Gdf-8 Propeptide Binds to GDF-8 and Antagonizes Biological Activity by Inhibiting GDF-8 Receptor Binding

Abstract

GDF-8 is a new member of the TGF-β superfamily which appears to be a negative regulator of skeletal muscle mass. Factors which regulate the biological activity of GDF-8 have not yet been identified. However, the biological activities of TGF-β superfamily members, TGF-β1,-β2 and-β3, can be inhibited by noncovalent association with TGF-β1,-β2 and β3 propeptides cleaved from the amino-termini of the TGF-β precursor proteins. In contrast, the propeptides of other TGF-β superfamily members do not appear to be inhibitory. In this investigation, we demonstrate that purified recombinant GDF-8 propeptide associates with purified recombinant GDF-8 to form a noncovalent complex, as evidenced by size exclusion chromatography and chemical crosslinking analysis. Furthermore, we show that GDF-8 propeptide inhibits the biological activity of GDF-8 assayed on A204 rhabdomyosarcoma cells transfected with a (CAGA)₁₂ reporter construct. Finally, we demonstrate that GDF-8 propeptide inhibits specific GDF-8 binding to L6 myoblast cells. Collectively, these data identify the GDF-8 propeptide as an inhibitor of GDF-8 biological activity.     

VEGF受体结合抑制肽的筛选及其特性鉴定

目的　筛选能抑制VEGF与血管内皮细胞表面受体结合的VEGF模拟短肽 ,探讨小分子短肽作为血管内皮细胞生长抑制剂、肿瘤疫苗的可行性。方法　以抗 VEGF单克隆抗体分别对噬菌体随机 7肽、12肽库进行筛选。通过硝酸纤维膜斑点印迹法进行阳性克隆鉴定。用噬菌体克隆免疫小鼠检验这种短肽可否模拟VEGF的抗原决定簇诱发机体产生能与VEGF结合的抗体 ,研究小鼠抗血清、噬菌体表达的 7肽和 12肽以及人工合成 7肽对人脐静脉血管内皮细胞 (HUVEC)生长的抑制作用。结果　对肽库进行 3轮筛选后 ,噬菌体克隆具有良好的富集效果。硝酸纤维膜斑点印迹法证实阳性率达到 10 0 % ,测序结果显示噬菌体上的短肽有共同序列 :GWYYDAL、VASAVFYSALVE。这两种噬菌体短肽免疫小鼠能激发产生高效价的抗 VEGF抗体。噬菌体表达的 7肽和 12肽对HUVEC的生长有明显的抑制作用。由短肽GWYYDAL阳性克隆免疫的抗血清对VEGF诱导的内皮细胞生长起抑制作用 ,人工合成的 7肽 (GWYYDAL)呈剂量依赖性阻拮VEGF促HUVEC增殖作用。结论　从噬菌体肽库中筛选到的噬菌体短肽GWYYDAL、VASAVFYSALVE模拟了VEGF的抗原表位 ,为研究VEGF受体结合抑制肽和设计肿瘤疫苗奠定了实验基础。     

Suppression of Toll-like receptor 4 activation by endogenous oxidized phosphatidylcholine, KOdiA-PC by inhibiting LPS binding to MD2

Objective

Activation of Toll-like receptor 4 (TLR4) triggers immune and inflammatory events by sensing endogenous danger signals as well as invading pathogens and contributes to the development of chronic inflammatory diseases. In this study, we investigated effect of 1-palmitoyl-2-(5-keto-6-octenedioyl)-sn-glycero-3-phosphocholine (KOdiA-PC), an oxidized phosphatidylcholine, on TLR4 activation and the underlying regulatory mechanism.

Methods

RAW264.7 macrophages were used for the study. The levels of TNF-α, IFN-β, and COX-2 mRNA and protein were determined by quantitative PCR and ELISA, respectively. Activation of TLR4-signaling was examined by immunoblot and luciferase reporter assays. In vitro binding assay was performed to determine LPS binding to MD2. Macrophage migration was analyzed using a transwell-culture system.

Results

KOdiA-PC prevented the activation of TLR4-signaling components including ERK, JNK, p38, NF-κB, and IRF3 leading to decrease of TNF-α, IFN-β, and COX-2 expression. In vitro binding assay revealed that KOdiA-PC interrupted LPS binding to MD2, a TLR4 co-receptor. Consistently, KOdiA-PC suppressed LPS-induced macrophage migration.

Conclusion

The results demonstrate that KOdiA-PC can modulate TLR4 activation by regulating ligand-receptor interaction. Therefore, endogenously generated, oxidized phospholipids may play a role in resolving inflammation by terminating TLR activation and macrophage recruitment to the inflamed site.     

BCL‐XL binds and antagonizes RASSF6 tumor suppressor to suppress p53 expression

RASSF6, a member of the tumor suppressor Ras‐association domain family proteins, induces apoptosis in the caspase‐dependent and caspase‐independent manners. RASSF6 interacts with MDM2 and stabilizes p53. BCL‐XL is a prosurvival member of BCL‐2 family proteins. BCL‐XL directly inhibits proapoptotic BAX and BAK. BCL‐XL also traps tBID, a proapoptotic activator BH3‐only protein, and sequesters p53. In addition, BCL‐XL regulates the mitochondrial membrane permeability via voltage‐dependent anion channel. In these manners, BCL‐XL plays an antiapoptotic role. We report the interaction of BCL‐XL with RASSF6. BCL‐XL inhibits the interaction between RASSF6 and MDM2 and suppresses p53 expression. Consequently, BCL‐XL antagonizes RASSF6‐mediated apoptosis. Thus, the inhibition of RASSF6‐mediated apoptosis also underlies the prosurvival role of BCL‐XL.     

Relationship of cholecystokinin receptor binding to regulation of biological functions in pancreatic acini

Cholecystokinin (CCK) was conjugated to 125I-Bolton-Hunter reagent (125I-BH-CCK), and the binding of this ligand to CCK receptors in isolated mouse pancreatic acini was correlated with the regulation by CCK of both amylase release and the transport of 2-deoxyglucose and alpha-aminoisobutyric acid. Stimulation of amylase release by CCK was biphasic. At low CCK concentrations (less than 200 pM), amylase release was progressively stimulated, whereas at higher CCK concentrations (greater than 200 pM), amylase release was progressively reduced. In contrast, stimulation of 2-[3H]deoxyglucose transport and inhibition of alpha-[3H]aminoisobutyric acid transport were monophasic, being one-half maximal at 0.85 and 0.44 nM, respectively. Under incubation conditions identical to those employed for measuring biological functions, the binding of 125I-BH-CCK to receptors in acini was rapid and reversible. Competition-inhibition curves and Scatchard plots of equilibrium binding were compatible with two orders of binding sites. Employing a computer program for analysis of multiple binding sites, a high-affinity, low-capacity binding component having a Kd of 26 pM and a lower-affinity, higher-capacity binding component having a component Kd of 2.2 nM were resolved. Regulation of 2-[3H]deoxyglucose and alpha-[3H]aminoisobutyric acid uptake appeared, therefore, to be the result of fractional occupancy of the lower-affinity CCK receptors. Regulation of amylase released was more complex and appeared to be due to the concomitant occupancy of both the high- and low-affinity CCK receptors.     

Inhibition of urokinase activity and prevention of urokinase receptor binding by monoclonal antibodies

Two murine monoclonal antibodies produced against human urokinase-type plasminogen activator were characterized with respect to their antigen-binding specificity and their effects on urokinase activity and urokinase receptor binding. One of the antibodies binds to the protease domain of urokinase (Kass = 2.1 X 10(7) M-1). Antibody binding inhibits catalysis of plasminogen activation. It does not, however, affect amidolytic activity of urokinase towards the chromogenic substrate D-Val-Leu-Arg-p-nitroanilide. The antibody thus appears to interfere with plasminogen binding without directly affecting catalytically active amino acid residues of the enzyme. The other antibody binds to the aminoterminal fragment of urokinase (Kass = 1.0 X 10(7) M-1) and prevents binding of the enzyme to high affinity receptors on human granulocytes. Binding of this antibody neither influences plasminogen activation nor the amidolytic activity of urokinase. Both antibodies are potentially useful for the further analysis and manipulation of urokinase function.     

Suramin blocks the binding of interleukin-1 to its receptor and neutralizes IL-1 biological activities

This report demonstrates the ability of the anti-cancer drug suramin to interfere with the binding of interleukin (IL)-1 to its receptor and to inhibit IL-1-induced biological activities. In a radioreceptor cell based assay, suramin inhibits the binding of IL-1 α to several murine cell lines expressing predominantly type I and type II IL-1 receptors. Affinity cross-linking experiments using IL-1 α and EL-4.6.1 cells confirms that suramin inhibits the binding of the ligand to the kDa IL-1 type I receptor. In contrast, suramin fails to displace significantly prebound IL-1. In a cell-free system, suramin prevents the binding of IL-1 α and IL-1 β to murine and human recombinant soluble type I IL-1 receptors. For example, the IC₅₀ for suramin inhibiting IL-1 α and IL-1 β binding to soluble human IL-1 receptor were 204 μM and 186 μM, respectively. The suramin analogues, NF-058 and NF-103 (which bear the same number of sulfate groups as suramin), are between three-and ten-fold less active than suramin in inhibiting IL-1 binding to EL-4.6.1 cells, and to recombinant soluble IL-1 receptor. Furthermore, in a dose-dependent manner suramin prevents several IL-1 mediated biological responses, including thymocyte proliferation, PGE-2 synthesis and IL-6 production. The inhibitory effect of the drug can be significantly reversed by the addition of excess cytokine. Taken together, the results indicate that suramin is a competitive IL-1 receptor antagonist. Because IL-1 participates in a broad range of immunological and inflammatory functions, the data suggest that suramin administration may influence important activities beyond those associated strictly with tumor inhibition.     

Characterization of a monoclonal antibody inhibiting the biological activity of human chorionic gonadotrophin

A mouse monoclonal antibody (MCA), raised against human chorionicgonadotrophin (HCG) and coded 130A, was characterized with severaltypes of immunoassay and with in-vitro and in-vivo bioassays.MCA 130A binds strongly to the intact HCG molecule but lessso to either -or -subunit; The dissociation constant of theMCA-HCG complex was found to be in the order of 70 pmol/l. MCA130A inhibits the biological activity of HCG very effectively,both in vitro and in vivo. In a competitive immunoassay MCA130A binds 30 x better to HCG than to human luteinizing hormone(HLH). The selectivity in the bioassays was much higher, e.g.in vitro, 50% inhibition of HLH-stimulated testosterone productionrequires 2600 x as much MCA as is needed for inhibiting HCG-stimulatedproduction. Possible reasons for this difference in selectivityare discussed     

Correlation between epitopes on hemagglutinin of measles virus and biological activities: passive protection by monoclonal antibodies is related to their hemagglutination inhibiting activity

Measles virus monoclonal antibodies (MoAbs) were used to investigate the structure of the hemagglutinin (H) antigen, in order to study the regions of the molecule implicated in protection. Using a competition binding assay, three overlapping domains were defined, and these have been correlated with the biological activities of their corresponding MoAb. All of the anti-H MoAbs, with a single exception, neutralized virus infectivity in vitro. We investigated their capacity in passive protection using a measles virus-mouse model, in which inoculated newborn mice died of an acute encephalitis. The course of the disease was monitored after passive administration of the MoAbs, and from their activity, these MoAbs could be divided in three groups: I--protective, II--inducer of a retarded disease, III--nonprotective. The isotype of the antibody did not play a direct role in determining the course of the disease. Moreover, we were able to correlate protection with biological activity of the MoAbs. Only the MoAbs which inhibit hemagglutination activity (HI) protected against the acute disease. Measles MoAbs which neutralize canine distemper virus (CDV) in vitro failed to passively protect CDV-infected mice against disease. These results suggest an immune mechanism for in vivo protection different from that implicated in in vitro neutralization. Administration of one MoAb (55) led to a retarded neurological disease. Mice receiving lower quantities of other protective MoAbs did not display such disease. These results are discussed in relationship to immunization and protection.     

Myostatin (GDF-8) inhibits chondrogenesis and chondrocyte proliferation in vitro by suppressing Sox-9 expression

Here, we investigate a possible direct role for myostatin in chondrogenesis. First, we examined the effects of myostatin on the proliferation of bone marrow stromal cells (BMSCs) and epiphyseal growth plate (EGP) chondrocytes (EGPCs) isolated from myostatin-deficient mice. Results show that myostatin deficiency is associated with a significant (P?相似文献   

Myostatin (GDF-8) inhibits chondrogenesis and chondrocyte proliferation in vitro by suppressing Sox-9 expression

Here, we investigate a possible direct role for myostatin in chondrogenesis. First, we examined the effects of myostatin on the proliferation of bone marrow stromal cells (BMSCs) and epiphyseal growth plate (EGP) chondrocytes (EGPCs) isolated from myostatin-deficient mice. Results show that myostatin deficiency is associated with a significant (P < 0.001) increase in proliferation of both BMSCs (+25%) and EGPCs (+35%) compared with wild-type cells. Next, we examined the effects of myostatin treatment on chondrogenic differentiation of BMSCs. These experiments show that myostatin treatment starting at either 0 or 48 h induces a significant decrease in collagen type II protein synthesis by 31% (P < 0.001) and 25% (P < 0.05), respectively. Real-time PCR reveals significant (P < 0.01) down regulation of Sox9 mRNA expression with 10 and 100 ng/ml treatments. Together, these findings suggest that myostatin has direct effects on chondrogenesis, and may, therefore, represent a potential therapeutic target for improving bone repair.     

N G-nitro-l-arginine antagonizes endothelium-dependent dilator responses by inhibiting endothelium-derived relaxing factor release in the isolated rabbit heart

The effects of a recently described inhibitor of endothelial NO synthesis, N ^G-nitro-l-arginine (l-NNA), on the vasomotor responses to endothelium-dependent and independent vasodilators, and on the release of endothelium-derived relaxing factor (EDRF), were studied in the isolated saline-perfused rabbit heart. Infusion of l-NNA (30 M) resulted in a 52±12% increase in basal coronary perfusion pressure. The vasomotor responses to 1 M acetylcholine (ACh) and serotonin after l-NNA became biphasic, showing a small transient dilation followed by a pronounced vasoconstriction. In contrast, the dilation observed with sodium nitroprusside was not affected by l-NNA. None of the above-mentioned effects was elicited by the Stereo-isomer d-NNA. Similarly, an increase in the basal coronary perfusion pressure by endothelin-1 (0.3 nM) to the same level as observed with l-NNA did not alter the vasomotor responses to ACh and sodium nitroprusside. The increase in cyclic GMP (cGMP) in platelets passing through the coronary vascular bed was used as an index of EDRF release. Platelet cGMP amounted to 0.50±0.10 pmol/mg protein after passage through the coronary bed of the unstimulated heart. When platelets were injected during an ACh infusion (1 M), a 2.7 fold increase in cGMP was observed (P<0.01). After a 30-min infusion with l-NNA, the cGMP content of platelets passing through the unstimulated heart was reduced by 62%. Likewise, the ACh-induced increase in platelet cGMP was totally blocked. These results show that l-NNA inhibits EDRF release, and is thus a potent and selective inhibitor of EDRF-mediated dilation in the isolated rabbit heart.     

Mullerian inhibiting substance binding and uptake.

Mullerian inhibiting substance (MIS) is a 140,000 M(r) Sertoli cell derived glycoprotein with a critical regulatory role in the male fetus initiated presumably by ligand binding with receptor. To localize this binding species we performed time course incubations of cultured fetal rat lungs or control tissues with MIS, applied rabbit anti-MIS IgG, and fluorescein conjugated anti-rabbit IgG, and examined specimens with laser confocal microscopy. Punctate surface fluorescence followed by cytosolic and nuclear localization in lung consistent with specific adsorptive endocytosis was seen. Confocal imaging also detected MIS binding to the Mullerian duct in the urogenital ridge. Crosslinking of 125I-MIS with plasma membranes revealed a high molecular mass binder with signal displaceable by excess unlabeled ligand. These data support the hypothesis that a specific plasma membrane binding protein for MIS exists.     

Fesselin binds to actin and myosin and inhibits actin-activated ATPase activity

Fesselin is an actin binding protein that bundles actin filaments and accelerates nucleation of actin polymerization. The effect of fesselin on actin polymerization is regulated by Ca⁺⁺-calmodulin. Because actin filaments serve both structural and contractile functions we also examined the effect of fesselin on activation of myosin S1 ATPase activity. Fesselin inhibited the activation of S1-catalyzed ATP hydrolysis in a similar manner in both the presence and absence of tropomyosin. This inhibition was unaffected by Ca⁺⁺-calmodulin. Fesselin inhibited the binding of myosin-S1 to actin during steady-state ATP hydrolysis. Fesselin also displaced caldesmon from actin. S1 displaced fesselin from actin in the absence of nucleotide when the affinity of S1 for actin was much greater than the affinity of fesselin for actin. It is likely that fesselin and S1 share common binding sites on F-actin. We also observed that fesselin could bind to smooth muscle myosin with μM affinity. Fesselin shares some similarities to caldesmon in binding to several other proteins and having multiple potential functions. Parts of this work were presented in preliminary form at the 45th Annual Biophysical Society Meeting, Baltimore, MD, February 2004 and the 46th Annual Meeting, Long Beach, CA, February 2005.     

68A new way of targeting protease-receptor binding: Selection of uPA binding RNA aptamers inhibiting its association to its receptor uPAR

The binding of urokinase-type plasminogen activator (uPA) to its cell surface receptor (uPAR) has been implicated in tumour spread. We have employed a new method for developing inhibitors of the binding. Systematic evolution of ligands by exponential enrichment (SELEX) is an approach to selecting for RNA or DNA oligonucleotides with specific properties. Selections can be performed from a large pool of random sequences (10¹⁵) of RNA or DNA oligonucleotides which fold into three-dimensional structures. It is then possible to select RNA or DNA oligonucliotides binding with high affinities to targets of interest. These binding specific oligonucleotides are called aptamers. In a SELEX experiment, we used a library of serum-stable 2'-fluoro-pyrimidine modified RNA oligonucleotides to select for RNA aptamers capable of binding to urokinase-type plasminogen activator (uPA). Analysed by surface plasmon resonance (SPR), the selected aptamers bind to the amino terminal fragment of human uPA with K_D -values in the low nanomolar range. Cell binding assays showed that the selected aptamers were able to inhibit the binding of the amino terminal fragment of uPA to the receptor uPAR expressed on U937 cells. The same conclusion was reached by SPR analyse. The aptamers showed no inhibition of uPA's proteolytic activity. Based on computerised secondary structure predictions, it was possible to reduce size of the aptamers down to 49 nucleotides without losing the inhibitory properties. uPA-binding aptamers could be a promising tool for interfering with the pathophysiological functions of the plasminogen activation system. The inhibition of the uPA-uPAR interaction will inhibit plasminogen activation on the cells surface without interfering with the other function of uPAR and uPA. Aptamers are also interesting tools for analytic and imaging purpose because of the small size and high affinities.     

Cyclothiazide binding to the GABAA receptor

In order to explore the molecular interaction between cyclothiazide (CTZ) and gamma-aminobutyric acidA (GABAA) receptors, possibly underlying inhibition of GABAA receptor currents, [3H]-CTZ was synthesized. Binding of [3H]-CTZ to rat brain synaptic membranes could be observed only in the presence of the GABAA receptor antagonist (-)[1S,9R]-bicuculline methiodide (BMI) (EC(50,BMI)=500+/-80microM). GABA decreased [(3)H]-CTZ binding induced by the presence 300microM and 3mM BMI with IC(50,GABA) values of 300+/-50microM and 5.0+/-0.7mM, respectively. Binding of CTZ to [3H]-CTZ labeled sites was characterized by IC(50,CTZ) values of 0.16+/-0.03muM ([BMI]=300microM) and 7.0+/-0.5microM ([BMI]=3mM). Binding of the diastereomeric fraction [3H]-(3R,1'S,4'S,5'R+3S,1'R,4'R,5'S)-CTZ induced by 3mM BMI was quantitatively the more significant in cerebrocortical and hippocampal membranes. It was characterized by IC(50,CTZ)=80+/-15nM and IC(50,GABA)=13+/-3mcapital EM, Cyrillic. In the absence of BMI, CTZ (1mM) significantly decreased GABA-induced enhancement of [3H]-flunitrazepam binding. Our findings suggest that functional inhibition may occur through binding of CTZ to an allosteric site of GABAA receptors. This allosteric site is possibly emerged in the receptor conformation, stabilized by BMI binding.     

抗人Toll样受体4单克隆抗体的制备及其生物活性研究

目的运用单克隆抗体技术制备抗人Toll样受体4(TLR4)单克隆抗体(mhTLR4抗体),并鉴定其生物学活性。方法以重组人TLR4(rhTLR4)作为抗原,腹腔注射免疫Balb/c小鼠。分离小鼠脾脏B淋巴细胞与骨髓瘤细胞融合并培养,挑选阳性杂交瘤细胞,扩大培养,制备和鉴定mhTLR4其生物学效应。结果经3次免疫后的小鼠血清中抗rhTLR4抗体的效价具有较高水平的表达。1∶500的小鼠免疫血清可明显抑制人PBMC的TNF-α表达。分离小鼠脾细胞与骨髓瘤细胞进行融合,获得3株阳性单克隆杂交瘤细胞。其中2株经过增殖培养、纯化浓缩制备出较高产量的鼠抗人TLR4单克隆抗体。生物学活性测定表明,产生的抗rhTLR4单克隆抗体,能够明显地阻断脂多糖(LPS)诱导的人PBMC细胞对TNF-α的表达。结论本文制备的鼠抗人TLR4单克隆抗体,经鉴定具有较高的阻断内毒素信号传导的效应。为进一步研制人源化抗TLR4抗体、研制新一代抗内毒素靶向药物奠定了基础。