A remote substrate docking mechanism for the tec family tyrosine kinases

During T cell signaling, Itk selectively phosphorylates a tyrosine within its own SH3 domain and a tyrosine within PLCgamma1. We find that the remote SH2 domain in each of these substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. Additionally, we detect a stable interaction between the substrate SH2 domains and the kinase domain of Itk and find that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation. On the basis of these results, we show that the kinetic parameters of a generic peptide substrate of Itk are significantly improved via fusion of the peptide substrate to the SH2 domain of PLCgamma1. This work is the first characterization of a substrate docking mechanism for the Tec kinases and provides evidence of a novel, phosphotyrosine-independent regulatory role for the ubiquitous SH2 domain.     

Canonical heterotrimeric G proteins regulating mating and virulence of Cryptococcus neoformans

Perturbation of pheromone signaling modulates not only mating but also virulence in Cryptococcus neoformans, an opportunistic human pathogen known to encode three Galpha, one Gbeta, and two Ggamma subunit proteins. We have found that Galphas Gpa2 and Gpa3 exhibit shared and distinct roles in regulating pheromone responses and mating. Gpa2 interacted with the pheromone receptor homolog Ste3alpha, Gbeta subunit Gpb1, and RGS protein Crg1. Crg1 also exhibited in vitro GAP activity toward Gpa2. These findings suggest that Gpa2 regulates mating through a conserved signaling mechanism. Moreover, we found that Ggammas Gpg1 and Gpg2 both regulate pheromone responses and mating. gpg1 mutants were attenuated in mating, and gpg2 mutants were sterile. Finally, although gpa2, gpa3, gpg1, gpg2, and gpg1 gpg2 mutants were fully virulent, gpa2 gpa3 mutants were attenuated for virulence in a murine model. Our study reveals a conserved but distinct signaling mechanism by two Galpha, one Gbeta, and two Ggamma proteins for pheromone responses, mating, and virulence in Cryptococcus neoformans, and it also reiterates that the link between mating and virulence is not due to mating per se but rather to certain mating-pathway components that encode additional functions promoting virulence.     

Inflammation-dependent downregulation of miR-194-5p contributes to human intervertebral disc degeneration by targeting CUL4A and CUL4B

Inflammation is one of the major causes of intervertebral disc degeneration (IDD). Emerging evidence has revealed that increase in the levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), can activate a variety of signaling pathways, eventually resulting in IDD. Here, we show that the two cullin family genes, CUL4A and CUL4B, but not other cullins, are specifically overexpressed in IDD samples compared with healthy controls, and the CUL4A and CUL4B levels are positively correlated with the severity of IDD. In vitro analyses in human osteoblast cells (hFOB1.19), nucleus pulposus cells (hNPCs), and annulus fibrosus cells (hAFCs) indicated that treatment with IL-6 and TNF-α can increase CUL4A and CUL4B levels. By performing a microRNA-based microarray analysis, we found a set of microRNAs (miRNAs) that were differentially expressed in IDD samples compared with samples from healthy controls. Of these miRNAs, miR-194-5p, was significantly downregulated in IDD samples and could bind to the three prime untranslated regions (3′-UTRs) of both CUL4A and CUL4B, thereby downregulating their expression. The in vitro overexpression or downregulation of miR-194-5p, with a miR-194-5p-mimic or with anti-miR-194-5p, can cause the repression or induction of both CUL4A and CUL4B, respectively. Interestingly, treatment with IL-6 and TNF-α inhibitors in primary hNPCs and hAFCs that were isolated from patients with IDD led to the downregulation of CUL4A and CUL4B. Together, these findings provide insight into how the inflammation-dependent downregulation of miR-194-5p contributes to the pathogenesis of IDD, which may aid in the development of new therapeutic approaches for IDD by directly targeting miR-194-5p or CUL4A and CUL4B.     

Unique Protein Profiles of Extracellular Vesicles as Diagnostic Biomarkers for Early and Advanced Non‐Small Cell Lung Cancer

Extracellular vesicles (EVs) mediate intercellular communication via transferring proteins and other biological molecules and have been recently investigated as biomarkers of disease. Sensitive and specific biomarkers are required for lung cancer diagnosis and prognosis. The present study screens for abnormal EV proteins in non‐small cell lung cancer (NSCLC) using a quantitative proteomics strategy involving LC‐MS/MS to identify ideal biomarkers for NSCLC diagnosis. EVs are enriched from the sera of early and advanced NSCLC patients and healthy controls and from cell culture supernatants of lung adenocarcinoma and bronchial epithelial cell lines. In the sera and supernatants, 279 and 632 differentially expressed proteins, respectively, are associated with signaling pathways including extracellular membrane–receptor interaction, focal adhesion, and regulation of the actin cytoskeleton. Thirty‐two EV proteins are identified at the intersection of differentially expressed proteins between the NSCLC groups and cell lines. Based on bioinformatics analysis, in silico immunohistochemical, and PRM verification, fibronectin is selected for following in vitro studies and validation with an independent cohort. Fibronectin on EVs is estimated to perform well in the diagnosis of NSCLC patients based on AUC, showing great potential for clinical use and demonstrating the efficacy of this method for EV‐associated biomarker screening.     

香栓菌的平喘及抗氧化活性研究

本文对香栓菌子实体提取物的平喘作用和抗氧化活性进行研究。平喘药理实验采用鸡卵清蛋白（OVA）致敏法建立小鼠过敏性哮喘模型,灌胃给药香栓菌水提物（高240mg/kg、中120mg/kg、低60mg/kg剂量）和石油醚提取物（高24mg/kg、中12mg/kg、低6mg/kg剂量）。比较各组小鼠行为变化,检测IgE、TNF-α、IL-1β、IL-6、IL-4和IFN-γ等指标,分类计数血液及支气管肺泡灌洗液（BALF）中的嗜酸性粒细胞数量,分析肺组织病理变化;抗氧化实验采用香栓菌水提取物、乙酸乙酯提取物、氯仿提取物、石油醚提取物比较清除DPPH和ABTS自由基能力。平喘试验结果表明,香栓菌水提物中剂量（120mg/kg）能降低血中嗜酸性粒细胞（Eos）及血清中IgE含量,减少BALF中Eos,降低血清及肺组织匀浆中IL-4含量,提高IFN-γ/IL-4,减轻模型的病理改变;石油醚提取物高剂量（24mg/kg）能降低血中Eos和血清中IgE、TNF-α、IL-1、IL-6含量,减少肺组织炎症细胞浸润;抗氧化结果显示,香栓菌各层提取物均有不同程度的抗氧化能力,并呈现一定的量效关系,同时水提取物的抗氧化效果最显著。本文为深入研究香栓菌子实体对呼吸系统疾病的治疗提供了重要试验依据。     

腰大池持续引流术对老年重型颅脑损伤患者颅内压和并发症及预后的影响

目的:探讨腰大池持续引流术对老年重型颅脑损伤患者颅内压、并发症和预后的影响。方法:选取2014年8月～2018年8月期间成都医学院第一附属医院收治的老年重型颅脑损伤患者60例为研究对象。根据随机数字表法将患者分为对照组(n=30)与研究组(n=30),其中对照组术后给予常规的脱水对症治疗,研究组则在此基础上给予腰大池持续引流术。比较两组术后临床疗效以及并发症,术后3d、5d、7d颅内压,随访半年,观察两组患者预后。结果:研究组治疗后总有效率为90.00%,显著高于对照组患者的66.67%(P0.05)。两组患者术后3d、5d、7d颅内压均较术前降低,且研究组低于对照组(P0.05)。研究组脑梗死、脑水肿、硬膜下积液、癫痫、纵裂积液等并发症发生率均低于对照组(P0.05),而颅内感染比较差异无统计学意义(P0.05)。研究组良好例数高于对照组,而重残以及死亡例数均低于对照组,差异有统计学意义(P0.05);而两组轻残、植物生存例数比较差异无统计学意义(P0.05)。结论:腰大池持续引流术治疗老年重型颅脑损伤患者,疗效满意,可显著改善患者颅内压,减少并发症发生率,改善患者预后,适于临床推广。     

Pharmacological Identification of a Guanidine-Containing β-Alanine Analogue with Low Micromolar Potency and Selectivity for the Betaine/GABA Transporter 1 (BGT1)

The γ-aminobutyric acid (GABA) transporters (GATs) are key membrane transporter proteins involved in the termination of GABAergic signaling at synapses in the mammalian brain and proposed drug targets in neurological disorders such as epilepsy. To date, four different GAT subtypes have been identified: GAT1, GAT2, GAT3 and the betaine/GABA transporter 1 (BGT1). Owing to the lack of potent and subtype selective inhibitors of the non-GAT1 GABA transporters, the physiological role and therapeutic potential of these transporters remain to be fully understood. Based on bioisosteric replacement of the amino group in β-alanine or GABA, a series of compounds was generated, and their pharmacological activity assessed at human GAT subtypes. Using a cell-based [³H]GABA uptake assay, several selective inhibitors at human BGT1 were identified. The guanidine-containing compound 9 (2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid hydrochloride) displayed more than 250 times greater potency than the parent compound β-alanine at BGT1 and is thus the most potent inhibitor reported to date for this subtype (IC₅₀ value of 2.5 µM). In addition, compound 9 displayed about 400, 16 and 40 times lower inhibitory potency at GAT1, GAT2 and GAT3, respectively. Compound 9 was shown to be a substrate for BGT1 and to have an overall similar pharmacological profile at the mouse orthologue. Compound 9 constitutes an interesting pharmacological tool for specifically investigating the cellular pharmacology of BGT1 and is the first small-molecule substrate identified with such a high selectivity for BGT1 over the three other GAT subtypes.     

张家口土壤蜘蛛群落结构及多样性研究

1996年8月中旬,在张家口地区4种生境中共捕获土壤蜘蛛461头,隶属于12科54种(属)。研究表明:4种生境中的土壤蜘蛛群落相似性很小,群落结构组成不同,丰富度、多样性指数、均匀度、优势度也不同。植被类型、疏密程度是影响群落结构的主要生态因子,农事活动是另一个重要影响因素。