Search

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell- to- cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic- di- AMP (c- di- AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c- di- AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c- di- AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c- di- AMP inactivation and lack of c- di- AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c- di- AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c- di- AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery. [ABSTRACT FROM AUTHOR]

ABSTRACT The rck open reading frame (ORF) on the pefI-srgC operon encodes an outer membrane protein responsible for invasion of nonphagocytic cell lines and resistance to complement-mediated killing. Until now, the rck ORF was only detected on the virulence plasmids of three serovars of Salmonella subsp. enterica (i.e., Bovismorbificans, Enteritidis, and Typhimurium). The increasing number of Salmonella genome sequences allowed us to use a combination of reference sequences and whole-genome multilocus sequence typing (wgMLST) data analysis to probe the presence of the operon and of rck in a wide array of isolates belonging to all Salmonella species and subspecies. We established the presence of partial or complete operons in 61 subsp. enterica serovars as well as in 4 other subspecies with various syntenies and frequencies. The rck ORF itself was retrieved in 36 subsp. enterica serovars and in two subspecies with either chromosomal or plasmid-borne localization. It displays high conservation of its sequence within the genus, and we demonstrated that most of the allelic variations identified did not alter the virulence properties of the protein. However, we demonstrated the importance of the residue at position 38 (at the level of the first extracellular loop of the protein) in the invasin function of Rck. Altogether, our results highlight that rck is not restricted to the three formerly identified serovars and could therefore have a more important role in virulence than previously expected. Moreover, this work raises questions about the mechanisms involved in rck acquisition and about virulence plasmid distribution and evolution. IMPORTANCE The foodborne pathogen Salmonella is responsible for a wide variety of pathologies depending on the infected host, the infecting serovars, and its set of virulence factors. However, the implication of each of these virulence factors and their role in the specific host-pathogen interplay are not fully understood. The significance of our research is in determining the distribution of one of these factors, the virulence plasmid-encoded invasin and resistance to complement killing protein Rck. In addition to providing elements of reflection concerning the mechanisms of acquisition of specific virulence genes in certain serotypes, this work will help to understand the role of Rck in the pathogenesis of Salmonella.

Mitogen Activated Protein (MAP) kinases are a category of serine/threonine kinases that have been demonstrated to regulate intracellular events including stress responses, developmental processes, and cancer progression Although many MAP kinases have been extensively studied in various disease processes, MAP3K19 is an understudied kinase whose activities have been linked to lung disease and fibroblast development. In this manuscript, we use bioinformatics databases starBase, GEPIA, and KMPlotter, to establish baseline expressions of MAP3K19 in different tissue types and its correlation with patient survival in different cancers.

Abstract Background Infectious bursal disease (IBD) is an acute contagious immunosuppressive disease which lead to acute bursal injury and immune dysfunction in poultry. It has caused heavy economic losses in the commercial poultry industry for many years in worldwide. Attenuated live vaccine has widely used in poultry showing some promising signs against IBDV infection. But it has defects such as generating enhanced virulence and immunosuppression prohibits. Therefore, the development of mucosal vaccines using the food-grade lactic acid bacterium is necessary. Here, we construct a recombinant Lactococcus co-expressing the major IBDV antigens VP2 and RCK protein of Salmonella enterica to prevent IBD. Results The recombinant fusion protein VP2-RCK was expressed in a soluble and stable form in the cytoplasm of the recombinant Lactococcus lactis. Animal experiments showed that: (1) the survival rates of the injected immunization inactivated recombinant LAB group and oral immunization live recombinant LAB group were 100% and 80%, respectively; (2) ELISA titers of all serum samples from all experimental groups were negative, but high amounts of specific neutralizing antibodies were detected (1:210 to 1:212); and (3) the bursas of the injected immunization inactivated recombinant LAB group did not suffer damage, as confirmed by clinical observation and bursal histopathological examination. Our results indicate that r-L. lactis-OptiVP2-RCK induces a specific neutralizing-antibody-mediated immune response that confers full protection against very-virulent IBDV (vvIBDV) challenge. Conclusion Lactococcus lactis NZ3900 strain and its matching plasmid pNZ8149 could express the recombinant fusion protein VP2-RCK in a soluble form in the cytoplasm. The protective efficacy of r-L. lactis-OptiVP2-RCK (100%) was better than r-L. lactis-OptiVP2 (0%) which prove RCK protein played its unique role. The neutralizing antibodies titers against infectious bursal disease virus via one-time vaccination with inactivated r-L. lactis-OptiVP2-RCK could reach 1:210 to 1:212, but ELISA titers of all serum samples were negative. For this phenomenon, perhaps because of the change of delivery pathway or the spatial structure of fusion protein. We need further study to test these hypotheses.

Pathogens that colonize deep tissues and spread systemically encounter the innate host resistance mechanism of complement‐mediated lysis and complement opsonization leading to engulfment and degradation by phagocytic cells. Yersinia and Salmonella species have developed numerous strategies to block the antimicrobial effects of complement. These include recruitment of complement regulatory proteins factor H, C4BP, and vitronectin (Vn) as well as interference in late maturation events such as assembly of C9 into the membrane attack complex that leads to bacterial lysis. This review will discuss the contributions of various surface structures (proteins, lipopolysaccharide, and capsules) to evasion of complement‐mediated immune clearance of the systemic pathogens Yersiniae and Salmonellae. Bacterial proteins required for recruitment of complement regulatory proteins will be described, including the details of their interaction with host regulatory proteins, where known. The potential role of the surface proteases Pla (Yersinia pestis) and PgtE (Salmonella species) on the activity of complement regulatory proteins will also be addressed. Finally, the implications of complement inactivation on host cell interactions and host cell targeting for type 3 secretion will be discussed. [ABSTRACT FROM AUTHOR]

Dolomite is used as basic lining for rotary cement kiln due its high refractoriness, corrosion resistance against basic environments and high coating performance, but its poor hydration resistance limits its use. In this work, Egyptian dolomite was converted into outstanding refractory magnesia-calcium zirconate composite (M-CZ) by the addition of 37.8–47.8 wt% zirconia. The mixtures were milled, uni-axially formed and fired at a temperature of 1400–1550 °C for 2 h. Thermal analysis, phase composition, microstructure, densification parameters, and other technological properties of the fired specimens were investigated. It was found that sintered M-CZ composite with bulk density (3.95 g/cm 3 ), cold crushing strength (170 MPa), and high coating ability can be obtained by firing Egyptian dolomite with 37.8 wt% zirconia at temperature 1500 °C with the formation of belite as a secondary product. This composite can be nominated to be produced on a large industrial scale. [ABSTRACT FROM AUTHOR]

Potassium channels ubiquitously exist in nearly all kingdoms of life and perform diverse but important functions. Since the first atomic structure of a prokaryotic potassium channel (KcsA, a channel from Streptomyces lividans) was determined, tremendous progress has been made in understanding the mechanism of potassium channels and channels conducting other ions. In this review, we discuss the structure of various kinds of potassium channels, including the potassium channel with the pore-forming domain only (KcsA), voltage-gated, inwardly rectifying, tandem pore domain, and ligand-gated ones. The general properties shared by all potassium channels are introduced first, followed by specific features in each class. Our purpose is to help readers to grasp the basic concepts, to be familiar with the property of the different domains, and to understand the structure and function of the potassium channels better. [ABSTRACT FROM AUTHOR]

Abstract: The adenovirus E4 11k protein, product of E4 ORF3, is required in infection for processes including normal accumulation of viral late mRNAs. 11k restructures both the nucleus and cytoplasm of infected cells by relocalizing specific host cell target proteins, most strikingly components of nuclear PML oncogenic domains. It is likely that in many cases relocalization inactivates target proteins to produce 11k''s effects, although the mechanism and targets for stimulation of late mRNA accumulation is unknown. We have identified a new set of proteins relocalized by 11k: at least five protein components of cytoplasmic mRNA processing bodies (p-bodies) are found in 11k-induced cytoplasmic aggresomes, sites where proteins are inactivated or destroyed. One of these p-body proteins, RNA helicase Ddx6, binds 11k, suggesting a mechanism for relocalization. Because p-bodies are sites for mRNA degradation, their modification by 11k may provide an explanation for the role of 11k in viral late mRNA accumulation. [Copyright &y& Elsevier]

Abstract: RCK is a cytoplasmic regulatory domain of calcium-gated potassium channels. Binding of Ca2+ by RCK leads to channel activation through a series of yet unknown conformational changes. Structures of the K+ channel, MthK, and its cytoplasmic RCK domain revealed two binding sites for Ca2+ per dimer. We determined the crystal structure of RCK in complex with Cd2+ at 2.2Å resolution. Cd2+ activates MthK more efficiently, and binds at the same binding sites for Ca2+ but with reduced coordination number. Two additional binding sites for Cd2+ are found per dimer; one on the main Rossman-fold lobe, and the other on the small lobe of RCK. Using patch-clamp experiments, we demonstrate that Cd2+ binding to these novel sites enhances activation by Cd2+ and not by Ca2+. The structure reveals a large negatively charged surface patch in the proximity of the Ca2+/Cd2+ binding sites, charge neutralization of which appears to promote the channel open state. [Copyright &y& Elsevier]

Abstract: Attempts to explore the structure and function of Kch, a putative potassium channel of Escherichia coli have yielded varying results; potassium-associated functions have been found in vivo but not in vitro. Here the kch gene is shown to produce two proteins, full-length Kch and the large C-terminal cytosolic domain (the RCK domain). Further, these two proteins are associated at the initial stages of purification. Previous structural studies of full-length Kch claim that the isolated protein forms large aggregates that are not suitable for analysis. The results presented here show that the purified protein sample, although heterogeneous, has one major population with a mass of about 400kDa, implying the presence of two Kch tetramers in a complex form. A three dimensional reconstruction at 25Å based on electron microscopy data from negatively stained particles, revealed a 210Å long and 95Å wide complex in which the two tetrameric Kch units are linked by their RCK domains, giving rise to a large central ring of density. The formation of this dimer of tetramers on expression or during purification, may explain why attempts to reconstitute Kch into liposomes for activity measurements have failed. [Copyright &y& Elsevier]

The Arabidopsis SDS (SOLO DANCERS) and RCK (ROCK-N-ROLLERS) genes are important for male meiosis, but it is still unknown whether they represent conserved functions in plants. We have performed phylogenetic analyses of SDS and RCK and their respective homologs, and identified their putative orthologs in poplar and rice. Quantitative real-time RT-PCR analysis indicated that rice SDS and RCK are expressed preferentially in young flowers, and transgenic RNAi rice lines with reduced expression of these genes exhibited normal vegetative development, but showed significantly reduced fertility with partially sterile flowers and defective pollens. SDS deficiency also caused a decrease in pollen amounts. Further cytological examination of male meiocytes revealed that the SDS deficiency led to defects in homolog interaction and bivalent formation in meiotic prophase I, and RCK deficiency resulted in defective meiotic crossover formation. These results indicate that rice SDS and RCK genes have similar functions to their Arabidopsis orthologs. Because rice and Arabidopsis, respectively, are members of monocots and eudicots, two largest groups of flowering plants, our results suggest that the functions of SDS and RCK are likely conserved in flowering plants.Cell Research (2009) 19:768–782. doi: 10.1038/cr.2009.52; published online 5 May 2009 [ABSTRACT FROM AUTHOR]

Large conductance voltage and Ca2+-dependent K+ channels (BKCa) are activated by both membrane depolarization and intra-cellular Ca2+. Recent studies on bacterial channels have proposed that a Ca2+-induced conformational change within specialized regulators of K+ conductance (RCK) domains is responsible for channel gating. Each pore-forming a subunit of the homotetrameric BKCa channel is expected to contain two intracellular RCK domains. The first RCK domain in BKCa channels (RCK1) has been shown to contain residues critical for Ca2+ sensitivity, possibly participating in the formation of a Ca2+-binding site. The location and structure of the second RCK domain in the BKca channel (RCK2) is still being examined, and the presence of a high-affinity Ca2+-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BKCa and prokaryotic RCK domains that reveal the location of a second RCK domain in human BKCa channels (hSIoRCK2). hSIoRCK2 includes a high-affinity Ca2+-binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSIoRCK2 undergoes a Ca2+-induced change in conformation, associated with an α-to-β structural transition. We also show that the Ca bowl is an essential element for the Ca2+-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca2+-dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSIoRCK1 and hSloRCK2 domains is discussed. [ABSTRACT FROM AUTHOR]

Recent studies in Saccharomyces cerevisiae have unveiled that meiotic recombination crossovers are formed by two genetically distinct pathways: a major interference-sensitive pathway and a minor interference-insensitive pathway. Several proteins, including the MSH4/MSH5 heterodimer and the MER3 DNA helicase, are indispensable for the interference-sensitive pathway. MSH4 homologs have been identified in mice and Arabidopsis and shown to be required for normal levels of crossovers, suggesting that the function of MSH4 may be conserved among major eukaryotic kingdoms. However, it is not known whether an MER3-like function is also required for meiosis in animals and plants. We have identified an Arabidopsis gene that encodes a putative MER3 homolog and is preferentially expressed in meiocytes. T-DNA insertional mutants of this gene exhibit defects in fertility and meiosis. Detailed cytological studies indicate that the mutants are defective in homolog synapsis and crossover formation, resulting in a reduction of bivalents and in the formation of univalents at late prophase I. We have named this gene ROCK-N-ROLLERS ( RCK) to reflect the mutant phenotype of chromosomes undergoing the meiotic ‘dance’ either in pairs or individually. Our results demonstrate that an MER3-like function is required for meiotic crossover in plants and provide further support for the idea that Arabidopsis, like the budding yeast, possesses both interference-sensitive and insensitive pathways for crossover formation. [ABSTRACT FROM AUTHOR]

Although Kch of Escherichia coli is thought to be a K+ channel by sequence homology, there is little evidence that it actually conducts K+ ions in vitro or in vivo. We isolated gain-of-function (GOF) Kch mutations that render bacteria specifically sensitive to K+ ions. Millimolar added K+, but not Na+ or sorbitol, blocks the initiation or continuation of mutant growth in liquid media. The mutations are mapped at the RCK (or KTN) domain, which is considered to be the cytoplasmic sensor controlling the gate. Additional mutations directed to the K+-fitter sequence rescue the GOF mutant. The apparent K+-specific conduction through the 'loose-cannon' mutant channel suggests that the wild-type Kch channel also conducts, albeit in a regulated manner. Changing the internal ATG does not erase the GOF toxicity, but removes kch's short second product, suggesting that it is not required for channel function in vivo. The mutant phenotypes are better explained by a perturbation of membrane potential instead of internal K+ concentration. Possible implications on the normal function of Kch are discussed. [ABSTRACT FROM AUTHOR]

Bacterial outer membrane vesicles (OMVs) enriched with bioactive proteins, toxins, and virulence factors play a critical role in host-pathogen and microbial interactions. The two-component system PhoP-PhoQ (PhoPQ) of Salmonella enterica orchestrates the remodeling of outer membrane lipopolysaccharide (LPS) molecules and concomitantly upregulates OMV production. In this study, we document a novel use of nanoparticle tracking analysis to determine bacterial OMV size and number. Among the PhoPQ-activated genes tested, pagC expression had the most significant effect on the upregulation of OMV production. We provide the first evidence that PhoPQ-mediated upregulation of OMV production contributes to bacterial survival by interfering with complement activation. OMVs protected bacteria in a dose-dependent manner, and bacteria were highly susceptible to complement-mediated killing in their absence. OMVs from bacteria expressing PagC bound to complement component C3b in a dose-dependent manner and inactivated it by recruiting complement inhibitor Factor H. As we also found that Factor H binds to PagC, we propose that PagC interferes with complement-mediated killing of Salmonella in the following two steps: first by engaging Factor H, and second, through the production of PagC-enriched OMVs that divert and inactivate the complement away from the bacteria. Since PhoPQ activation occurs intracellularly, the resultant increase in PagC expression and OMV production is suggested to contribute to the local and systemic spread of Salmonella released from dying host cells that supports the infection of new cells. IMPORTANCE Bacterial outer membrane vesicles (OMVs) mediate critical bacterium-bacterium and host-microbial interactions that influence pathogenesis through multiple mechanisms, including the elicitation of inflammatory responses, delivery of virulence factors, and enhancement of biofilm formation. As such, there is a growing interest in understanding the underlying mechanisms of OMV production. Recent studies have revealed that OMV biogenesis is a finely tuned physiological process that requires structural organization and selective sorting of outer membrane components into the vesicles. In Salmonella, outer membrane remodeling and OMV production are tightly regulated by its PhoPQ system. In this study, we demonstrate that PhoPQ-regulated OMV production plays a significant role in defense against host innate immune attack. PhoPQ-activated PagC expression recruits the complement inhibitor Factor H and degrades the active C3 component of complement. Our results provide valuable insight into the combination of tools and environmental signals that Salmonella employs to evade complement-mediated lysis, thereby suggesting a strong evolutionary adaptation of this facultative intracellular pathogen to protect itself during its extracellular stage in the host.

Background: Infectious bursal disease (IBD) is an acute contagious immunosuppressive disease which lead to acute bursal injury and immune dysfunction in poultry. It has caused heavy economic losses in the commercial poultry industry for many years in worldwide. Attenuated live vaccine has widely used in poultry showing some promising signs against IBDV infection. But it has defects such as generating enhanced virulence and immunosuppression prohibits. Therefore, the development of mucosal vaccines using the food-grade lactic acid bacterium is necessary. Here, we construct a recombinant Lactococcus co-expressing the major IBDV antigens VP2 and RCK protein of Salmonella enterica to prevent IBD. Results: The recombinant fusion protein VP2-RCK was expressed in a soluble and stable form in the cytoplasm of the recombinant Lactococcus lactis. Animal experiments showed that: (1) the survival rates of the injected immunization inactivated recombinant LAB group and oral immunization live recombinant LAB group were 100% and 80%, respectively; (2) ELISA titers of all serum samples from all experimental groups were negative, but high amounts of specific neutralizing antibodies were detected (1:210 to 1:212); and (3) the bursas of the injected immunization inactivated recombinant LAB group did not suffer damage, as confirmed by clinical observation and bursal histopathological examination. Our results indicate that r-L. lactis-OptiVP2-RCK induces a specific neutralizing-antibody-mediated immune response that confers full protection against very-virulent IBDV (vvIBDV) challenge. Conclusion: Lactococcus lactis NZ3900 strain and its matching plasmid pNZ8149 could express the recombinant fusion protein VP2-RCK in a soluble form in the cytoplasm. The protective efficacy of r-L. lactis-OptiVP2-RCK (100%) was better than r-L. lactis-OptiVP2 (0%) which prove RCK protein played its unique role. The neutralizing antibodies titers against infectious bursal disease virus via one-time vaccination with inactivated r-L. lactis-OptiVP2-RCK could reach 1:210 to 1:212, but ELISA titers of all serum samples were negative. For this phenomenon, perhaps because of the change of delivery pathway or the spatial structure of fusion protein. We need further study to test these hypotheses. [ABSTRACT FROM AUTHOR]

Development of a cell-based functional assay for large-conductance calcium-activated potassium (BK(Ca)) channels is challenging because of the unique requirement of both voltage and high concentrations of Ca²⁺ for activation of these channels. Here, we describe a new cell-based assay system that utilizes a hyperactive mutant BK(Ca) channel. The hyperactive mutant was generated by introducing two point-mutations into the cytosolic flexible interface between the two RCK domains of the wild-type BK(Ca) channel. The mutant channel exhibited a large negative shift in its conductance-voltage relationship, which indicates activation by modest depolarization at resting concentrations of intracellular Ca²⁺. Unlike the wild-type BK(Ca) channel, the hyperactive mutant did not require a concomitant increase of intracellular Ca²⁺ for activation. Despite the observed shift in its voltage activation profile, activity of the mutant channel was further potentiated by a known BK(Ca) channel activator. When tested in a commercially available cell-based K⁺ channel assay, cell-lines stably expressing the hyperactive BK(Ca) channel generated a strong fluorescence signal under conditions that are typical for voltage-gated K⁺ channels. In summary, cell-lines expressing the hyperactive mutant BK(Ca) channel represent a new cell-based assay system for investigation of BK(Ca) channels that can be used to screen for novel modulators of these channels., (Copyright © 2013 Elsevier B.V. All rights reserved.)