Interplay between calcium, diacylglycerol, and phosphorylation in the spatial and temporal regulation of PKCalpha-GFP

The function of protein kinase C (PKC) is closely regulated by its subcellular localization. We expressed PKCalpha fused to green fluorescent protein (PKCalpha-GFP) and examined its translocation in living and permeabilized cells of the human parotid cell line, HSY-EB. ATP induced an oscillatory translocation of PKCalpha-GFP to and from the plasma membrane that paralleled the appearance of repetitive Ca2+ spikes. Staurosporine attenuated the relocation of PKCalpha-GFP to the cytosol and caused a stepwise accumulation of PKCalpha-GFP at the plasma membrane during ATP stimulation. Diacylglycerol enhanced the amplitude and duration of the ATP-induced oscillatory translocation of PKCalpha-GFP. Ionomycin induced a transient translocation of PKCalpha-GFP to the plasma membrane despite the continuous elevation of cytosolic Ca2+. The ionomycin-induced transient translocation of PKCalpha-GFP was prolonged by staurosporine, diacylglycerol, and phorbol myristate acetate. Experiments using permeabilized cells showed that staurosporine or the elimination of ATP and Mg2+ decreases the rate of dissociation of PKCalpha-GFP from the membrane. Diacylglycerol slowed the dissociation of PKCalpha-GFP from the membrane regardless of the Ca2+ concentration. The effect of diacylglycerol was attenuated by ATP plus Mg2+ at low concentrations of Ca2+ (<500 nm) but not at high concentrations of Ca2+ (>1000 nm). These data suggest a complex interplay between Ca2+, diacylglycerol, and phosphorylation in the regulation of the membrane binding of PKCalpha.     

Adiponectin inhibits Toll-like receptor family-induced signaling

Recent studies have shown that adiponectin, an adipocyte-derived cytokine, acts as a potent inhibitor of inflammatory responses. It has been also demonstrated that bacterial and viral signalings in host cells are triggered via Toll-like receptor (TLR) molecules. Therefore, in the present study, we investigated whether globular adiponectin (gAd) would be able to inhibit TLR-mediated nuclear factor-kappaB (NF-kappaB) signaling in mouse macrophages (RAW264). gAd predominantly bound to the AdipoR1 receptor and suppressed TLR-mediated NF-kappaB signaling. gAd-mediated inhibition of TLR-mediated IkappaB phosphorylation and NF-kappaB activation was eliminated by the pretreatment of cycloheximide. Also their inhibitions of gAd were blocked by preincubation of the cells with an antibody against AdipoR1, but not with an antibody against AdipoR2. Taken together, these findings indicate that adiponectin negatively regulates macrophage-like cell response to TLR ligands via an unknown endogenous product(s).     

Effects of intracellular Mn on the radiation resistance of the halophilic archaeon Halobacterium salinarum

Ionizing radiation (IR) is of particular interest in biology because its exposure results in severe oxidative stress to the cell’s macromolecules. Our recent work with extremophiles supports the idea that IR resistance is most likely achieved by a metabolic route, effected by manganese (Mn) antioxidants. Biochemical analysis of “super-IR resistant” mutants of H. salinarum, evolved over multiple cycles of exposure to high doses of IR, confirmed the key role for Mn antioxidants in the IR resistance of this organism. Analysis of the proteome of H. salinarum “super-IR resistant” mutants revealed increased expression for proteins involved in energy metabolism, replenishing the cell with reducing equivalents depleted by the oxidative stress inflicted by IR. Maintenance of redox homeostasis was also activated by the over-expression of coenzyme biosynthesis pathways involved in redox reactions. We propose that in H. salinarum, increased tolerance to IR is a combination of metabolic regulatory adjustments and the accumulation of Mn-antioxidant complexes.     

Overexpression of BUNDLE SHEATH DEFECTIVE 2 improves the efficiency of photosynthesis and growth in Arabidopsis

Bundle Sheath Defective 2, BSD2, is a stroma‐targeted protein initially identified as a factor required for the biogenesis of ribulose 1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) in maize. Plants and algae universally have a homologous gene for BSD2 and its deficiency causes a RuBisCO‐less phenotype. As RuBisCO can be the rate‐limiting step in CO₂ assimilation, the overexpression of BSD2 might improve photosynthesis and productivity through the accumulation of RuBisCO. To examine this hypothesis, we produced BSD2 overexpression lines in Arabidopsis. Compared with wild type, the BSD2 overexpression lines BSD2ox‐2 and BSD2ox‐3 expressed 4.8‐fold and 8.8‐fold higher BSD2 mRNA, respectively, whereas the empty‐vector (EV) harbouring plants had a comparable expression level. The overexpression lines showed a significantly higher CO₂ assimilation rate per available CO₂ and productivity than EV plants. The maximum carboxylation rate per total catalytic site was accelerated in the overexpression lines, while the number of total catalytic sites and RuBisCO content were unaffected. We then isolated recombinant BSD2 (rBSD2) from E. coli and found that rBSD2 reduces disulfide bonds using reductants present in vivo, for example glutathione, and that rBSD2 has the ability to reactivate RuBisCO that has been inactivated by oxidants. Furthermore, 15% of RuBisCO freshly isolated from leaves of EV was oxidatively inactivated, as compared with 0% in BSD2‐overexpression lines, suggesting that the overexpression of BSD2 maintains RuBisCO to be in the reduced active form in vivo. Our results demonstrated that the overexpression of BSD2 improves photosynthetic efficiency in Arabidopsis and we conclude that it is involved in mediating RuBisCO activation.     

RICE SALT SENSITIVE3 binding to bHLH and JAZ factors mediates control of cell wall plasticity in the root apex

Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of plants, in accordance with their sessile lifestyle. This is linked to the balance between plasticity and rigidity of cells in the root apex, and thus is coordinated with the control of cell wall properties. However, mechanisms underlying such harmonization are not well understood, in particular under stressful conditions. We have recently demonstrated that RICE SALT SENSITIVE3 (RSS3), a nuclear factor that mediates restrictive expression of jasmonate-induced genes, plays an important role in root elongation under saline conditions. In this study, we report that loss-of-function of RSS3 results in changes in cell wall properties such as lignin deposition and sensitivity to a cellulose synthase inhibitor, concomitant with altered expression of genes involved in cell wall metabolism. Based on these and previous phenotypic observations of the rss3 mutant, we propose that RSS3 plays a role in the coordinated control of root elongation and cell wall plasticity in the root apex.     

Proteomic identification of differentially expressed genes during differentiation of cynomolgus monkey (Macaca fascicularis) embryonic stem cells to astrocyte progenitor cells in vitro

Understanding astrocytogenesis is valuable for the treatment of nervous system disorders, as astrocytes provide structural, metabolic and defense support to neurons, and regulate neurons actively. However, there is limited information about the molecular events associated with the differentiation from primate ES cells to astrocytes. We therefore investigated the differentially expressed proteins in early astrocytogenesis, from cynomolgus monkey ES cells (CMK6 cell line) into astrocyte progenitor (AstP) cells via the formation of primitive neural stem spheres (Day 4), mature neural stem spheres (NSS), and neural stem (NS) cells in vitro, using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). We identified 66 differentially expressed proteins involved in these five differentiation stages. Together with the results of Western blotting, RT-PCR, and a search of metabolic pathways related to the identified proteins, these results indicated that collapsin response mediator protein 2 (CRMP2), its phosphorylated forms, and cellular retinoic acid binding protein 1 (CRABP1) were upregulated from ES cells to Day 4 and NSS cells, to which differentiation stages apoptosis-associated proteins such as caspases were possibly related; Phosphorylated CRMP2s were further upregulated but CRABP1 was downregulated from NSS cells to NS cells, during which differentiation stage considerable axon guidance proteins for development of growth cones, axon attraction, and repulsion were possibly readied; Nonphosphorylated CRMP2 was downregulated but CRABP1 was re-upregulated from NS cells to AstP cells, in which differentiation stage reorganization of actin cytoskeleton linked to focal adhesion was possibly accompanied. These results provide insight into the molecular basis of early astrocytogenesis in monkey.     

Differences in the activity pattern of the wild boar Sus scrofa related to human disturbance

Over the last century, human activity has caused significant changes to the activity patterns of many wildlife species. The wild boar is one species known to change its activity pattern with the intensity of human disturbance. We conducted camera trap surveys in two study sites, Shingo and Himuro, in Tochigi, central Japan. We investigated effects of two types of human disturbance on the activity pattern of a wild boar population: ‘direct’ disturbance related to hunting activity and ‘indirect’ disturbance related to daily human activity. In the hunting season, relative abundance indices (RAI) of wild boars significantly decreased, and the proportion of activity at night increased compared with the nonhunting season. RAI of wild boars at night decreased with increasing distance from the settlement, while RAI of wild boars during the day did not. Relative proportion of activity at night was higher in cameras at 0–200 m from the settlements, while no significant pattern was found in cameras far from settlements. Both direct and indirect effects of human activity had a significant effect on the activity pattern of wild boars. A decrease in human activity may result in the rapid expansion of wild boar populations, and re-evaluation of the human factor is important for more intelligent management of wild boar populations and to solve the human–wildlife conflict.     

Disability Mediates the Impact of Common Conditions on Perceived Health

Background

We examined the extent to which disability mediates the observed associations of common mental and physical conditions with perceived health.

Methods and Findings

WHO World Mental Health (WMH) Surveys carried out in 22 countries worldwide (n = 51,344 respondents, 72.0% response rate). We assessed nine common mental conditions with the WHO Composite International Diagnostic Interview (CIDI), and ten chronic physical with a checklist. A visual analog scale (VAS) score (0, worst to 100, best) measured perceived health in the previous 30 days. Disability was assessed using a modified WHO Disability Assessment Schedule (WHODAS), including: cognition, mobility, self-care, getting along, role functioning (life activities), family burden, stigma, and discrimination. Path analysis was used to estimate total effects of conditions on perceived health VAS and their separate direct and indirect (through the WHODAS dimensions) effects.Twelve-month prevalence was 14.4% for any mental and 51.4% for any physical condition. 31.7% of respondents reported difficulties in role functioning, 11.4% in mobility, 8.3% in stigma, 8.1% in family burden and 6.9% in cognition. Other difficulties were much less common. Mean VAS score was 81.0 (SD = 0.1). Decrements in VAS scores were highest for neurological conditions (9.8), depression (8.2) and bipolar disorder (8.1). Across conditions, 36.8% (IQR: 31.2–51.5%) of the total decrement in perceived health associated with the condition were mediated by WHODAS disabilities (significant for 17 of 19 conditions). Role functioning was the dominant mediator for both mental and physical conditions. Stigma and family burden were also important mediators for mental conditions, and mobility for physical conditions.

Conclusions

More than a third of the decrement in perceived health associated with common conditions is mediated by disability. Although the decrement is similar for physical and mental conditions, the pattern of mediation is different. Research is needed on the benefits for perceived health of targeted interventions aimed at particular disability dimensions.     

PspA Family Distribution,Antimicrobial Resistance and Serotype of Streptococcus pneumoniae Isolated from Upper Respiratory Tract Infections in Japan

Background

The protection against pneumococcal infections provided by currently available pneumococcal polysaccharide conjugate vaccines are restricted to the limited number of the serotypes included in the vaccine. In the present study, we evaluated the distribution of the pneumococcal capsular type and surface protein A (PspA) family of pneumococcal isolates from upper respiratory tract infections in Japan.

Methods

A total of 251 S. pneumoniae isolates from patients seeking treatment for upper respiratory tract infections were characterized for PspA family, antibiotic resistance and capsular type.

Results

Among the 251 pneumococci studied, the majority (49.4%) was identified as belonging to PspA family 2, while most of the remaining isolates (44.6%) belonged to family 1. There were no significant differences between the distributions of PspA1 versus PspA2 isolates based on the age or gender of the patient, source of the isolates or the isolates’ susceptibilities to penicillin G. In contrast, the frequency of the mefA gene presence and of serotypes 15B and 19F were statistically more common among PspA2 strains.

Conclusion

The vast majority of pneumococci isolated from the middle ear fluids, nasal discharges/sinus aspirates or pharyngeal secretions represented PspA families 1 and 2. Capsular serotypes were generally not exclusively associated with certain PspA families, although some capsular types showed a much higher proportion of either PspA1 or PspA2. A PspA-containing vaccine would potentially provide high coverage against pneumococcal infectious diseases because it would be cross-protective versus invasive disease with the majority of pneumococci infecting children and adults.