A Disintegrin and Metalloprotease 10 in neuronal maturation and gliogenesis during cortex development

The multiple-layer structure of the cerebral cortex is important for its functions. Such a structure is generated based on the proliferation and differentiation of neural stem/progenitor cells. Notch functions as a molecular switch for neural stem/progenitor cell fate during cortex development but the mechanism remains unclear. Biochemical and cellular studies showed that Notch receptor activation induces several proteases to release the Notch intracellular domain （NICD）. A Disintegrin and Metalloprotease 10 （ADAM10） might be a physiological rate-limiting $2 enzyme for Notch activation. Nestin-driven conditional ADAM10 knockout in mouse cortex showed that ADAM10 is cdtical for maintenance of the neural stem cell population during early embryonic cortex development. However, the expression pattern and function of ADAM10 during later cerebral cortex development remains poorly understood. We performed in situ hybridization for ADAMIO mRNA and immunofluorescent analysis to determine the expression of ADAM10 and NICD in mouse cortex from embryonic day 9 （E14.5） to postnatal day 1 （P1）. ADAM10 and NICD were highly co-localized in the cortex of E16.5 to P1 mice. Comparisons of expression patterns of ADAM10 with Nestin （neural stem cell marker）, Tujl （mature neuron marker）, and S100β （gila marker） showed that ADAM10 expression highly matched that of S10013 and partially matched that of Tujl at later embryonic to early postnatal cortex developmental stages. Such expression patterns indicated that ADAM10-Notch signaling might have a critical function in neuronal maturation and gliogenesis during cortex development.

Matrix metalloproteases and their tissue inhibitors in non-alcoholic liver fibrosis of human immunodeficiency virus-infected patients

AIM To investigate the relationships among diverse metalloproteases(MMPs) and their tissue inhibitors(TIMPs) and non-alcoholic liver fibrosis in human immunodeficiency virus(HIV)-infected patients.METHODS Single nucleotide polymorphisms(SNPs) in MMPs, TNF-α and CCR5 genes, and serum levels of MMPs and TIMPs were determined in HIV-infected individuals with/out hepatitis C virus(HCV) coinfection. A total of 158 patients were included, 57 of whom were HCVcoinfected. All patients drank < 50 g ethanol/day. Diverse SNPs(MMP-1-1607 1G/2G, MMP-8-799C/T, MMP-9-1562 C/T, MMP-13-77A/G, TNF-α-308 G/A,CCR5-?32), and serum levels of MMPs(2, 3, 8, 9 and 10) and TIMPs(1, 2 and 4) were assessed. Liver fibrosis was determined by transient elastometry, although other non-invasive markers of fibrosis were also considered. Significant liver fibrosis(F ≥ 2) was defined by a transient elastometry value ≥ 7.1 kP a.RESULTS A total of 34 patients(21.5%) had liver fibrosis ≥ F2. MMP-2 and TIMP-2 serum levels were higher in patients with liver fibrosis ≥ F2(P = 0.02 and P = 0.03, respectively) and correlated positively with transient elastometry values(P = 0.02 and P = 0.0009, respectively), whereas MMP-9 values were negatively correlated with transient elastometry measurements(P = 0.01). Multivariate analyses showed that high levels of MMP-2(OR = 2.397; 95%CI: 1.191-4.827, P = 0.014) were independently associated with liver fibrosis ≥ F2 in the patients as a whole. MMP-2(OR = 7.179; 95%CI: 1.210-42.581, P = 0.03) and male gender(OR = 10.040; 95%CI: 1.621-62.11, P = 0.013) were also independent predictors of fibrosis ≥ F2 in the HCV-infected subgroup. Likewise, MMP-2, TIMP-2 and MMP-9 were independently associated with transient elastometry values and other non-invasive markers of liver fibrosis. None of the six SNPs evaluated had any significant association with liver fibrosis ≥ F2.CONCLUSION Certain MMPs and TIMPs, particularly MMP-2, seems to be associated with non-alcoholic liver fibrosis in HIVinfected patients with/without HCV coinfection.

Tissue inhibitors of metalloproteases strike a nerve

Signals of touch,pressure,pain,temperature,position,and vibration are transmitted from the peripheral nervous system（PNS）to the dorsal horn of the segmental spinal cord via pseudounipolar dorsal root ganglia（DRG）neurons.Sensory information gathering relies on functional integrity of DRG neurons and can be interrupted by PNS injury due to trauma,disease or exposure to drugs, toxins or viral pathogens. Despite the high regenerative capacity of DRG neurons, sensory recovery after PNS injury is often incomplete and severe neuropathic pain may last for years. Although numerous mechanisms of PNS injury have been established, neuropathic pain is refractory to therapy, contributing to the physical and emotional suffering of patients and the enormous economic burden to society.

Comparison of Properties of Tumor Necrosis Factor-α Converting Enzyme (TACE) and Some Matrix Metalloproteases (MMPs) in Catalytic Domains

The crystal structural data of TACE, MMP-1, MMP-2, MMP-3 and MMP-9 were obtained from PDB database, and then their catalytic domains＇ properties including conformation, molecular surface hydrophobicity and electrostatic potential were analyzed and compared by using Insight II molecular modeling software. It was found that the conformation and molecular surface hydrophobicity of catalytic domains of TACE and MMPs were not obviously different, but the molecular surface electrostatic potential of catalytic domain of TACE＇ and MMPs had obvious differences. The findings are helpful in the Rational Drug Design of TACE selective inhibitor.

The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat

Wheat(Triticum aestivum L.)is an important staple crop for global human.The necrotrophic fungus Rhizoctonia cerealis is the causal pathogen of sharp eyespot,a devastating disease of wheat.Herein,we identified RcMEP1,a zinc metalloproteaseencoding gene from R.cerealis genomic sequences,and characterized its pathogenesis function.RcMEP1 expressed at markedly-high levels during R.cerealis infection process to wheat.The predicted protein RcMEP1 comprises of 287 amino acid residues and contains a signal peptide and a M43 metalloprotease domain harboring the active site motif(HEVGHWLGLYH).The assays of Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana leaves indicated that RcMEP1 is an apoplastic elicitor of cell death,and that the predicted signal peptide functions and is required for secretion and cell death-induction.The purified RcMEP1 protein and its M43 domain peptide were individually able to induce plant cell death and H2 O2 accumulation,and to inhibit expression of host chitinases when infiltrated into wheat and N.benthamiana leaves,while the M43 domain-deleting peptide and negative control lacked the capacity.Moreover,compared with the control pretreatment,the purified RcMEP1 protein or its M43-domain peptide resulted in enhanced pathogenesis in the inoculated wheat,whereas the M43 domain-deleting peptide failed.These results suggest that RcMEP1 acted as an important pathogenicity factor during R.cerealis infection to wheat and that its signal peptide and M43 domain are required for the secretion and pathogenesis of RcMEP1.This study provides insights into pathogenesis role of M43 domain-containing metalloproteases during R.cerealis infection to wheat.

Cloning of catalase gene and antioxidant genes in Scophthalmus maximus response to metalloprotease of Vibrio anguillarum stress

Metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic eff ects in turbot(Scophthalmus maximus).The toxicological eff ect partly results from oxidative damage due to the production of excessive reactive oxygen species(ROS).Catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx)are major antioxidant enzymes induced by various oxidative stresses and can scavenge peroxides generated in cells.To evaluate the eff ects of metalloprotease-induced ROS on the antioxidation defense mechanism of S.maximus head kidney cells,the cDNA of CAT gene(designated as SmCAT)was cloned and characterized.SmCAT comprises a 1584-bp coding sequence that encodes a protein containing 527 amino acids with a poly(A)tail.Bioinformatics analysis revealed an active site signature sequence,a heme-ligand signature sequence,and three catalytic amino acid residues.The deduced SmCAT amino acid sequence shares a sequence similarity of 66.1%-92.4%with those of other species.Phylogenetic analysis revealed that SmCAT is classifi ed with CAT of other fi shes.Quantitative real-time PCR analysis showed that SmCAT was extensively expressed in all tested tissues,especially in blood.The expression of SmCAT,SmMnSOD,and SmGPx were inhibited signifi cantly in head kidney cells treated with metalloprotease from 12 to 24 h.In 6 to 24 h metalloprotease-treated groups compared to that of the untreated group,it was found that the production of ROS was markedly increased,and the mitochondrial membrane potential was decreased considerably.Hoechst 33342 staining revealed the presence of apoptotic bodies when the cells were incubated with 8.0 or 40.0μg/mL metalloprotease for 12 and 24 h.Hence,the toxic eff ects of metalloprotease are associated with the down-regulation of antioxidant enzyme expression and increased ROS levels,which trigger the activation of apoptosis in the head kidney cells of turbot.Our fi ndings provide a better understanding on the mechanism of metalloprotease-induced apoptosis in fi sh.

Detection of Vibrio anguillarum and Its Virulent Metalloprotease Using the Method of ELISA

A method detecting pathogenic Vibrio anguillarum and its virulent metalloprotease is reported in this paper. The metalloprotease is isolated from extracellular product (ECP) of V.anguillarum by the cellophane plate technique and purified by gel filtration and ion exchange chromatography. Anti sera are prepared by injecting V.anguillarum cells and metalloprotease into the rabbits. Slide Agglutination Assay is used to detect V.anguillarum in the infection experiment and Enzyme Linked Immunosorbent Assay (ELISA) is carried out to detect concentration of metalloprotease. The result shows that bacterium strain M3 is able to diffuse into the viscera of infected fish through the blood circulating system 10 hours after intramuscular infection, and ELASA is a sensitive method to detect the metalloprotease with detectable amount of 7.8ng. The aim of this study is to establish a sensitive and specific method to observe the infection of V.anguillarum in the host.

Identification and Characterization of Peptides Mimicking the Epitopes of Metalloprotease of Schistosoma Japonicum

In an attempt to isolate and characterize peptides mimicking epitopes of metalloprotease and explore their immunological protection against Schistosoma japonicum （S. japonicum）, polyclonal anti-metalloprotease sera was prepared to screen a 12-mer random peptide library to isolate phages binding specially to antisera IgG. Then, phage ELISA, animal immunization, DNA sequencing, Western blotting and enzymatic activity neutralizing analysis were used to characterize the selected phage clones. All of ten randomly picked clones were shown to be positive. Five peptides of different amino acid sequences deduced from DNA sequences were obtained and two of them （peptides 2 and 3） could induce significant reduction （31.0% and 31.8%, respectively） in worm burden and high reduction （52.6% and 54.9%, respectively） in liver eggs per gram （LEPG）, while, unexpectedly, others （peptides 1, 4 and 5） could not elicit enough protection against infection of S. japonicum. Peptides 2 and 3 could be recognized by S. japonicum infected mouse sera （IMS） and could elicit neutralizing Abs. The results show that peptides 2 and 3 are antigenic and immunogenic. They are true mimics of epitopes of metalloprotease and useful as novel vaccine candidates against S.japonicum. Cellular ＆ Molecular Immunology. 2005;2（3）：219-223.

去整合素金属蛋白酶10和高迁移率族蛋白B1在声门型喉癌患者中的表达及预后分析

目的探讨去整合素金属蛋白酶10(a disintegrin and metalloprotease 10,ADAM10)和高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1)与声门型喉癌患者病理特征及预后关系分析。方法回顾性收集2017年3月~2020年12月于南京医科大学附属南京医院确诊及治疗的声门型喉癌患者50例(观察组),另取相对喉癌组织切缘0.5cm以上部位标本作为对照组。观察并比较ADAM10和HMGB1在两组中的阳性表达率,分析其阳性表达与声门型喉癌患者的病理特征关系。单因素分析影响声门型喉癌预后的危险因素,Cox多因素回归分析声门型喉癌患者不良预后的独立危险因素。结果ADAM10和HMGB1在观察组的阳性表达率均高于对照组,差异均有统计学意义(P<0.05)。声门型喉癌组织中的ADAM10与淋巴结转移和T分级差异比较有统计学意义,而与年龄、性别、饮酒史、吸烟史、分化程度差异比较无统计学意义(P>0.05);HMGB1与分化程度差异比较有统计学意义(P<0.05),而与年龄、性别、饮酒史、吸烟史、淋巴结转移、T分级差异比较无统计学意义(P>0.05)。单因素分析结果表明,淋巴结转移、T分级、分化程度、ADAM10、HMGB1是患者预后的影响因素。Cox多因素回归分析结果表明,淋巴结转移、T3+T4分级、低分化程度、ADAM10阳性、HMGB1阳性为声门型喉癌患者预后不良的独立影响因素(P<0.05)。结论ADAM10和HMGB1可作为声门型喉癌不良预后的风险评估指标。

Blocking postsynaptic density-93 binding to C-X3-C motif chemokine ligand 1 promotes microglial phenotypic transformation during acute ischemic stroke

We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357–395 of C-X3-C motif chemokine ligand 1(CX3 CL1) to induce microglia polarization. More importantly, the peptide Tat-CX3 CL1(comprising amino acids 357–395 of CX3 CL1) disrupts the interaction between postsynaptic density-93 and CX3 CL1, reducing neurological impairment and exerting a protective effect in the context of acute ischemic stroke. However, the mechanism underlying these effects remains unclear. In the current study, we found that the pro-inflammatory M1 phenotype increased and the anti-inflammatory M2 phenotype decreased at different time points. The M1 phenotype increased at 6 hours after stroke and peaked at 24 hours after perfusion, whereas the M2 phenotype decreased at 6 and 24 hours following reperfusion. We found that the peptide Tat-CX3 CL1(357–395 aa) facilitates microglial polarization from M1 to M2 by reducing the production of soluble CX3 CL1. Furthermore, the a disintegrin and metalloprotease domain 17(ADAM17) inhibitor GW280264 x, which inhibits metalloprotease activity and prevents CX3 CL1 from being sheared into its soluble form, facilitated microglial polarization from M1 to M2 by inhibiting soluble CX3 CL1 formation. Additionally, Tat-CX3 CL1(357–395 aa) attenuated long-term cognitive deficits and improved white matter integrity as determined by the Morris water maze test at 31–34 days following surgery and immunofluorescence staining at 35 days after stroke, respectively. In conclusion, Tat-CX3 CL1(357–395 aa) facilitates functional recovery after ischemic stroke by promoting microglial polarization from M1 to M2. Therefore, the Tat-CX3 CL1(357–395 aa) is a potential therapeutic agent for ischemic stroke.

ADAM9特异性siRNA对肾透明细胞癌786-0细胞ADAM9基因表达及体外侵袭能力的影响

目的:探讨去整合素金属蛋白酶9(ADAM9)特异性siRNA对人肾透明细胞癌(RCCC)786-0细胞中ADAM9基因表达及体外侵袭能力的影响。方法:设计合成ADAM9特异性siRNA。将786-0细胞分4组处理,分别为正常对照组(正常培养786-0细胞)、空脂质体转染组、阴性转染组(转染无义siRNA)和ADAM9 siRNA转染组。转染24、48、72 h后,采用RT-PCR法检测细胞ADAM9 mRNA的表达;转染48 h后,采用Western blot法检测细胞ADAM9蛋白的表达,并用Transwell法检测细胞侵袭能力。结果:转染24、48、72 h后,ADAM9 siRNA转染组786-0细胞ADAM9 mRNA的表达显著低于其他3组(F=47.945、180.456、60.978,P均<0.001);转染48 h后,ADAM9 siRNA转染组786-0细胞ADAM9蛋白表达显著降低(F=142.816,P<0.001),穿膜细胞数显著减少(F=45.389,P<0.001)。结论:ADAM9特异性siRNA能够有效沉默786-0细胞中ADAM9的表达并降低其体外侵袭能力。

解聚素金属蛋白酶17及变异型分化簇44在喉癌和喉咽癌组织中的表达

喉癌约占头颈恶性肿瘤的7.9%~35%[1],喉咽癌约占头颈部恶性肿瘤的1.4%~5.0%[1]。解聚素金属蛋白酶17（A disintegrin and metalloprotease 17,ADAM17）,最初由Black等[2]和Moss等[3]两个独立的小组同时发现,又称肿瘤坏死因子α转换酶（tumor necrosis factorαconveting enzyme,TACE）。

Decorin treatment of spinal cord injury

The scarring response after a penetrant central nervous system injury results from the interaction between invading leptominingeal/pericyte-derived fibroblasts and endogenous reactive astrocytes about the wound margin. Extracellular matrix and scar-derived axon growth inhibitory mole- cules fill the lesion site providing both a physical and chemical barrier to regenerating axons. Dec orin, a small leucine-rich chondroitin-dermatan sulphate proteoglycan expressed by neurons and astrocytes in the central nervous system, is both anti-fibrotic and anti-inflammatory and attenu- ates the formation and partial dissolution of established and chronic scars. Here, we discuss the potential of using Decorin to antagonise scarring in the central nervous system.

An in vitro and in silico study of anti-dermatophytic activity of gossypol from fruits of Thespesia populnea(L.)Sol.ex Correa

Objective:To isolate,purify,and characterize gossypol from the fruits of Thespesia populnea(L)Sol.ex Correa,test its antidermatophytic activity,identify its targets on the dermatophyte,and confirm the binding of gossypol with the fungal target by molecular docking study.Methods:Gossypol from Thespesia populnea was characterized by high performance liquid chromatography,liquid chromatographmass spectrometry,Fourier transform infrared spectroscopy,and nuclear magnetic resonance.The anti-dermatophytic activity of gossypol was tested against four different dermatophytes,viz.Trichophyton mentagrophytes,Trichophyton rubrum,Microsporum canis,and Microsporum gypseum.Trichophyton mentagrophytes was selected for further studies.The inhibitory mode of action of gossypol on Trichophyton mentagrophytes was determined by analyzing the modulation of gene expression in various pathways of the dermatophyte.Results:Gossypol inhibited all the dermatophytes.The minimum inhibitory concentrations were 12.5μg/mL for Trichophyton mentagrophytes and Microsporum canis and 25μg/mL for Trichophyton rubrum and Microsporum gypseum.The minimum fungicidal concentrations were 50μg/mL for Trichophyton mentagrophytes,100μg/mL for Microsporum canis and Trichophyton rubrum,and 200μg/mL for Microsporum gypseum.Gossypol inhibited the mRNA expression of metalloprotease(MEP4)and isocitrate lyase(ICL).The binding of gossypol with the enzymes was confirmed by molecular docking studies.The best docking poses were found and the low binding energies were recorded with the two target enzymes.Conclusions:Gossypol is a potential antifungal agent and can be further explored as an anti-dermatophytic drug.

TIMP-1 and its potential diagnostic and prognostic value in pulmonary diseases

Tissue inhibitors of metalloproteases(TIMPs)have caught the attention of many scientists due to their role in various physiological and pathological processes.TIMP-1,2,3,and 4 are known members of the TIMPs family.TIMPs exert their biological effects by,but are not limited to,inhibiting the activity of metalloproteases(MMPs).The balance between MMPs and TIMPs is critical for maintaining homeostasis of the extracellular matrix(ECM),while the imbalance between MMPs and TIMPs can lead to pathological changes,such as cancer.In this re-view,we summarized the current knowledge of TIMP-1 in several pulmonary diseases namely,acute lung injury(ALI)/acute respiratory distress syndrome(ARDS),pneumonia,asthma,chronic obstructive pulmonary disease(COPD),cystic fibrosis,and pulmonary fibrosis.Considering the potential of TIMP-1 serving as a non-invasive di-agnostic and/or prognostic biomarker,we also reviewed the circulating TIMP-1 levels in translational and clinical studies.

创伤弧菌和副溶血弧菌双重LAMP检测方法的建立及初步应用

为建立创伤弧菌和副溶血弧菌的双重环介导等温扩增（loop—mediated isothermal amplification，LAMP）检测方法，本试验以创伤弧菌metalloprotease基因和副溶血弧菌ompA基因为靶点设计LAMP扩增引物，并在内引物间添加不同的酶切位点，通过对反应时间和温度的优化及酶切反应，成功建立了双重LAMP检测方法，并对其检测特异性、灵敏度和实际应用性进行了研究。结果显示，62℃反应45min时可同时检测到2种弧菌的存在，通过限制性内切酶酶切，可准确区分2种弧菌。该方法比普通PCR检测方法灵敏度高10^2～10^3倍。选择17株细菌菌株作为检测模板，发现除创伤弧菌和副溶血弧菌反应结果呈阳性外，其他均为阴性。以大菱鲆作为实验动物，检测双重LAMP技术的实际应用可能，该方法可准确检测并鉴定出组织和血液中感染的细菌种类，其灵敏度可达374～410CFU／g（mL）。SYBR Green Ⅰ是一种结合于DNA双链小沟中的荧光染料，反应产物加入该染料后，极易用肉眼判别。本试验成功建立了创伤弧菌和副溶血弧菌的双重LAMP检测方法，该方法可用于水产养殖中病原菌的早期诊断。

Glioma associated microglia/macrophages, a potential pharmacological target to promote antitumor inflammatory immune response in the treatment of glioblastoma

Glioma associated microglia/macrophages (GAMs) constitute the largest proportion of glioma infiltrating cells, par-ticularly in high grade tumors (i.e., glioblastoma). Once inside the tumors, GAMs usually acquire a specific pheno-type of activation that favors tumor growth, angiogenesis and promotes the invasion of normal brain parenchyma. Therefore, treatments that limit or prevent GAMs' recruitment at the tumor site or modulate their immune activa-tion promoting antitumor activities are expected to exert beneficial effects in glioblastoma. In the present paper, we aim at the revision of pharmacological strategies that interfere with GAMs' function and are currently proposed as an alternative/additional option to current approved cytotoxic regimens.

ADAM9 decreases in castration resistant prostate cancer and is a prognostic factor for overall survival

Background A disintegrin and metalloprotease 9 （ADAM9） is a membrane-anchored enzyme which is considered to be involved in some diseases including tumor. However, the role of ADAM9 in castration resistant prostate cancer （CRPC） is not clear. This study aimed to explore the different expressions on protein and messenger RNA （mRNA） level of ADAM9 between hormonal sensitive prostate cancer （HSPC） and CRPC tissue, and find the correlation with prognosis.