YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA

sigmaE is an alternative sigma factor involved in a pathway of extracytoplasmic stress responses in Escherichia coli. Under normal growth conditions, sigmaE activity is down-regulated by the membrane-bound anti-sigmaE protein, RseA. Extracytoplasmic stress signals induce degradation of RseA by two successive proteolytic events: DegS-catalyzed first cleavage at a periplasmic site followed by YaeL-mediated second proteolysis at an intramembrane region. Normally, the second reaction (site-2 proteolysis) only occurs after the first cleavage (site-1 cleavage). Here, we show that YaeL variants with the periplasmic PDZ domain deleted or mutated allows unregulated cleavage of RseA and consequent sigmaE activation. It was also found that a glutamine-rich region in the periplasmic domain of RseA was required for the avoidance of the YaeL-mediated proteolysis in the absence of site-1 cleavage. These results indicate that multiple negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine-rich region) determine the strict dependence of the site-2 proteolysis on the site-1 cleavage.     

A novel G protein alpha subunit in embryo of the ascidian,Halocynthia roretzi

A cDNA clone encoding a novel G protein alpha subunit, HrGalpha(n) was isolated from the larvae of ascidian, Halocynthia roretzi. In contrast with overall amino acid identity (63%) with G protein alpha subunit of G(i) or G(o) subclass, HrGalpha(n) has a unique amino acid sequence, which lacks a residue for pertussis toxin substrate, but retains for cholera toxin substrate for ADP-ribosylation. The sequence characteristics and molecular phylogenetic analysis suggest that HrGalpha(n) defines a novel subclass within G(i) class of G protein alpha subunits. The zygotic expression of HrGalpha(n) was first detected at the 64-cell stage and observed in all blastomeres except for B7.4, B7.5 and B7.6 cells till the 110-cell stage. As progress of the developmental stages, the expression of HrGalpha(n) became restricted and was observed in the muscle, mesenchyme and a part of trunk lateral cells in tailbud embryos. With HrGalpha(n)-GFP fusion-gene construct it was showed that the genomic fragment containing 2674 bp upstream of the putative translation start site of HrGalpha(n) contained the regulatory sequence responsible for the expression in the muscle and mesenchyme cells, and that the regulatory sequence functioned also in Ciona intestinalis. Our results suggest a possible involvement of HrGalpha(n) in the signaling system regulates the cell fate during the embryogenesis of the ascidian.     

Pathogenic complement activation in collagen antibody-induced arthritis in mice requires amplification by the alternative pathway

Immune complex-induced inflammation can be mediated by the classical pathway of complement. However, using mice genetically deficient in factor B or C4, we have shown that the collagen Ab-induced model of arthritis requires the alternative pathway of complement and is not dependent on the classical pathway. We now demonstrate that collagen Ab-induced arthritis is not altered in mice genetically deficient in either C1q or mannose-binding lectins A and C, or in both C1q and mannose-binding lectins. These in vivo results prove the ability of the alternative pathway to carry out pathologic complement activation in the combined absence of intact classical and lectin pathways. C3 activation was also examined in vitro by adherent collagen-anti-collagen immune complexes using sera from normal or complement-deficient mice. These results confirm the ability of the alternative pathway to mediate immune complex-induced C3 activation when C4 or C1q, or both C1q and mannose-binding lectins, are absent. However, when all three activation pathways of complement are intact, initiation by immune complexes occurs primarily by the classical pathway. These results indicate that the alternative pathway amplification loop, with its ability to greatly enhance C3 activation, is necessary to mediate inflammatory arthritis induced by adherent immune complexes.     

Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis

During apoptotic execution, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert highly condensed form that is fragmented and packaged into apoptotic bodies. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cell-free system and time-lapse imaging. We demonstrated that the population of nuclei undergoing apoptosis in vitro appears to follow a reproducible program of nuclear condensation, suggesting the existence of an ordered biochemical pathway. This enabled us to define three stages of apoptotic chromatin condensation: stage 1 ring condensation; stage 2 necklace condensation; and stage 3 nuclear collapse/disassembly. Electron microscopy revealed that neither chromatin nor detectable subnuclear structures were present inside the stage 1 ring-condensed structures. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectable DNase activity. However, DNase(s) were required for stage 2 necklace condensation. Finally, we demonstrated that hydrolyzable ATP is required for stage 3 nuclear collapse/disassembly. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis.     

Functional diversification of kir7.1 in cichlids accelerated by gene duplication

Mutation in the inward rectifier potassium channel gene, kir7.1, was previously identified as being responsible for the broader stripe zebrafish skin pattern mutant, jaguar/obelix. An amino acid substitution in this channel causes a broader stripe pattern than that of wild type zebrafish. In this study we analyzed cichlid homologs of the zebrafish kir7.1 gene. We identified two kinds of homologous genes in cichlids and named them cikir7.1 and cikir7.2. Southern hybridization using cichlid genome revealed that cichlids from the African Great Lakes, South America and Madagascar have two copies of the gene. Cichlids from Sri Lanka, however, showed only one band in this experiment. Database analysis revealed that only one copy of the kir7.1 gene exists in the genomes of the teleosts zebrafish, tetraodon, takifugu, medaka and stickleback. The deduced amino acid sequence of cikir7.1 is highly conserved among African cichlids, whereas that of cikir7.2 has several amino acid substitutions even in conserved transmembrane domains. Gene expression analysis revealed that cikir7.1 is expressed specifically in brain and eye, and cikir7.2 in testis and ovary; zebrafish kir7.1, however, is expressed in brain, eye, skin, caudal fin, testis and ovary. These results suggest that gene duplication of the cichlid kir7.1 occurred in a common ancestor of the family Cichlidae, that the function of parental kir7.1 was then divided into two genes, cikir7.1 and cikir7.2, and that the evolutionary rate of cikir7.2 might have been accelerated, thereby effecting functional diversification in the cichlid lineage. Thus, the evolution of kir7.1 genes in cichlids provides a typical example of gene duplication--one gene is conserved while the other becomes specialized for a novel function.     

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.     

Altered KLOTHO and NF-κB-TNF-α Signaling Are Correlated with Nephrectomy-Induced Cognitive Impairment in Rats

Renal insufficiency can have a negative impact on cognitive function. Neuroinflammation and changes in klotho levels associate with chronic kidney disease (CKD) and may play a role in the development of cognitive impairment (CI). The present study evaluates the correlation of cognitive deficits with neuroinflammation and soluble KLOTHO in the cerebral spinal fluid (CSF) and brain tissue of nephrectomized rats (Nx), with 5/6 renal mass ablation. Nx and sham Munich Wistar rats were tested over 4 months for locomotor activity, as well as inhibitory avoidance or novel object recognition, which started 30 days after the surgery. EMSA for Nuclear factor-κB and MILLIPLEXMAP or ELISA kit were used to evaluate cytokines, glucocorticoid and KLOTHO levels. Nx animals that showed a loss in aversive-related memory and attention were included in the CI group (Nx-CI) (n=14) and compared to animals with intact learning (Nx-M n=12 and Sham n=20 groups). CSF and tissue samples were collected 24 hours after the last behavioral test. The results show that the Nx-groups have increased NF-κB binding activity and tumor necrosis factor-alpha (TNF-α) levels in the hippocampus and frontal cortex, with these changes more pronounced in the Nx-CI group frontal cortex. In addition, the Nx-CI group showed significantly increased CSF glucocorticoid levels and TNF-α /IL-10 ratio compared to the Sham group. Klotho levels were decreased in Nx-CI frontal cortex but not in hippocampus, when compared to Nx-M and Sham groups. Overall, these results suggest that neuroinflammation mediated by frontal cortex NF-κB, TNF-α and KLOTHO signaling may contribute to Nx-induced CI in rats.     

Implications for Social Support on Prolonged Sleep Difficulties among a Disaster-Affected Population: Second Report from a Cross-Sectional Survey in Ishinomaki,Japan

Study Objectives

This study aimed to investigate the role of social factors, especially social support for sleep, among victims living at home around 1–2 years after the Great East Japan Earthquake and tsunami.

Design

A cross-sectional household survey was conducted between May and December 2012 (14–21 months after the disaster) in the Ishinomaki area, Japan. Univariate and multivariate logistic regression models were used to examine the association between social factors, including social support, and prolonged sleep difficulties (persisting over 1 month). Social support was divided into three functions: emotional, informational, and instrumental support.

Participants

Data were obtained on 2,593 individuals who were living at home after the disaster.

Results

The prevalence of prolonged sleep difficulties was 6.9% (5.8% male, 7.7% female). This study showed that lack of social support has a stronger association with prolonged sleep difficulties than non-modifiable or hardly modifiable consequences caused directly by the disaster, i.e., severity of home damage, change in family structure and income. Among the three dimensions of social support, lack of emotional support showed the strongest association with prolonged sleep difficulties.

Conclusions

Social support, especially emotional support, may positively affect sleep among victims living at home around 1–2 years after a disaster.     

Activity of bacteria in water of hot springs from Southern and Central Kamchatskaya geothermal provinces, Kamchatka Peninsula, Russia

The hot-spring waters of numerous hot springs at the Kamchatka Peninsula, Russia differ in their chemical characteristics and elemental composition. Total bacterial abundance (TBA) as well as enzymatically active and respiring bacteria was enumerated in water samples collected from the Nalychevskie, Oksinskie, Apapelskie, and Dachnye hot springs. 5-Carboxyfluorescein diacetate activity was detected in all water samples and comprised 29-65% of the TBA as determined by 4',6-diamidino-2-phenylindol staining. The respiratory activity of iron-oxidizing bacteria was assayed by 5-cyano-2,3-ditolyltetrazolium chloride reduction. Respiring cells accounted for 9-14% of the TBA, indicating a positive correlation with the number of iron-oxidizing bacteria from the hot-spring biomats. Enumeration of heterotrophic bacteria revealed a high-density bacterial population only in the water of the Apapelskie hot spring, which has a temperature of 36 degrees C. Therefore, it appears that heterotrophic and iron-oxidizing bacteria inhabiting the hot-spring waters are essential for the geochemical processes occurring in hydrothermal systems.