Synaptobrevin cleavage by the tetanus toxin light chain is linked to the inhibition of exocytosis in chromaffin cells

Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells

Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilized adrenal chromaffin cells

The molecular events underlying the inhibition of exocytosis by tetanus toxin were investigated in permeabilized adrenal chromaffin cells. We found that replacement of amino acid residues within the putative zinc binding domain of the tetanus toxin light chain such as of histidine (position 233) by cysteine or valine, or of glutamate (position 234) by glutamine completely abolished the effect of the light chains on Ca2+ induced catecholamine release. Dipicolinic acid, a strong chelating agent for zinc, also prevented the effect of the tetanus toxin light chain. Zn2+ and, less potently Cu2+ and Ni2+, but not Cd2+ and Co2+, restored the activity of the neurotoxin. These data show that zinc and the putative zinc binding domain constitute the active site of the tetanus toxin light chain. Neither captopril, an inhibitor of synaptobrevin cleavage nor peptides spanning the site of synaptobrevins cleaved by the tetanus toxin in neurons, prevented the inhibition of Ca2+ induced catecholamine release by the tetanus toxin light chain. This suggests that synaptobrevins are not a major target of tetanus toxin in adrenal chromaffin cells

Diurnal variation and size dependence of the hygroscopicity of organic aerosol at a forest site in Wakayama, Japan: their relationship to CCN concentrations

Formation of biogenic secondary organic aerosol (BSOA) and its subsequent evolution can modify the hygroscopicity of the organic aerosol component (OA) in the forest atmosphere, and affect the concentrations of cloud condensation nuclei (CCN) there. In this study, size-resolved aerosol hygroscopic growth at 85&thinsp;% relative humidity and size-resolved aerosol composition were measured using a hygroscopic tandem differential mobility analyzer and an aerosol mass spectrometer, respectively, at a forest site in Wakayama, Japan, in August and September 2015. The hygroscopicity parameter of OA (κorg) presented daily minima in the afternoon hours, and it also showed an increase with the increase in particle dry diameter. The magnitudes of the diurnal variations in κorg for particles with dry diameters of 100 and 300&thinsp;nm were on average 0.091 and 0.096, respectively, and the difference in κorg between particles with dry diameters of 100 and 300&thinsp;nm was on average 0.056. The relative contributions of the estimated fresh BSOA and regional OA to total OA could explain 40&thinsp;% of the observed diurnal variations and size dependence of κorg. The hygroscopicity parameter of fresh BSOA was estimated to range from 0.089 to 0.12 for particles with dry diameters from 100 to 300&thinsp;nm. Compared with the use of time- and size-resolved κorg, the use of time- and size-averaged κorg leads to under- and over-estimation of the fractional contribution of OA to CCN number concentrations in the range from −5.0&thinsp;% to 26&thinsp;%. This indicates that the diurnal variations and size dependence of κorg strongly affect the overall contribution of OA to CCN concentrations. The fractional contribution of fresh BSOA to CCN number concentrations could reach 0.28 during the period of intensive BSOA formation. The aging of the fresh BSOA, if it occurs, increases the estimated contribution of BSOA to CCN number concentrations by 52&thinsp;%–84&thinsp;%.</p

Role of glycosaminoglycans of biglycan in BMP-2 signaling

Recently we have reported that biglycan (BGN) promotes osteoblast differentiation and that this function is due in part to its ability to positively modulate bone morphogenetic protein (BMP) functions. In this study we investigated the role of glycosaminoglycans (GAGs) of BGN in this function using in vitro and in vivo models. C2C12 myogenic cells were treated or untreated with BMP-2 alone or in combination with glycanated, partially glycanated or de-glycanated BGN, and the effects on BMP signaling and function were assessed by Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the effect of de-glycanation of BGN on BMP-2 induced osteogenesis was investigated employing a rat mandible defect model. The defects were filled with collagen scaffolds loaded with glycanated, partially glycanated or de-glycanated BGN alone or in combination with a sub-optimal dose of BMP-2 (subBMP). In in vitro experiments, BMP signaling and function were the greatest when BMP-2 was combined with de-glycanated BGN among the groups tested. In the rat mandible experiments, μCT analyses revealed that the newly formed bone was significantly increased only when subBMP was combined with de-glycanated BGN. The data indicate that the GAG component of BGN functions as a suppressor for the BGN-assisted BMP function

Experimental characterization of the fracture properties of pultruded GFRP structural elements

Pultruded Fiber Reinforced Polymers (FRPs) are a class of novel composite materials with remarkable strength (comparable or even greater to that of steel) and resistance to environmental effects. However, the strongly orthotropic behavior of these materials and the relatively high deformability and spatial variability in mechanical properties bring challenges to the widespread adoption of these elements in structural applications. To this end, the orientation and distribution of the fibers are the most influential parameters that affect both the ultimate strength and stiffness of the specimens. This work presents an experimental campaign conducted on GFRP specimens in uniaxial tension and 3-point bending; coupon specimens with three different fibers orientations (namely 0, 45, and 90 degrees) were tested to characterize the ultimate strength and failure modes Results of such experimental campaign are first presented, and detailed statistical measures of the so-obtained strength values are presented with the ultimate goal of characterizing the variability in mechanical properties in commercially available profiles

Identification of effector candidate genes of Rhizoctonia solani AG-1 IA expressed during infection in Brachypodium distachyon

Rhizoctonia solani is a necrotrophic phytopathogen belonging to basidiomycetes. It causes rice sheath blight which inflicts serious damage in rice production. The infection strategy of this pathogen remains unclear. We previously demonstrated that salicylic acid-induced immunity could block R. solani AG-1 IA infection in both rice and Brachypodium distachyon. R. solani may undergo biotrophic process using effector proteins to suppress host immunity before necrotrophic stage. To identify pathogen genes expressed at the early infection process, here we developed an inoculation method using B. distachyon which enables to sample an increased amount of semi-synchronous infection hyphae. Sixty-one R. solani secretory effector-like protein genes (RsSEPGs) were identified using in silico approach with the publicly available gene annotation of R. solani AG-1 IA genome and our RNA-sequencing results obtained from hyphae grown on agar medium. Expression of RsSEPGs was analyzed at 6, 10, 16, 24, and 32 h after inoculation by a quantitative reverse transcription-polymerase chain reaction and 52 genes could be detected at least on a single time point tested. Their expressions showed phase-specific patterns which were classified into 6 clusters. The 23 RsSEPGs in the cluster 1-3 and 29 RsSEPGs in the cluster 4-6 are expected to be involved in biotrophic and necrotrophic interactions, respectively

The Role of Ellis‐Van Creveld 2(EVC2) in Mice During Cranial Bone Development

EvC syndrome is a type of autosomal‐recessive chondrodysplasia. Previous case studies in patients suggest abnormal craniofacial development, in addition to dwarfism and tooth abnormalities. To investigate how craniofacial development is affected in EvC patients, surface models were generated from micro‐CT scans of control mice, Evc2 global mutant mice and Evc2 neural crest‐specific mutant mice. The anatomic landmarks were placed on the surface model to assess the morphological abnormalities in the Evc2 mutants. Through analyzing the linear and angular measurements between landmarks, we identified a smaller overall skull, shorter nasal bone, shorter frontal bone, and shorter cranial base in the Evc2 global mutants. By comparing neural crest‐specific Evc2 mutants with control mice, we demonstrated that the abnormalities within the mid‐facial regions are not accounted for by the Evc2 mutation within these regions. Additionally, we also identified disproportionate length to width ratios in the Evc2 mutants at all levels from anterior to posterior of the skull. Overall, this study demonstrates a more comprehensive analysis on the craniofacial morphological abnormalities in EvC syndrome and provides the developmental insight to appreciate the impact of Evc2 mutation within the neural crest cells on multiple aspects of skull deformities. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:46–55, 2018. © 2017 Wiley Periodicals, Inc.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141635/1/ar23692_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141635/2/ar23692.pd