Three lysine residues in the common β chain of the interleukin-5 receptor are required for Janus kinase (JAK)-dependent receptor ubiquitination, endocytosis, and signaling

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory diseases including allergic asthma and hypereosinophilic syndrome. Eosinophil physiology is critically dependent on IL-5 and the IL-5 receptor (IL-5R), composed of a ligand binding α chain (IL-5Rα), and a common β chain, βc. Previously, we demonstrated that the βc cytoplasmic tail is ubiquitinated and degraded by proteasomes following IL-5 stimulation. However, a complete understanding of the role of βc ubiquitination in IL-5R biology is currently lacking. By using a well established, stably transduced HEK293 cell model system, we show here that in the absence of ubiquitination, βc subcellular localization, IL-5-induced endocytosis, turnover, and IL-5R signaling were significantly impaired. Whereas ubiquitinated IL-5Rs internalized into trafficking endosomes for their degradation, ubiquitination-deficient IL-5Rs accumulated on the cell surface and displayed blunted signaling even after IL-5 stimulation. Importantly, we identified a cluster of three membrane-proximal βc lysine residues (Lys(457), Lys(461), and Lys(467)) whose presence was required for both JAK1/2 binding to βc and receptor ubiquitination. These findings establish that JAK kinase binding to βc requires the presence of three critical βc lysine residues, and this binding event is essential for receptor ubiquitination, endocytosis, and signaling.     

Endoplasmic reticulum stress induces inverse regulations of major functions in portal myofibroblasts during liver fibrosis progression

Portal myofibroblasts (PMF) form a sub-population of highly proliferative and proangiogenic liver myofibroblasts that derive from portal mesenchymal progenitors. Endoplasmic reticulum (ER) stress was previously shown to modulate fibrogenesis, notably in the liver. Our aim was to determine if ER stress occurred in PMF and affected their functions. PMF were obtained after their expansion in vivo from bile duct-ligated (BDL) rats and referred to as BDL PMF. Compared to standard PMF obtained from normal rats, BDL PMF were more myofibroblastic, as assessed by higher alpha-smooth muscle actin expression and collagen 1 production. Their proangiogenic properties were also higher, whereas their proliferative and migratory capacities were lower. CHOP expression was detected in the liver of BDL rats, at the leading edge of portal fibrosis where PMF accumulate. BDL PMF displayed ER dilatation and an overexpression of the PERK pathway downstream targets, Chop, Gadd34 and Trb3, in comparison with standard PMF. In vitro, the induction of ER stress by tunicamycin in standard PMF, caused a decrease in their proliferative and migratory activity, and an increase in their proangiogenic activity, without affecting their myofibroblastic differentiation. Conversely, the treatment of BDL PMF with the PERK inhibitor GSK2656157 reduced ER stress, which caused a decrease in their angiogenic properties, and restored their proliferative and migratory capacity. In conclusion, PMF develop ER stress as they expand with the progression of fibrosis, which further increases their proangiogenic activity, but also inhibits their proliferation and migration. This phenotypic switch may restrict PMF expansion while they support angiogenesis.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

秸秆带状覆盖对半干旱雨养区冬小麦田地温和产量的影响

为了明确秸秆带状覆盖对西北半干旱雨养区冬小麦田地温和产量形成的影响,于2013-2015年连续进行2年定位试验,设不覆盖露地(CK)、全膜覆土穴播(PM)、秸秆带状覆盖(覆盖带和种植带各30 cm,播种3行,SM₁)、(覆盖带和种植带各40 cm,播种4行,SM₂)、(覆盖带和种植带各50 cm,播种5行,SM₃)5个处理.结果表明: 各处理在不同生育时期、不同土层土壤温度存在显著差异.与CK相比,SM₁、SM₂和SM₃处理全生育期0～25 cm土层土壤温度分别比CK显著降低1.0～1.3 ℃、0.7～0.9 ℃和0.7～1.1 ℃.不同时期比较,覆盖处理存在增温和降温的双重效应,秸秆覆盖在苗期-越冬期具有提高地温的作用,返青期-成熟期具有降温效应,增温效应覆膜>秸秆覆盖,而降温效应秸秆覆盖>覆膜.同时,秸秆覆盖降低了全生育期土壤有效积温和日变化幅度,全生育期有效积温较CK降低3.4～33.5 ℃·d,土壤温差较CK降低0.6～2.0 ℃;秸秆覆盖在越冬期平均温度比CK高0.2～0.3 ℃、负积温比CK高0.4～17.0 ℃·d.秸秆覆盖较CK增产11.9％～19.5％,处理间单位面积穗数的差异是引起产量差异的主要结构因素.因此,秸秆带状覆盖适宜在西北雨养区旱地冬小麦产区推广应用.     

Discovery and SAR analysis of phenylbenzo[d][1,3]dioxole-based proprotein convertase subtilisin/kexin type 9 inhibitors

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapeutic target for the development of cholesterol-lowering drugs. In the discovery of PCSK9/LDLR (low-density lipoprotein receptor) protein-protein interaction (PPI) impairing small molecules, a total of 47 phenylbenzo[d][1,3] dioxole-based compounds were designed and synthesised. The result revealed that the 4-chlorobenzyl substitution in the amino group is important for the PPI disrupting activity. In the hepatocyte-based functional tests, active compounds such as A12, B1, B3, B4 and B14, restored the LDLR levels on the surface of hepatic HepG2 cells and increased extracellular LDL uptake in the presence of PCSK9. It is notable that molecule B14 exhibited good performance in all the evaluations. Collectively, novel structures targeting PCSK9/LDLR PPI have been developed with hypolipidemic potential. Further structural modification of derived active compounds is promising in the discovery of lead compounds with improved activity for the treatment of hyperlipidaemia-related disorders.     

Interleukin-22 Mediates Early Host Defense against Rhizomucor pusilluscan Pathogens

Background

In recent years, the fungal infectious disease zygomycosis has increased in incidence worldwide, especially among the immunodeficient population. Despite the rates of zygomycosis-related death and deformation being very high, the mechanism(s) by which the fungal pathogens cause these severe manifestations remain unknown.

Methods

Using the associated Rhizomucor variabilis species, which can selectively induce cutaneous zygomycosis in otherwise healthy individuals, we investigated the host mechanisms of infection-related responses, including cytokine and chemokine expression as well as contributions of particular T cell subsets. siRNA specifically targeting IL-22,IL-17 and IFN-γ were used to down-regulate expression of those molecules.

Results

In mouse models of infection, IL-22 was implicated in development of Rhizomucor spp.-induced skin lesions. In cultured human peripheral blood monocytes, R. pusilluscan, which is often found in immunodeficient patients, induced the production of IL-22, while R. variabilis did not. Moreover, Rhizomucor spp.-induced secretion of Il-22 from CCR6⁺CCR4⁺CCR10⁺ cells was down-regulated by knockdown of IL-22 related signaling receptors, RORC and ARH.

Conclusion

Our data strongly suggest that avoidance of IL-22 may be one mechanism by which mucor species produce morbidity and mortality in infected individuals.     

Protein Kinase LTRPK1 Influences Cold Adaptation and Microtubule Stability in Rice

Rice LTRPK1, which encodes a member of the casein kinase I family, has been reported to be involved in root development, hormone response, and metabolic processes. Here we further show that LTRPK1 participates in stress resistance by regulating cytoskeleton rearrangement and formation of cold tolerance and adaptation. Semiquantitative RT-PCR analysis revealed enhanced expression of LTRPK1 in plants subject to low-temperature stress at 4 °C, suggesting a role in low-temperature-related cell responses and signal transduction pathways. Further analysis of LTRPK1-deficient transgenic plants showed that under low-temperature treatment, the growth rate of transgenic plant primary roots, which is commonly used as an indicator for cold stress response abilities, was less inhibited than that of control plants. Moreover, damage to the plasma membrane of root cells in LTRPK1-deficient plants was greater than that of controls as measured by relative electrical conductivity (REC). The malondialdehyde (MDA) content of LTRPK1-deficient plants also increased over that of the control, indicating increased plasma membrane permeability. Further immunofluorescence localization observations indicated that microtubules of transgenic plants subject to low temperature disassembled more rapidly, whereas the control plant microtubules in most cells of the root elongation zone kept their normal habitus, which suggested that LTRPK1-deficient plants had reduced capacity to resist low-temperature stress through regulation of microtubule assembly. These results demonstrate involvement of LTRPK1 in low-temperature stress and provide new insight for rice breeding and germplasm innovation to improve crop cold tolerance.     

A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress

RING-finger proteins (RFP) function as ubiquitin ligases and play key roles in plant responses to biotic and abiotic stresses. However, little information is available on the regulation of RFP expression. Here, we isolate and characterize the RFP promoter sequence from the disease-resistant Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1. Promoter-GUS fusion assays revealed that defense signaling molecules, powdery mildew infection, and heat stress induce VpRFP1 promoter activity. By contrast, the RFP1 promoter isolated from Vitis vinifera was only slightly induced by pathogen infection and heat treatment. By promoter deletion analysis, we found that the ?148 bp region of the VpRFP1 promoter was the core functional promoter region. We also found that, in Arabidopsis, VpRFP1 expressed under its own promoter activated defense-related gene expression and improved disease resistance, but the same construct using the VvRFP1 promoter slightly improve disease resistance. Our results demonstrated that the ?148 bp region of the VpRFP1 promoter plays a key role in response to pathogen and heat stress, and suggested that expression differences between VpRFP1 and VvRFP1 may be key for the differing disease resistance phenotypes of the two Vitis genotypes.