PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.     

Small GTPase Rab4 regulates Ca2+-induced alpha-granule secretion in platelets

Upon activation, platelets release many active substances stored in alpha- and dense-core granules. However, the molecular mechanisms governing regulated exocytosis are not yet fully understood. Here, we have established an assay system using permeabilized platelets to analyze the Ca(2+)-induced exocytosis of both types of granules, focusing on RabGTPases. Incubation with Rab GDP dissociation inhibitor, an inhibitory regulator of RabGTPases, reduced membrane-bound RabGTPases extensively, and caused strong inhibition of the Ca(2+)-induced secretion of von Willebrand factor (vWF) stored in alpha-granules, but not that of [(3)H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 co-fractionated with vWF and P-selectin (an alpha-granule marker) upon separation of platelet organelles by density gradient centrifugation. Incubation of the permeabilized platelets with cell extracts expressing the dominant negative mutant of His-tagged Rab4S22N, but not with those of similar mutant His-Rab3BT36N, inhibited the vWF secretion, whereas neither of the cell extracts affected the [(3)H]5-HT secretion. Importantly, the inhibition of vWF secretion was rescued by depleting the cell extracts of the His-Rab4S22N with nickel beads. Thus, in platelets, the regulatory mechanisms governing alpha- and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca(2+)-induced exocytosis of alpha-granules.     

‘Only Fathers Smoking’ Contributes the Most to Socioeconomic Inequalities: Changes in Socioeconomic Inequalities in Infants’ Exposure to Second Hand Smoke over Time in Japan

BackgroundExposure to second hand smoke (SHS) is one of the major causes of premature death and disease among children. While socioeconomic inequalities exist for adult smoking, such evidence is limited for SHS exposure in children. Thus, this study examined changes over time in socioeconomic inequalities in infants’ SHS exposure in Japan.MethodsThis is a repeated cross-sectional study of 41,833 infants born in 2001 and 32,120 infants born in 2010 in Japan from nationally representative surveys using questionnaires. The prevalence of infants’ SHS exposure was determined and related to household income and parental education level. The magnitudes of income and educational inequalities in infants’ SHS exposure were estimated in 2001 and 2010 using both absolute and relative inequality indices.ResultsThe prevalence of SHS exposure in infants declined from 2001 to 2010. The relative index of inequality increased from 0.85 (95% confidence interval [CI], 0.80 to 0.89) to 1.47 (95% CI, 1.37 to 1.56) based on income and from 1.22 (95% CI, 1.17 to 1.26) to 2.09 (95% CI, 2.00 to 2.17) based on education. In contrast, the slope index of inequality decreased from 30.9 (95% CI, 29.3 to 32.6) to 20.1 (95% CI, 18.7 to 21.5) based on income and from 44.6 (95% CI, 43.1 to 46.2) to 28.7 (95% CI, 27.3 to 30.0) based on education. Having only a father who smoked indoors was a major contributor to absolute income inequality in infants’ SHS exposure in 2010, which increased in importance from 45.1% in 2001 to 67.0% in 2010.ConclusionsThe socioeconomic inequalities in infants’ second hand smoke exposure increased in relative terms but decreased in absolute terms from 2001 to 2010. Further efforts are needed to encourage parents to quit smoking and protect infants from second hand smoke exposure, especially in low socioeconomic households that include non-smoking mothers.     

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.     

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children Estimated by a Test-Negative Case-Control Design Study Based on Influenza Rapid Diagnostic Test Results

We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics with a fever of 38°C or over and had undergone an IRDT were enrolled in this study. Patients with positive IRDT results were recorded as cases, and patients with negative results were recorded as controls. Between November 2013 and March 2014, a total of 4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405 were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46% (95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95% CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better protection against influenza A infection than a single dose did. VE against hospitalization influenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.     

Extended High Circular Polarization in the Orion Massive Star Forming Region: Implications for the Origin of Homochirality in the Solar System

We present a wide-field (∼6′ × 6′) and deep near-infrared (K _s band: 2.14 μm) circular polarization image in the Orion nebula, where massive stars and many low-mass stars are forming. Our results reveal that a high circular polarization region is spatially extended (∼0.4 pc) around the massive star-forming region, the BN/KL nebula. However, other regions, including the linearly polarized Orion bar, show no significant circular polarization. Most of the low-mass young stars do not show detectable extended structure in either linear or circular polarization, in contrast to the BN/KL nebula. If our solar system formed in a massive star-forming region and was irradiated by net circularly polarized radiation, then enantiomeric excesses could have been induced, through asymmetric photochemistry, in the parent bodies of the meteorites and subsequently delivered to Earth. These could then have played a role in the development of biological homochirality on Earth.     

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.     

Locomotion ability variation among instars of the bean bug, Riptortus pedestris (Fabricius) (Heteroptera: Alydidae) nymphs

The nymphal locomotion ability (walking distance) of the stenophagous bean bug Riptortus pedestris (Fabricius) was studied in each instar. We measured the walking distance using two systems. The walking distance in photophase was measured for 6 h using a tracking system with a charge coupled device (CCD) camera and computer software. The daily activity of nymphs was measured by an actograph system counting the number of infrared beam intercepts. The actograph data were converted to distance using a linear regression against the data of the tracking system. The longevity of nymphs without food was also studied to estimate the potential walking distance. Using both the tracking and actograph systems, it was determined that first instars walked less than the other instars (only 10.7 m within 6 h). The second to fifth instars could move 20-25 m within 6 h, and this distance did not differ among instar. This indicates that first instars seldom move after hatching in the field. The walking distance for 24 h varied and was greatest for the third instars (80.8 m). The potential longevity of nymphs was found to increase with instar age. Potential locomotion ability (walking distance for 24 hxpotential longevity) was high in the third to fifth instars (approximately 340 m). The potential locomotion ability for the second instars was relatively low compared with the elder instars (approximately 180 m). From these results, nymphs of R. pedestris seem to adapt by identifying feeding site locations after hatching and elder instars may be able to find a novel feeding site after the degradation of previous habitat.