共查询到20条相似文献,搜索用时 15 毫秒
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Masaaki Sawa Hirotaka Tateishi Kazuhiro Mizuno Hiroshi Harada Mayumi Oue Hiroshi Tsujiuchi Yasuji Furutani Shiro Kato 《Bioorganic & medicinal chemistry letters》2004,14(24):1121
A series of tryptamine derivatives with modified sulfonamide were designed, synthesized, and evaluated for their ability to stimulate cAMP accumulation in CHO cells expressing the cloned human β3-adrenergic receptor (AR). For this series of compounds, our objective was to symmetrize the α-position of the tryptamine moiety maintaining its activity and reducing the cost of production. Compound 11h, having m-aminobenzene, exhibited excellent agonistic activity for β3-AR with excellent subtype selectivity. 相似文献
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Wataru Sakamoto Osamu Nishikaze 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,633(3):305-309
α1-Antitrypsin, α2-macroglobulin and low-molecular weight kininogen were isolated from human serum and kallikreins from human urine and saliva.α1-Antitrypsin and α2-macroglobulin inhibited the activity of trypsin in releasing kinin from low-molecular weight kininogen, due to their binding with the enzyme, but did non inhibit or bind with urinary and salivary kallikreins. 相似文献
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Franoise Maupas-Schwalm Aurlie Bedel Nathalie Aug Marie-Hlne Grazide Elodie Mucher Jean-Claude Thiers Robert Salvayre Anne Ngre-Salvayre 《Cellular signalling》2009,21(12):1925-1934
Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin αvβ3, evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the αvβ3 blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin αvβ3 interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin αvβ3, uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin αvβ3 and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells. 相似文献
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Zvi Schwartz Bryan F. Bell Liping Wang Ge Zhao Rene Olivares-Navarrete Barbara D. Boyan 《The Journal of steroid biochemistry and molecular biology》2007,103(3-5):606
Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. β1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)2D3 in a surface-dependent manner. To determine if β1 has a role in mediating osteoblast response, we silenced β1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human β1 to block ligand binding. β1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, β1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)2D3. Anti β1 antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1α,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)2D3. The results also show that β1 mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)2D3. 相似文献
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Enhanced expression of transforming growth factor-β1(TGF-β1) demonstrated in human colonic mucosa of patients with ulcerative colitis (UC), indicates its possible significance in the pathogenesis of this disease. The aim of this study was to evaluate plasma TGF-β1concentration in patients with different degrees of colonic mucosal injury, as a possible indicator of ulcerative colitis activity. TGF-β1concentration was measured with an enzyme immunoassay (EIA) in plasma of 45 patients with endoscopically confirmed UC. Values observed in UC patients (40.5±15.9 ng/ml) were significantly higher than in healthy people (18.3±11.6 ng/ml) and higher than in patients with irritable colon syndrome (ICS), (20.5±13.6 ng/ml). The highest plasma TGF-β1(58.6±112.1 ng/ml) was in patients with the severe UC course. TGF-β1level analysed in all UC patients revealed significant positive correlation with scored degree of mucosal injury (r=0.396;P<0.01). Among other possible laboratory markers of the disease activity, only C-reactive protein concentration demonstrated significant correlation. Enhanced production of TGF-β1can be related to inflammation activity. Measurement of plasma TGF-β1may be considered as a biomarker of the disease activity. 相似文献
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The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC α1 and α2 mRNAs is developmentally regulated in differentiating C2Cl2 myogenic cells. The α1 mRNA is not detectable in the myoblast form of C2Cl2 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the α2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation. 相似文献
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Jian Hua Liang Jing Du Lian Deng Xu Tao Jiang Shuang Hao Jing Bi Bo Jiang 《Neurochemistry international》2009,55(8):741-746
It has been reported that catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa, protected cells from damage induced by a variety of toxic stimulus such as LPS, MPP+ and rotenone. Here, we further evaluated the effect of catalpol against Aβ1–42-induced apoptosis in primary cortical neuron cultures. In the present study, the primary cortical neuron culture treated with Aβ1–42 was severed as cell model of Alzheimer's disease (AD) in vitro. By exposure to Aβ1–42 (5 μM) for 72 h in cultures, neuronal apoptosis occurred characterized by enhancement of activities of caspases and reactive oxygen species (ROS) as well as Bax increase, loss of mitochondrial membrane potential and cytochrome c release. Pretreatment with catalpol (0.5 mM) for 30 min prior to Aβ1–42 treatment attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial membrane potential and, cytochrome c release to some extent, but also through regulating the activity and cleavage of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Aβ1–42 through a mitochondrial-dependent caspase pathway. 相似文献
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Arturo Andrade Alejandro Sandoval Ricardo Gonzlez-Ramírez Diane Lipscombe Kevin P. Campbell Ricardo Felix 《Cell calcium》2009,46(4):282-292
The auxiliary CaVα2δ-1 subunit is an important component of voltage-gated Ca2+ (CaV) channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly important in the case of the neuronal L-type CaV channels where these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the CaVα2δ-1 subunit on the functional expression and the pharmacology of recombinant L-type CaV1.3 channels in HEK-293 cells. Co-expression of CaVα2δ-1 significantly increased macroscopic currents and conferred the CaV1.3α1/CaVβ3 channels sensitivity to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, CaVα2δ-1 subunits harboring point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane δ domain of the protein, reduced functionality in terms of enhancement of CaV1.3α1/CaVβ3 currents. In addition, co-expression of the δ domain drastically inhibited macroscopic currents through recombinant CaV1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the CaVα2δ-1 auxiliary subunit may interact with CaV1.3 channels and regulate their functional expression. 相似文献
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Zuzana Dostalova Xiaojuan Zhou Aiping Liu Xi Zhang Yinghui Zhang Rooma Desai Stuart A. Forman Keith W. Miller 《Protein science : a publication of the Protein Society》2014,23(2):157-166
Gamma‐aminobutyric acid type A receptors (GABAARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABAARs determine their function and pharmacological profile. GABAARs are heteropentamers of subunits, and (α1)2(β3)2(γ2L)1 is a common subtype. Biochemical and biophysical studies of GABAARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABAAR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)3GK–1D4 GABAAR. These cells achieved expression levels of 70–90 pmol [3H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [3H]flunitrazepam to [3H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABAARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ~30%. Typical purifications yielded 1.0–1.5 nmoles of [3H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [3H]muscimol binding were maintained in the purified state. 相似文献
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Receptors for α2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled α2-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α2-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat α1-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-α1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor. 相似文献
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Susumi Hatakeyama Satoshi Nagashima Naoko Imai Keisuke Takahashi Jun Ishihara Atsuko Sugita Takeshi Nihei Hitoshi Saito Fumiaki Takahashi Noboru Kubodera 《The Journal of steroid biochemistry and molecular biology》2007,103(3-5):222
1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71), an analog of active vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)2D3. ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)2D3. Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)2D3, an epimer of 1,25(OH)2D3 at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)2D3 at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D3 analogs were found to be 1,25(OH)2D3 > ED-71 ≥ 3-epi-1,25(OH)2D3 3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays. 相似文献
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Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998 相似文献
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Adele R. Thomas Ryno J. Naud Willem Oelofsen Takako Naganuma Koji Muramoto 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2001,129(4)
This study reports the isolation and partial characterisation of the ostrich serpin, α2AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich α2AP was purified using
lysine–Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI–Sepharose chromatographies. It revealed a Mr of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine α2AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human α2AP, DFP and EACA. Ostrich plasminogen was highly purified after
lysine–Sepharose chromatography and showed a Mr of 92 K, a total of 775 amino acids and its N-terminal sequence showed 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a Mr of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40°C, respectively. 相似文献
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Barbara D. Boyan Kevin L. Wong Mimi Fang Zvi Schwartz 《The Journal of steroid biochemistry and molecular biology》2007,103(3-5):467
Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1α,25(OH)2D3 and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1α,25(OH)2D3 regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1α,25(OH)2D3-binding protein ERp60, phospholipase A2 (PLA2), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1α,25(OH)2D3 (10−8 M), which binds ERp60, activating PLA2, and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-β1 stored in the ECM as large latent TGF-β1 complexes, consisting of latent TGF-β1 binding protein, latency associated peptide, and latent TGF-β1. Others have shown that MMP-2 specifically activates TGF-β2. TGF-β1 regulates 1α,25(OH)2D3-production, providing a mechanism for local control of growth factor activation. 1α,25(OH)2D3 activates PKCα in the PM via ERp60-signaling through PLA2, lysophospholipid production, and PLCβ. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCζ. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1α,25(OH)2D3, PKCζ activity is decreased and PKCα is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling. 相似文献
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Simon T. Barry Steven B. Ludbrook Elaine Murrison Carmel M. T. Horgan 《Experimental cell research》2000,258(2):342
The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN. 相似文献