ABSTRACT
Chronic visceral pain can occur in many disorders, the most common of which is irritable bowel syndrome (IBS). Moreover, depression is a frequent comorbidity of chronic visceral pain. The P2X7 receptor is crucial in inflammatory processes and is closely connected to developing pain and depression. Gallic acid, a phenolic acid that can be extracted from traditional Chinese medicine, has been demonstrated to be anti-inflammatory and anti-depressive. In this study, we investigated whether gallic acid could alleviate comorbid visceral pain and depression by reducing the expression of the P2X7 receptor. To this end, the pain thresholds of rats with comorbid visceral pain and depression were gauged using the abdominal withdraw reflex score, whereas the depression level of each rat was quantified using the sucrose preference test, the forced swimming test, and the open field test. The expressions of the P2X7 receptor in the hippocampus, spinal cord, and dorsal root ganglion (DRG) were assessed by Western blotting and quantitative real-time PCR. Furthermore, the distributions of the P2X7 receptor and glial fibrillary acidic protein (GFAP) in the hippocampus and DRG were investigated in immunofluorescent experiments. The expressions of p-ERK1/2 and ERK1/2 were determined using Western blotting. The enzyme-linked immunosorbent assay was utilized to measure the concentrations of IL-1ß, TNF-α, and IL-10 in the serum. Our results demonstrate that gallic acid was able to alleviate both pain and depression in the rats under study. Gallic acid also reduced the expressions of the P2X7 receptor and p-ERK1/2 in the hippocampi, spinal cords, and DRGs of these rats. Moreover, gallic acid treatment decreased the serum concentrations of IL-1ß and TNF-α, while raising IL-10 levels in these rats. Thus, gallic acid may be an effective novel candidate for the treatment of comorbid visceral pain and depression by inhibiting the expressions of the P2X7 receptor in the hippocampus, spinal cord, and DRG.
Subject(s)
Visceral Pain , Animals , Depression/drug therapy , Gallic Acid/pharmacology , Hyperalgesia/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2X7/genetics , Tumor Necrosis Factor-alpha/metabolism , Visceral Pain/drug therapyABSTRACT
Niemann-Pick disease, type C1 (Npc1), is an atypical lysosomal storage disorder caused by autosomal recessive inheritance of mutations in Npc1 gene. In the Npc1 mutant mice (Npc1-/- ), the initial manifestation is enlarged spleen, concomitant with free cholesterol accumulation. Telocytes (TCs), a novel type of interstitial cell, exist in a variety of tissues including spleen, presumably thought to be involved in many biological processes such as nursing stem cells and recruiting inflammatory cells. In this study, we found that the spleen is significantly enlarged in Npc1-/- mice, and the results from transmission electron microscopy examination and immunostaining using three different TCs markers, c-Kit, CD34 and Vimentin revealed significantly increased splenic TCs in Npc1-/- mice. Furthermore, hematopoietic stem cells and macrophages were also elevated in Npc1-/- spleen. Taken together, our data indicate that splenic TCs might alleviate the progress of splenic malfunction via recruiting hematopoietic stem cells and macrophages.
Subject(s)
Proteins/metabolism , Spleen/metabolism , Telocytes/metabolism , Animals , Antigens, CD/metabolism , Antigens, CD34/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Cell Count , Fluorescent Antibody Technique , Intracellular Signaling Peptides and Proteins , Macrophages/metabolism , Male , Mice, Mutant Strains , Nanog Homeobox Protein/metabolism , Niemann-Pick C1 Protein , Pluripotent Stem Cells/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Spleen/pathology , Spleen/ultrastructure , Telocytes/pathology , Telocytes/ultrastructure , Vimentin/metabolismABSTRACT
The bone marrow-derived mesenchymal stem cells (bmMSCs) have been widely used in cell transplant therapy, and the proliferative ability of bmMSCs is one of the determinants of the therapy efficiency. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) as a transmembrane protein is responsible for binding, internalizing and degrading oxidized low density lipoprotein (ox-LDL). It has been identified that LOX-1 is expressed in endothelial cells, vascular smooth muscle cells, cardiomyocytes, fibroblasts and monocytes. In these cells, low concentration of ox-LDL (<40 µg/mL) stimulates their proliferation via LOX-1 activation. However, it is poor understood that whether LOX-1 is expressed in bmMSCs and which role it plays. In this study, we investigated the status of LOX-1 expression in bmMSCs and its function on bmMSC proliferation. Our results showed that primary bmMSCs exhibiting a typical fibroblast-like morphology are positive for CD44 and CD90, but negative for CD34 and CD45. LOX-1 in both mRNA and protein levels is highly expressed in bmMSCs. Meanwhile, bmMSCs exhibit a strong potential to take up ox-LDL. Moreover, LOX-1 expression in bmMSCs is upregulated by ox-LDL with a dose- and time-dependent manner. Presence of ox-LDL also enhances the proliferation of bmMSCs. Knockdown of LOX-1 expression significantly inhibits ox-LDL-induced bmMSC proliferation. These findings indicate that LOX-1 plays a role in bmMSC proliferation.
Subject(s)
Bone Marrow Cells/metabolism , Cell Proliferation , Lipoproteins, LDL/metabolism , Mesenchymal Stem Cells/metabolism , Scavenger Receptors, Class E/metabolism , Animals , Bone Marrow Cells/cytology , Cells, Cultured , Endothelial Cells/metabolism , Humans , Lipoproteins, LDL/immunology , Mesenchymal Stem Cells/cytology , Mice , Scavenger Receptors, Class E/immunologyABSTRACT
CONTEXT: First-principles calculations based on the generalized gradient approximation gradient and the Perdew-Burke-Ernzerhof function (GGA-PBE generalized function) are carried out on the intrinsic and lithium-doped black phosphine systems to investigate the effects of different uniaxial tensile deformations on the electronic and optical properties of the systems. It is shown that the structural stability of the intrinsic and lithium-doped systems decreases with increasing tensile deformation, and all systems are most stable at 0% tensile deformation. The intrinsic black phosphazene system is a direct band gap semiconductor, and its band gap increases and then decreases with tensile deformation and reaches a maximum value of 1.086 eV at 4%. Lithium doping closes the band gap of the black phosphazene system, which is metallic in nature, but the band gap is opened up when the tensile deformation is 4-6%. From the density of states analysis, the density of states of all systems is basically contributed by the s and p orbitals, with little contribution from the d orbitals, and the contribution from the p orbitals is dominant. From the analysis of optical properties, the increase of tensile deformation causes the absorption peaks of the intrinsic system to redshift then blueshift then redshift, causes the absorption peaks of the lithium-doped system to redshift, and causes the reflection peaks of all systems to redshift. In addition, lithium doping blueshifts the absorption and reflection peaks of the systems compared to the intrinsic black phosphazene system. METHODS: Using the CASTEP section of the Materials Studio software, first-principle calculations based on density functional theory are done on the top-site doped lithium atoms of monolayer black phosphine under uniaxial stretching deformation in the a-direction, and the generalized gradient approximation gradients and Perdew-Burke-Ernzerhof functions (GGA-PBE generalized functionals) are used for the optimization and approximation process. The optimization parameters are set for the supercell structure: its plane-wave truncation energy is set to 400 eV, its Brillouin zone K-point grid is set to 3*3*3, its self-consistent field iteration accuracy convergence value is 2.0e-6 eV/atom, the convergence basis of its structural optimization is 0.02 eV/ Å, and the convergence of the stress value is 0.05 gpa. During the optimization period, the interaction force between atoms is 0.03 eV/ Å and the atomic displacement is less than 0.001 Å. To eliminate the effect of interlayer forces, a vacuum layer with a thickness of 15 Å is placed in its vertical direction (i.e., c-axis direction).
ABSTRACT
Members of the ADAM (a disintegrin and metalloprotease) family are type I transmembrane proteins involved in biological processes of proteolysis, cell adhesion, cell-matrix interaction, as well as in the intracellular signaling transduction. In the present study, expression patterns of seven members of the ADAM family were investigated at the early stages of the developing cochlea by in situ hybridization. The results show that each individual ADAM is expressed and regulated in the early developing cochlea. ADAM9, ADAM10, ADAM17, and ADAM23 are initially and widely expressed in the otic vesicle at embryonic day 2.5 (E2.5) and in the differential elements of the cochlear duct at E9, while ADAM12 is expressed in acoustic ganglion cells at E7. ADAM22 is detectable in cochlear ganglion cells as early as from E4 and in the basilar papilla from E7. Therefore, the present study extends our previous results and suggests that ADAMs also play a role in the early cochlear development.
Subject(s)
ADAM Proteins/metabolism , Cochlea/embryology , Cochlea/metabolism , Animals , Chick Embryo , Chickens , Gene Expression Regulation, Developmental , Immunohistochemistry , In Situ HybridizationABSTRACT
Bone marrow-derived mesenchymal stem cells (bmMSCs) are the most important cell source for stem cell transplant therapy. The migration capacity of MSCs is one of the determinants of the efficiency of MSC-based transplant therapy. Our recent study has shown that low concentrations of oxidized low-density lipoprotein (ox-LDL) can stimulate proliferation of bmMSCs. In this study, we investigated the effects of ox-LDL on bmMSC migration and adhesion, as well as the related mechanisms. Our results show that transmigration rates of bmMSCs and cell-cell adhesion between bmMSCs and monocytes are significantly increased by treatments with ox-LDL in a dose- and time-dependent manner. Expressions of ICAM-1, PECAM-1, and VCAM-1 as well as the levels of intracellular Ca(2+) are also markedly increased by ox-LDL in a dose-dependent manner. Cytoskeleton analysis shows that ox-LDL treatment benefits to spreading of bmMSCs and organization of F-actin fibers after being plated for 6 hours. More interestingly, treatments with ox-LDL also markedly increase expressions of LOX-1, MCP-1, and TGF- ß ; however, LOX-1 antibody and MCP-1 shRNA markedly inhibit ox-LDL-induced migration and adhesion of bmMSCs, which suggests that ox-LDL-induced bmMSC migration and adhesion are dependent on LOX-1 activation and MCP-1 expression.
Subject(s)
Bone Marrow Cells/cytology , Chemokine CCL2/metabolism , Gene Expression Regulation , Lipoproteins, LDL/metabolism , Mesenchymal Stem Cells/cytology , Scavenger Receptors, Class E/metabolism , Actins/metabolism , Animals , Calcium/metabolism , Cell Adhesion , Cell Movement , Cytoskeleton/metabolism , Flow Cytometry , Intercellular Adhesion Molecule-1/metabolism , Mice , Mice, Inbred C57BL , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Transforming Growth Factor beta/metabolism , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Sweet taste is a primary sensation for the preference and adaption of primates to diet, which is crucial for their survival and fitness. It is clear now that the sweet perception is mediated by a G protein-coupled receptor (GPCR)-sweet taste receptor T1R2/T1R3, and many behavioral or physiological experiments have described the diverse sweet taste sensitivities in primates. However, the structure-function relationship of T1R2s/T1R3s in primates, especially the molecular basis for their species-dependent sweet taste, has not been well understood until now. In this study, we performed a comprehensive sequence, structural and functional analysis of sweet taste receptors in primates to elucidate the molecular determinants mediating their species-dependent sweet taste recognition. Our results reveal distinct taxonomic distribution and significant characteristics (interaction, coevolution and epistasis) of specific key function-related residues, which could partly account for the previously reported behavioral results of taste perception in primates. Moreover, the prosimians Lemuriformes species, which were reported to have no sensitivity to aspartame, could be proposed to be aspartame tasters based on the present analysis. Collectively, our study provides new insights and promotes a better understanding for the diversity, function and evolution of sweet taste receptors in primates.
ABSTRACT
The delta-protocadherin (δ-Pcdh) family of transmembrane proteins belongs to the cadherin superfamily, which is involved in embryogenesis mediated by a homophilic binding during the embryonic development. In the present study, expression patterns of eight members of the δ-Pcdh family were investigated in the developing chicken cochlea by in situ hybridization. Our results provide a dynamical profile to show that the δ-Pcdhs are expressed spatially and temporally in the developing chicken cochleae. The earliest onset of the δ-Pcdh expression begins in the otic vesicle from embryonic incubation day (E) 3. From E11 onwards, the individual δ-Pcdh is expressed in different cell types of the cochlea. Protocadherin-1 (Pcdh1) is mainly expressed by spindle-shaped cells and acoustic ganglion cells; Pcdh7 and Pcdh17 are strongly expressed by supporting cells, cuboidal cells, hyaline cells and acoustic ganglion cells, and Pcdh9 is prominently expressed by homogene cells and acoustic ganglion cells; Pcdh8 was found to be transcribed in hair cells, spindle-shaped cells and acoustic ganglion cells; Pcdh10 mRNA is restricted to spindle-shaped cells and acoustic ganglion cells at later stages. mRNAs of Pcdh1, Pcdh18 and Pcdh19 are also expressed in blood vessels of the cochlea. The expression of the different δ-Pcdhs suggests a functional role for them during cochlear development.
Subject(s)
Cadherins/metabolism , Cochlea/embryology , Cochlea/metabolism , Gene Expression Regulation, Developmental/physiology , Animals , Chick Embryo , Cochlea/cytology , Gene Expression Profiling , Immunohistochemistry , In Situ HybridizationABSTRACT
Sweet-tasting protein is a kind of biomacromolecule that has remarkable sweetening power and is regarded as the promising sugar replacer in the future. Some sweet-tasting proteins has been used in foods and beverages. However, the structure and function relationship of these proteins is still elusive, and guidelines for their protein engineering is limited. It is well-known that the sweet-tasting proteins bind to and activate the sweet taste receptor T1R2/T1R3, thus eliciting their sweetness. The "wedge-model" for describing the interaction between sweet-tasting proteins and sweet taste receptor to elucidate their sweetness has been reported. In this perspective article, we revealed that the intramolecular interaction forces in sweet-tasting proteins is directly correlated to their properties (sweetness and stability). This intramolecular interaction pattern, named as "protein sector," refers to a small subset of residues forming physically connections, which cooperatively affect the function of the proteins. Based on the analysis of previous experimental data, we suggest that "protein sector" of sweet-tasting proteins is pivotal for their sweet properties, which are meaningful guidelines for the future protein engineering.
ABSTRACT
BACKGROUND/AIMS: The differentiation efficiency of bone marrow mesenchymal stem cells (BM-MSCs) is low in vivo after transplantation. Therefore, it is necessary to look for effective reagents for enhancing cardiac differentiation of BM-MSCs. It has been reported that cardiac differentiation of stem cells depends on the activation of extracellular signal-regulated protein 1/2 (ERK1/2) signaling. Oxidized low-density lipoprotein (ox-LDL) is a potent reagent for ERK1/2 activation. This indicates that ox-LDL may be a potential reagent to stimulate cardiac differentiation of stem cells. In this study, we investigated the effect of ox-LDL on cardiac differentiation of BM-MSCs and its relationship with ERK1/2 signaling. METHODS: BM-MSCs were isolated from mouse bone marrow, cultured in DMEM supplemented with 15% FBS, and passaged up to the 3rd passage. Following culture with 5 µg/mL ox-LDL for 3 weeks, the cardiac differentiation of the 3rd passage BM-MSCs was identified by immunostaining, Western blotting, and RT-PCR assays for measuring the expression of cardiac-specific markers. To further explore the role of ERK1/2 signaling in cardiac differentiation of BM-MSCs, we simultaneously exposed BM-MSCs to ERK1/2 inhibitor (U0126) and ox-LDL, and identified the cardiac differentiation again. RESULTS: The expressions of cardiac-specific markers including α-cardiac actin, α-MHC, ß-MHC, ANP, and BNP were markedly increased in BM-MSCs following treatment with ox-LDL (P < .05), which indicates a directional differentiation of BM-MSCs to cardiac cells. Further, ox-LDL could also activate ERK1/2 in BM-MSCs, and application of U0126 markedly inhibited ox-LDL-induced cardiac transformation of BM-MSCs. CONCLUSIONS: Ox-LDL induces cardiac differentiation of BM-MSCs via activation of ERK1/2 signaling.
Subject(s)
Bone Marrow Cells/drug effects , Cell Differentiation/drug effects , Lipoproteins, LDL/pharmacology , MAP Kinase Signaling System/drug effects , Mesenchymal Stem Cells/drug effects , Myocytes, Cardiac/drug effects , Animals , Butadienes/pharmacology , Cell Survival/drug effects , Enzyme Inhibitors/pharmacology , Mice , Mice, Inbred C57BL , Nitriles/pharmacologyABSTRACT
Pax3 and Pax7 are closely related transcription factors that are widely expressed in the developing nervous system and somites. In the CNS, both genes are expressed in the dorsal part of the neural tube during development. Pax3 and Pax7 are involved in the sonic hedgehog (Shh) signaling pathway and are inhibited by Shh overexpression. The present study confirms in vivo that Pax3 overexpression represses the expression of Pax7, whereas Pax7 overexpression endogenously enhances and ectopically induces the expression of Pax3 in the developing chicken spinal cord. Overexpression of Pax3 and Pax7 represses the endogenous expression of cadherin-7, a member of the cadherin family of morphogenetic genes, and induces its ectopic expression. The present study also shows that overexpression of Pax3 and Pax7 changes the fate and morphology of cells in the neuroepithelial layer and induces the expression of postmitotic neuronal markers. We show that both Pax3 and Pax7 promote the differentiation of neural progenitor cells into neurons. Furthermore, the downregulation of Pax3 and Pax7 with specific shRNAs results in apoptosis in the developing spinal cord. Collectively, these results suggest that the transcription factors Pax3 and Pax7 play important roles in regulating morphogenesis and cell differentiation in the developing spinal cord.
Subject(s)
Avian Proteins/biosynthesis , Cadherins/biosynthesis , Cell Differentiation/physiology , Neurons/metabolism , PAX7 Transcription Factor/metabolism , Paired Box Transcription Factors/metabolism , Spinal Cord/metabolism , Animals , Chick Embryo , Chickens , Gene Expression Regulation, Developmental , Mice , Neurogenesis/physiology , PAX3 Transcription Factor , Protein Binding/physiology , Spinal Cord/cytology , Spinal Cord/embryologyABSTRACT
N-cadherin is a calcium-sensitive cell adhesion molecule that plays an important role in the formation of the neural circuit and the development of the nervous system. In the present study, we investigated the function of N-cadherin in cell-cell connection in vitro with HEK293T cells, and in commissural axon projections in the developing chicken spinal cord using in ovo electroporation. Cell-cell connections increased with N-cadherin overexpression in HEK293T cells, while cell contacts disappeared after co-transfection with an N-cadherin-shRNA plasmid. The knockdown of N-cadherin caused the accumulation of ß-catenin in the nucleus, supporting the notion that N-cadherin regulates ß-catenin signaling in vitro. Furthermore, N-cadherin misexpression perturbed commissural axon projections in the spinal cord. The overexpression of N-cadherin reduced the number of axons that projected alongside the contralateral margin of the floor plate, and formed intermediate longitudinal commissural axons. In contrast, the knockdown of N-cadherin perturbed commissural axon projections significantly, affecting the projections alongside the contralateral margin of the floor plate, but did not affect intermediate longitudinal commissural axons. Taken together, these findings suggest that N-cadherin regulates commissural axon projections in the developing chicken spinal cord.
Subject(s)
Axons/metabolism , Cadherins/genetics , Cadherins/metabolism , Gene Expression , Signal Transduction , Spinal Cord/metabolism , beta Catenin/metabolism , Animals , Chickens , Fluorescent Antibody Technique , Gene Knockdown Techniques , HEK293 Cells , HumansABSTRACT
Classic cadherins belong to the family of cadherin genes and play important roles in neurogenesis, neuron migration, and axon growth. In the present study, we compared the expression patterns of 10 classic cadherins (Cdh2, Cdh4, Cdh6, Cdh7, Cdh8, Cdh9, Cdh11, Cdh12, Cdh18, and Cdh20) in the developing chicken spinal cord (SP) by in situ hybridization. Our results indicate that each of the investigated cadherins exhibits a spatially restricted and temporally regulated pattern of expression. At early developmental stages (E2.5-E3), Cdh2 is expressed throughout the neuroepithelial layer. Cdh6 is strongly positive in the roof plate and later also in the floor plate. Cdh7, Cdh11, Cdh12, and Cdh20 are expressed in restricted regions of the basal plate of the SP. At intermediate stages of development (E4-E10), specific expression profiles are observed for all investigated cadherins in the differentiating mantle layer along the dorsoventral, mediolateral, and rostrocaudal dimensions. Expression profiles are especially diverse for Cdh2, Cdh4, Cdh8, Cdh11, and Cdh20 in the dorsal horn, while different pools of motor neurons exhibit signal for Cdh6, Cdh7, Cdh8, Cdh9, Cdh12, and Cdh20 in the ventral horn. Interestingly, subpopulations of cells in the dorsal root ganglion express combinations of different cadherins. In the surrounding tissues, such as the boundary cap cells and the notochord, the cadherins are also expressed differentially. The highly regulated spatiotemporal expression patterns of the classic cadherins indicate that these genes potentially play multiple and diverse roles during the development of the SP and its surrounding tissues.
ABSTRACT
The expression of the chicken delta-protocadherin (Pcdh) subfamily was investigated in the developing feather buds of the chicken. The expression profiles of the eight investigated Pcdhs in the cells and tissues of the feather buds differ from each other. Pcdh1, Pcdh7, Pcdh8 and Pcdh10 are differentially expressed in the epidermis of the feather bud. Expression of Pcdh1 and Pcdh10 is restricted to the periderm and Pcdh17 expression to the epidermis of interbud region. Pcdh19 is mostly expressed at the anterior side of epidermis as well as in the blood vessels of the feather buds. Furthermore, Pcdh9 and Pcdh18 both are regionally expressed in the dermis of the feather bud. These results suggest that Pcdhs may play a variety of roles during avian feather bud formation.
Subject(s)
Cadherins/biosynthesis , Feathers/growth & development , Gene Expression Regulation, Developmental , Animals , Cadherins/genetics , Chickens/genetics , Chickens/growth & development , Epidermis/growth & development , Epidermis/metabolism , Feathers/metabolismABSTRACT
Protocadherins constitute the largest subfamily of cadherin genes and are widely expressed in the nervous system. In the present study, we cloned eight members of the delta-protocadherin subfamily of cadherins (Pcdh1, Pcdh7, Pcdh8, Pcdh9, Pcdh10, Pcdh17, Pcdh18, and Pcdh19) from the chicken, and investigated their expression in the developing chicken spinal cord by in situ hybridization. Our results showed that each of the investigated delta-protocadherins exhibits a spatially restricted and temporally regulated pattern of expression. Pcdh1, Pcdh8, Pcdh18, and Pcdh19 are expressed in restricted dorsoventral domains of the neuroepithelial layer at early developmental stages (E2.5E4). In the differentiating mantle layer, specific expression profiles are observed for all eight delta-protocadherins along the dorsoventral, mediolateral, and rostrocaudal dimensions at intermediate stages of development (E6E10). Expression profiles are especially diverse in the motor column, where different pools of motor neurons exhibit signal for subsets of delta-protocadherins. In the dorsal root ganglion, subpopulations of cells express combinations of Pcdh1, Pcdh7, Pcdh8, Pcdh9, Pcdh10, and Pcdh17. The ventral boundary cap cells are positive for Pcdh7, Pcdh9, and Pcdh10. Signals for Pcdh8, Pcdh18, and Pcdh19 are found in the meninges. Surrounding tissues, such as the notochord, dermomyotome, and sclerotome also exhibit differential expression patterns. The highly regulated spatiotemporal expression patterns of delta-protocadherins suggest that they have multiple and diverse functions during development of the spinal cord and its surrounding tissues.
Subject(s)
Cadherins/biosynthesis , Neurogenesis/physiology , Spinal Cord/embryology , Spinal Cord/metabolism , Animals , Cadherins/analysis , Chick Embryo , In Situ HybridizationABSTRACT
In this study, the cDNA library of Chang-liver cells was immunoscreened using common ADAMs antibody to obtain ADAM related genes. We found one positive clone that was confirmed as a new gene by Blast, which is an uncharacterized helical and coil protein and processes protease activity, and named protease-related protein 1 (ARP1). The submitted GenBank accession number is AY078070. Molecular characterizations of ARP1 were analyzed with appropriate bioinformatics software. To analyse its expression and function, ARP1 was subcloned into glutathione S-transferase fusion plasmid pGEX-2T and expressed by E. coli system. The in vitro expression product of ARP1 was recognized by common ADAMs antibody with western blot. Interestingly, ARP1 cleaves gelatine at pH9.5, which suggests it is an alkaline protease. Semi-quantitative RT-PCR result indicates that ARP1 mRNA is strongly transcribed in the liver and the treated Chang-liver cells.