loop secondary structures, length distribution, organ of expression, sense/antisense orientation and characterization from their expressed sequence tags (ESTs). Moreover, fifteen miRNAs along with housekeeping gene were randomly selected and subjected to RT-PCR expressional validation. A total of 32927 targets are also predicted and annotated for these newly profiled wheat miRNAs. These targets are found to involve in 50 gene ontology (GO) enrichment terms and significant processes. Some of the significant targets are RNA-dependent DNA replication (GO:0006278), RNA binding (GO:0003723), nucleic acid binding (GO:0003676), DNA-directed RNA polymerase activity (GO:0003899), magnesium ion transmembrane transporter activity (GO:0015095), antiporter activity (GO:0015297), solute:hydrogen antiporter activity (GO:0015299), protein kinase activity (GO:0004672), ATP binding (GO:0005524), regulation of Rab GTPase activity (GO:0032313) Rab GTPase activator activity (GO:0005097), regulation of signal transduction (GO:0009966) and phosphoprotein phosphatase inhibitor activity (GO:0004864). These findings will be helpful to manage this economically important grain plant for desirable traits through miRNAs regulation. [ABSTRACT FROM AUTHOR]

Neurons develop elaborate morphologies that provide a model for understanding cellular architecture. By studying C. elegans sensory dendrites, we previously identified genes that act to promote the extension of ciliated sensory dendrites during embryogenesis. Interestingly, the nonciliated dendrite of the oxygen-sensing neuron URX is not affected by these genes, suggesting it develops through a distinct mechanism. Here, we use a visual forward genetic screen to identify mutants that affect URX dendrite morphogenesis. We find that disruption of the MAP kinase MAPK-15 or the βH-spectrin SMA-1 causes a phenotype opposite to what we had seen before: dendrites extend normally during embryogenesis but begin to overgrow as the animals reach adulthood, ultimately extending up to 150% of their normal length. SMA-1 is broadly expressed and acts non-cell-autonomously, while MAPK-15 is expressed in many sensory neurons including URX and acts cell-autonomously. MAPK-15 acts at the time of overgrowth, localizes at the dendrite ending, and requires its kinase activity, suggesting it acts locally in time and space to constrain dendrite growth. Finally, we find that the oxygen-sensing guanylate cyclase GCY-35, which normally localizes at the dendrite ending, is localized throughout the overgrown region, and that overgrowth can be suppressed by overexpressing GCY-35 or by genetically mimicking elevated cGMP signaling. These results suggest that overgrowth may correspond to expansion of a sensory compartment at the dendrite ending, reminiscent of the remodeling of sensory cilia or dendritic spines. Thus, in contrast to established pathways that promote dendrite growth during early development, our results reveal a distinct mechanism that constrains dendrite growth throughout the life of the animal, possibly by controlling the size of a sensory compartment at the dendrite ending. [ABSTRACT FROM AUTHOR]

Calcium-dependent protein kinases (CPKs) play an essential role in the regulation of pollen tube growth. Although CPK genes have been identified in maize, and some have been functionally characterized, the molecular function of ZmCPKs associated with pollen tube development remains less well studied. Here, we report that a pollen-specific CPK, ZmCPK32, is involved in the regulation of pollen germination and tube extension. ZmCPK32 exhibited CPK activity and was localized on the plasma membrane and punctate internal membrane compartments via N-terminal acylation. In situ hybridization and real-time PCR revealed that ZmCPK32 transcripts accumulated in pollen and expression was dramatically upregulated during shedding. To elucidate the function of this gene, we transiently expressed a ZmCPK32-GFP fusion protein in tobacco pollen using microparticle bombardment. ZmCPK32 accumulation inhibited pollen germination and reduced pollen tube growth, but this effect was abolished when the kinase-inactive variant was expressed, indicating that kinase activity is critical for its regulatory function. In addition, the plasma membrane localization of ZmCPK32 is essential for regulating polar growth, as pollen expressing the cytosol-localized kinase displayed reduced tube length but germinated well. Moreover, the constitutively active form of ZmCPK32 enhanced the reduction in the germination rate, indicating that the specific activation of ZmCPK32 via calcium ions at the cortical growth point is essential for regulating appropriate germination. The results suggest that ZmCPK32 is functionally associated with pollen tube growth, and could represent a potential target for breeding male-sterile maize. [ABSTRACT FROM AUTHOR]

Four full-sibling intact male Miniature Poodles were evaluated at 4–19 months of age. One was clinically normal and three were affected. All affected dogs were reluctant to exercise and had generalised muscle atrophy, a stiff gait and a markedly elevated serum creatine kinase activity. Two affected dogs also showed poor development, learning difficulties and episodes of abnormal behaviour. In these two dogs, investigations into forebrain structural and metabolic diseases were unremarkable; electromyography demonstrated fibrillation potentials and complex repetitive discharges in the infraspinatus, supraspinatus and epaxial muscles. Histopathological, immunohistochemical and immunoblotting analyses of muscle biopsies were consistent with dystrophin-deficient muscular dystrophy. DNA samples were obtained from all four full-sibling male Poodles, a healthy female littermate and the dam, which was clinically normal. Whole genome sequencing of one affected dog revealed a >5 Mb deletion on the X chromosome, encompassing the entire DMD gene. The exact deletion breakpoints could not be experimentally ascertained, but we confirmed that this region was deleted in all affected males, but not in the unaffected dogs. Quantitative polymerase chain reaction confirmed all three affected males were hemizygous for the mutant X chromosome, while the wildtype chromosome was observed in the unaffected male littermate. The female littermate and the dam were both heterozygous for the mutant chromosome. Forty-four Miniature Poodles from the general population were screened for the mutation and were homozygous for the wildtype chromosome. The finding represents a naturally-occurring mutation causing dystrophin-deficient muscular dystrophy in the dog. [ABSTRACT FROM AUTHOR]

N. meningitidis induces extensive gene expression changes in human monocytes, suggesting that complex networks of signaling pathways are activated during meningococcal sepsis. These effects are modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). To further study changes in signal transduction suggested by mRNA data, we used kinase substrate arrays to identify composite kinase activities induced by lysates from a primary human monocyte model system. Cell lysates were prepared from monocytes treated with the following experimental conditions: 106 N. meningitidis/mL, 25 ng/mL IL-10, 106 N. meningitidis/mL in combination with 25 ng/mL IL-10, and vehicle. Lysates were subjected to kinase activity profiling with Tyrosine Kinase PamChip® arrays containing 144 kinase peptide substrates. In our experimental model, we were not able to detect a statistically significant large-scale change in ex vivo array peptide phosphorylation by lysates from monocytes treated for 15 minutes. Targets of the IL-10 anti-inflammatory response were not identified. A profound inhibition of array peptide phosphorylation by monocytes treated for 60 minutes was identified, suggesting low activity of a large number of kinases associated with different signaling pathways and immune cell functions, including STAT3 activity, Nf-κB and VEGF signaling, and PTEN signaling activity. The peptide representing ZBTB16, which was reduced in phosphorylation by lysates from all three experimental conditions, was in Ingenuity Pathway Analysis identified to be linked to reduced cytokine release and mRNA levels of tumor necrosis factor (TNF), IL-6, and CXCL10. Further studies should investigate changes in tyrosine kinase-mediated signal transduction in human immune cells, in order to evaluate the potential clinical application of kinome profiling in the study of systemic inflammatory responses to pathogens. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the potential effects of methyl donor supplementation of pregnant animals in the presence or absence of a concomitant lactation on the methylome of the offspring. Twenty Holstein cows, 10 nulliparous (non-lactating while pregnant) and 10 multiparous (lactating while pregnant) were blocked by parity and randomly assigned to an i.m. weekly injections of a placebo (CTRL) or a solution containing methyl donors (MET). After calving, 5 calves randomly selected from each treatment (two born to non-lactating and three to lactating dams) were blood-sampled to determine their full methylome. There were more than 2,000 CpG differentially methylated between calves born to CTRL and those born to MET, and also between calves born to lactating and non-lactating dams. Most of the differences affected genes involved in immune function, cell growth regulation and differentiation, kinase activity, and ion channeling. We conclude that the coexistence of pregnancy and lactation affects the methylome of the offspring, and that supplementation of methyl donors early in gestation has also consequences on the methylome. [ABSTRACT FROM AUTHOR]

Hepatic dysfunction is a feature of dengue virus (DENV) infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show apoptosis, which relates to the pathogenesis of DENV infection. However, how DENV induced liver injury is not fully understood. In this study, we aim to identify the factors that influence cell death by employing an apoptosis-related siRNA library screening. Our results show the effect of 558 gene silencing on caspase 3-mediated apoptosis in DENV-infected Huh7 cells. The majority of genes that contributed to apoptosis were the apoptosis-related kinase enzymes. Tumor necrosis factor superfamily member 12 (TNFSF12), and sphingosine kinase 2 (SPHK2), were selected as the candidate genes to further validate their influences on DENV-induced apoptosis. Transfection of siRNA targeting SPHK2 but not TNFSF12 genes reduced apoptosis determined by Annexin V/PI staining. Knockdown of SPHK2 did not reduce caspase 8 activity; however, did significantly reduce caspase 9 activity, suggesting its involvement of SPHK2 in the intrinsic pathway of apoptosis. Treatment of ABC294649, an inhibitor of SPHK2, reduced the caspase 3 activity, suggesting the involvement of its kinase activity in apoptosis. Knockdown of SPHK2 significantly reduced caspase 3 activity not only in DENV-infected Huh7 cells but also in DENV-infected HepG2 cells. Our results were consistent across all of the four serotypes of DENV infection, which supports the pro-apoptotic role of SPHK2 in DENV-infected liver cells. [ABSTRACT FROM AUTHOR]

Shigella dysenteriae (S.dysenteriae) the causative agent of bacillary dysentery invades the human colonic epithelium resulting in severe intestinal inflammatory response and epithelial destruction. However, the mechanism by which S.dysenteriae infection regulates proinflammatory cytokines during intestinal inflammation is still obscure. In this study, we evaluated whether the interaction of β-catenin and NF-κB regulates proinflammatory cytokines TNF-α and IL-8 by modulating GSK-3β activity during S.dysenteriae infection in rat ileal loop model. Here we demonstrated that S.dysenteriae infection stimulate β-catenin degradation which in turn decreased the association between NF-κB and β-catenin. Also, we showed that S.dysenteriae infection increased GSK-3β kinase activity which in turn phosphorylates β-catenin for its degradation by ubiquitination and upregulates IL-8 through NF-κB activation thereby leading to inflammation. Thus these findings revealed the role of β-catenin/ NF-κB and GSK-3β in modulating the inflammatory response during bacterial infection and also showed that β-catenin acts as a critical regulator of inflammation. [ABSTRACT FROM AUTHOR]

As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication. [ABSTRACT FROM AUTHOR]

The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1 or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation of a central transducer of signals that control environmental response and virulence programs. [ABSTRACT FROM AUTHOR]

A heavy-ion beam has been recognized as an effective mutagen for plant breeding and applied to the many kinds of crops including rice. In contrast with X-ray or γ-ray, the heavy-ion beam is characterized by a high linear energy transfer (LET). LET is an important factor affecting several aspects of the irradiation effect, e.g. cell survival and mutation frequency, making the heavy-ion beam an effective mutagen. To study the mechanisms behind LET-dependent effects, expression profiling was performed after heavy-ion beam irradiation of imbibed rice seeds. Array-based experiments at three time points (0.5, 1, 2 h after the irradiation) revealed that the number of up- or down-regulated genes was highest 2 h after irradiation. Array-based experiments with four different LETs at 2 h after irradiation identified LET-independent regulated genes that were up/down-regulated regardless of the value of LET; LET–dependent regulated genes, whose expression level increased with the rise of LET value, were also identified. Gene ontology (GO) analysis of LET-independent up-regulated genes showed that some GO terms were commonly enriched, both 2 hours and 3 weeks after irradiation. GO terms enriched in LET-dependent regulated genes implied that some factor regulates genes that have kinase activity or DNA-binding activity in cooperation with the ATM gene. Of the LET-dependent up-regulated genes, OsPARP3 and OsPCNA were identified, which are involved in DNA repair pathways. This indicates that the Ku-independent alternative non-homologous end-joining pathway may contribute to repairing complex DNA legions induced by high-LET irradiation. These findings may clarify various LET-dependent responses in rice. [ABSTRACT FROM AUTHOR]

Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the development of host immune responses that provide effective antitumor immunity against established ovarian cancer. [ABSTRACT FROM AUTHOR]

Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms, albeit lower than that observed in macrophages. The persistent transcriptome dysregulation in the microglia shared patterns with neurological disorders indicating that the associated persistent depressive symptoms share a common transcriptome basis. [ABSTRACT FROM AUTHOR]

The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and F1-elated protein inase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that ype ne rotein hosphatase 1 (TOPP1) and its regulatory protein, nhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. [ABSTRACT FROM AUTHOR]

FgfrL1 is the fifth member of the fibroblast growth factor receptor (Fgfr) family. Studies with FgfrL1 deficient mice have demonstrated that the gene plays an important role during embryonic development. FgfrL1 knock-out mice die at birth as they have a malformed diaphragm and lack metanephric kidneys. Similar to the classical Fgfrs, the FgfrL1 protein contains an extracellular part composed of three Ig-like domains that interact with Fgf ligands and heparin. However, the intracellular part of FgfrL1 is not related to the classical receptors and does not possess any tyrosine kinase activity. Curiously enough, the amino acid sequence of this domain is barely conserved among different species, with the exception of three motifs, namely a dileucine peptide, a tandem tyrosine-based motif YXXΦ and a histidine-rich sequence. To investigate the function of the intracellular domain of FgfrL1, we have prepared genetically modified mice that lack the three conserved sequence motifs, but instead contain a GFP cassette (FgfrL1ΔC-GFP). To our surprise, homozygous FgfrL1ΔC-GFP knock-in mice are viable, fertile and phenotypically normal. They do not exhibit any alterations in the diaphragm or the kidney, except for a slight reduction in the number of glomeruli that does not appear to affect life expectancy. In addition, the pancreas of both FgfrL1ΔC-GFP knock-in and FgfrL1 knock-out mice do not show any disturbances in the production of insulin, in contrast to what has been suggested by recent studies. Thus, the conserved motifs of the intracellular FgfrL1 domain are dispensable for organogenesis and normal life. We conclude that the extracellular domain of the protein must conduct the vital functions of FgfrL1. [ABSTRACT FROM AUTHOR]

Casein kinase 2 (protein kinase CK2) is a conserved eukaryotic serine/theronine kinase with multiple substrates and roles in the regulation of cellular processes such as cellular stress, cell proliferation and apoptosis. Here we report a detailed analysis of the Plasmodium falciparum CK2, PfCK2, demonstrating that this kinase, like the mammalian orthologue, is a dual specificity kinase able to phosphorylate at both serine and tyrosine. However, unlike the human orthologue that is auto-phosphorylated on tyrosine within the activation loop, PfCK2 shows no activation loop auto-phosphorylation but rather is auto-phosphorylated at threonine 63 within subdomain I. Phosphorylation at this site in PfCK2 is shown here to regulate the intrinsic kinase activity of PfCK2. Furthermore, we generate an homology model of PfCK2 in complex with the known selective protein kinase CK2 inhibitor, quinalizarin, and in so doing identify key co-ordinating residues in the ATP binding pocket that could aid in designing selective inhibitors to PfCK2. [ABSTRACT FROM AUTHOR]

LKB1 is a Ser/Thr kinase that plays an important role in controlling both energy metabolism and cell polarity in metazoan organisms. LKB1 is also a tumor suppressor, and homozygous, inactivating mutations are found in a wide range of human cancers. In lung cancer, inactivating mutations are found in 10 to 50%of cases, but the consequences of functional loss in this context are poorly understood. We report here that LKB1 is required for the maturation of apical junctions in the human bronchial epithelial cell line 16HBE14o- (16HBE). This activity is dependent on an interaction with the Rho guanine nucleotide exchange factor p114RhoGEF but is independent of LKB1 kinase activity. Together, LKB1 and p114RhoGEF control RhoA activity in these cells to promote apical junction assembly. [ABSTRACT FROM AUTHOR]

Directional cues guide growth cones. While molecules like UNC-6/netrin direct migrations along the dorsoventral axis of many organisms, it is unclear how anteroposterior guidance is achieved. We describe a physical interaction between VAB-8, a protein both necessary and sufficient for posteriorly directed migrations in C. elegans, and UNC-51, a conserved serine/threonine kinase that functions generally in axon outgrowth. We show that both proteins function in the CAN neurons to direct their axons posteriorly. Expression in the CANs of peptides predicted to interfere with interactions between UNC-51 and both VAB-8 and UNC-14, a second protein that interacts physically with UNC-51, disrupts CAN axon outgrowth. We provide genetic evidence that VAB-8 functions in an UNC-51 pathway for posteriorly directed CAN axon guidance and show that VAB-8 and UNC-14 can be targets of UNC-51 kinase activity. Taken together, our results suggest that VAB-8 and UNC-14 are substrates that mediate the function of UNC-51 in axon outgrowth. [ABSTRACT FROM AUTHOR]

Directional cues guide growth cones. While molecules like UNC-6/netrin direct migrations along the dorsoventral axis of many organisms, it is unclear how anteroposterior guidance is achieved. We describe a physical interaction between VAB-8, a protein both necessary and sufficient for posteriorly directed migrations in C. elegans, and UNC-51, a conserved serine/threonine kinase that functions generally in axon outgrowth. We show that both proteins function in the CAN neurons to direct their axons posteriorly. Expression in the CANs of peptides predicted to interfere with interactions between UNC-51 and both VAB-8 and UNC-14, a second protein that interacts physically with UNC-51, disrupts CAN axon outgrowth. We provide genetic evidence that VAB-8 functions in an UNC-51 pathway for posteriorly directed CAN axon guidance and show that VAB-8 and UNC-14 can be targets of UNC-51 kinase activity. Taken together, our results suggest that VAB-8 and UNC-14 are substrates that mediate the function of UNC-51 in axon outgrowth. [ABSTRACT FROM AUTHOR]

Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine-threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle- regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome. [ABSTRACT FROM AUTHOR]