Mitigation of oxidative stress and inflammatory factors, along with the antibrowning and antimicrobial effects of cassia seed microbial fermentation solution.

Introduction: Cassia seeds, originating from the mature seeds of leguminous cassia species, possess pharmacological effects attributed to their rich composition of various active ingredients, notably anthraquinones. While current research predominantly focuses on pharmaceutical extractions, there has been limited progress in fermentation studies. Methods: Our study aimed to enhance the content of active compounds such as anthraquinones, flavonoids, and polyphenols using microbial fermentation techniques. We specifically optimized a fermentation system through a single-factor experimental design. Results: The antioxidant properties of the fermentation solution were validated through assays involving HaCaT cells and zebrafish. We observed effective suppression of inflammatory reactions in both RAW264.7 cells and transgenic zebrafish by the fermentation solution. Moreover, significant inhibition of tyrosinase activity and melanin production was evident in B16-F10 cells and zebrafish. Positive outcomes were also obtained in antibacterial assays and chick embryo experiments. Discussion: These findings highlight the potential of cassia seed fermentation solution as a safe and eco-friendly material in food chemistry and biomedical sciences.

Screening, cloning, immobilization and application prospects of a novel ß-glucosidase from the soil metagenome.

The soil environment for straw return is a rich and valuable library containing many microorganisms and proteins. In this study, we aimed to screen a high-quality ß-glucosidase (BGL) from the soil metagenomic library and to overcome the limitation of the low extraction rate of resveratrol in Polygonum cuspidatum. This includes the construction of a soil metagenomic library, screening of BGL, bioinformatics analysis, cloning, expression, immobilization, enzymatic property analysis, and application for the transformation of polydatin. The results showed that the soil metagenomic library of straw return was successfully constructed, and a novel BGL was screened. The identified 1356 bp long BGL belonged to the glycoside hydrolase 1 (GH1) family and was named Bgl1356. After successful cloning and expression of Bgl1356, it was immobilized using chitosan. The optimum temperature of immobilized Bgl1356 was 50 °C, and the pH was 5. It exhibited good tolerance for various metal ions (CO2+, Ni2+, Cu2+, Mn2+, Na2+, Ca2+, and Ag+) and organic solvents (DMSO, Triton-X-10, and ethanol). Enzymatic kinetics assays showed that Bgl1356 had good affinity for the substrate, and the specific enzyme activity was 234.03 U/mg. The conversion rate of polydatin by immobilized Bgl1356 was 95.70 ± 1.08%, facilitating the production of high amounts of resveratrol. Thus, this paper reports a novel temperature-, organic solvent-, and metal ion-tolerant BGL that has good application prospects in the pharmaceutical industry.

Corrigendum: Improvement of antioxidant activity and active ingredient of Dendrobium officinale via microbial fermentation.

[This corrects the article DOI: 10.3389/fmicb.2023.1061970.].

Improvement of antioxidant activity and active ingredient of Dendrobium officinale via microbial fermentation.

This study used brewer's yeast to ferment Dendrobium officinale and single-factor and orthogonal experiments were conducted to determine the optimal fermentation conditions. The antioxidant capacity of Dendrobium fermentation solution was also investigated by in vitro experiments, which showed that different concentrations of fermentation solution could effectively enhance the total antioxidant capacity of cells. The fermentation liquid was found to contain seven sugar compounds including glucose, galactose, rhamnose, arabinose, and xylose using gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-Q-TOF-MS), with the highest concentrations of glucose and galactose at 194.628 and 103.899 µg/ml, respectively. The external fermentation liquid also contained six flavonoids with apigenin glycosides as the main structure and four phenolic acids including gallic acid, protocatechuic acid, catechol, and sessile pentosidine B.

NF-κB Activator 1 downregulation in macrophages activates STAT3 to promote adenoma-adenocarcinoma transition and immunosuppression in colorectal cancer.

BACKGROUND: Adenoma-adenocarcinoma transition is a key feature of colorectal cancer (CRC) occurrence and is closely regulated by tumor-associated macrophages (TAMs) and CD8+ T cells. Here, we investigated the effect of the NF-κB activator 1 (Act1) downregulation of macrophages in the adenoma-adenocarcinoma transition. METHODS: This study used spontaneous adenoma-developing ApcMin/+, macrophage-specific Act1-knockdown (anti-Act1), and ApcMin/+; anti-Act1 (AA) mice. Histological analysis was performed on CRC tissues of patients and mice. CRC patients' data retrieved from the TCGA dataset were analyzed. Primary cell isolation, co-culture system, RNA-seq, and fluorescence-activated cell sorting (FACS) were used. RESULTS: By TCGA and TISIDB analysis, the downregulation of Act1 expression in tumor tissues of CRC patients negatively correlated with accumulated CD68+ macrophages in the tumor. Relative expression of EMT markers in the tumor enriched ACT1lowCD68+ macrophages of CRC patients. AA mice showed adenoma-adenocarcinoma transition, TAMs recruitment, and CD8+ T cell infiltration in the tumor. Macrophages depletion in AA mice reversed adenocarcinoma, reduced tumor amounts, and suppressed CD8+ T cell infiltration. Besides, macrophage depletion or anti-CD8a effectively inhibited metastatic nodules in the lung metastasis mouse model of anti-Act1 mice. CRC cells induced activation of IL-6/STAT3 and IFN-γ/NF-κB signaling and the expressions of CXCL9/10, IL-6, and PD-L1 in anti-Act1 macrophages. Anti-Act1 macrophages facilitated epithelial-mesenchymal-transition and CRC cells' migration via CXCL9/10-CXCR3-axis. Furthermore, anti-Act1 macrophages promoted exhaustive PD1+ Tim3+ CD8+ T cell formation. Anti-PD-L1 treatment repressed adenoma-adenocarcinoma transition in AA mice. Silencing STAT3 in anti-Act1 macrophages reduced CXCL9/10 and PD-L1 expression and correspondingly inhibited epithelial-mesenchymal-transition and CRC cells' migration. CONCLUSIONS: Act1 downregulation in macrophages activates STAT3 that promotes adenoma-adenocarcinoma transition via CXCL9/10-CXCR3-axis in CRC cells and PD-1/PD-L1-axis in CD8+ T cells.

Effects of novel cellulase (Cel 906) and probiotic yeast fermentation on antioxidant and anti-inflammatory activities of vine tea (Ampelopsis grossedentata).

Vine tea (Ampelopsis grossedentata) is a plant resource with good nutritional and medicinal, and is widely consumed in China. This study aimed to develop a functional vine tea fermentation broth using microbial fermentation and cellulase degradation. First, the most suitable probiotics for vine tea fermentation were screened, and the fermentation conditions were optimized. Then, a new cellulase (Cel 906, MW076177) was added to evaluate the changes in the contents of effective substances and to study its efficacy. The results show that saccharomyces cerevisiae Y-401 was identified as the best strain, the optimal fermentation conditions were a time of 94.60 h, feeding concentration of 115.21 g/L, and temperature of about 34.97°C. The vine tea fermentation broth has a strong inhibitory ability on 2,2'-azinobis3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (99.73%), peroxyl (53.15%), superoxide anion radicals (84.13%), and 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) (92.48%). It has a decent inhibitory impact on the cell viability, tyrosinase activity (32.25%), and melanin synthesis (63.52%) of B16-F10 melanoma cells induced by α-MSH. Inflammatory cell recruitment was reduced in a zebrafish inflammation model. Therefore, this vine tea fermented broth has strong antioxidant, anti-melanoma, and anti-inflammatory effects, and has healthcare potential as a probiotic tea.

Expression, characterization, and immobilization of a novel SGNH esterase Est882 and its potential for pyrethroid degradation.

The widely-used pyrethroid pesticides have attracted public attention because of their potentials to cause environmental pollution and toxic effects on non-target organisms. Esterase is a kind of hydrolytic enzyme that can catalyze the cleavage or formation of ester bonds. it plays a pivotal role in the decomposition of pyrethroids and esters containing industrial pollutants through the hydrolysis of ester bonds. Here, a new esterase gene est882 was successfully screened, which encodes Est882, a SGNH family esterase composed of 294 amino acids. It was heterogeneously expressed, identified and immobilized. Multiple sequence alignment showed that Est882 had a typical GDS(X) conserved motif and a catalytic triad composed of Ser79, Asp269 and His275. Phylogenetic analysis showed that Est882 shall belong to a new esterase family. Biochemical characterization demonstrated that the optimum condition was 40°C and pH 9.0. Est882 immobilization was studied with mesoporous silica SBA-15 as the carrier and found to significantly improve the tolerance and stability of Est882. Its optimum pH increased to 10.0 and stabilized within pH 8.0-11.0. Free Est882 can effectively degrade various pyrethroids within 30 min, with a degradation rate above 80%. The immobilized Est882 yet degraded more than 70% of pyrethroids within 30 min. The present study indicated that Est882 has outstanding potential in bioremediation of a pyrethroid-polluted environment. These characteristics endow Est882 with potential values in various industrial applications and hydrolysis of pyrethroid residues.

Slit2/Robo1 Mitigates DSS-induced Ulcerative Colitis by Activating Autophagy in Intestinal Stem Cell.

Ulcerative colitis (UC) is a recurrent intestinal inflammatory disease. Slit2, a secreted protein, interacts with its receptor Robo1 to regulate the differentiation of intestinal stem cells and participate in inflammation and tumor development. However, whether Slit2/Robo1involved in the pathogenesis of UC is not known. We investigated Slit2/Robo1-mediated UC using a dextran sodium sulfate (DSS)-induced model. Eight-week-old male Slit2-Tg (Slit2 transgene) mice, Robo1/2+/- (Robo1+/- Robo2+/-) mice, and their WT littermates were allocated into two groups: (I) control group (n=10), of mice fed a normal diet and tap water and (II) DSS group (n=10), of mice fed a normal diet and drinking water with 2% DSS for 7 days. Colon tissues were collected and analyzed by qPCR, immunohistochemistry, western blot, and immunofluorescence. Slit2-Tg DSS mice showed less body weight loss, less blood in the stool, and less viscous stool compared to those of WTSlit DSS mice. Robo1/2+/- DSS mice displayed a heavier degree of blood in the stool and a more apparent viscosity of the stool compared to those of WTRobo1/2 DSS mice. Slit2 overexpression maintained Lgr5+ stem cell proliferation in the crypt after DSS treatment, significantly increased the LC3II/I ratio, and slightly stimulated p62 expression in the crypt compared to those of DSS-induced WTSlit mice. Robo1/2 partial knockout reduced the number of Lgr5+ stem cells, decreased the LC3II/I ratio, and markedly increased p62 expression in the crypt compare to those of DSS-treated WTRobo1/2 mice. Our findings suggest that Slit2/Robo1 mediates DSS-induced UC probably by activating the autophagy of Lgr5+ stem cells.

Author Correction: Phosphoinositide 3-Kinase (PI3K) Subunit p110δ Is Essential for Trophoblast Cell Differentiation and Placental Development in Mouse.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

The metabonomics study of P-selectin glycoprotein ligand-1 (PSGL-1) deficiency inhibiting the progression of atherosclerosis in LDLR-/- mice.

Atherosclerosis (AS) is a multi-factorial chronic disease commonly associated with the mechanisms of metabolism disorder, endothelial dysfunction and chronic inflammation. AS an inflammatory molecule, p-selectin glycoprotein ligand-1 (PSGL-1) played an important role in the inflammatory process of atherogenesis involving the recruitment of leukocyte and transmitting signals to activate leukocyte during the adhesion process. So far, there has been little study regarding the effects of PSGL-1 on AS progression and the metabolic regulation. In this report, we studied the effect of PSGL-1 deficiency on the formation and progression of AS and the metabolic regulation by use of LDLR-/-, PSGL-1-/- transgenic mice based on metabonomics. It was found that the PSGL-1 deficiency reduced the atherosclerotic plaque area, inflammatory cells infiltration and fiber hyperplasia during the AS development. The serum metabonomics study showed that the LDLR-/- ,PSGL-1-/- mice had higher levels of HDL, valine, acetate, pyruvate, choline, PC, GPC and glycine, and lower levels of LDL+VLDL and lactate at the early stage of atherosclerosis, while lactate, citrate and glutamine showed statistical significance at the late stage of atherosclerosis. These results showed that the PSGL-1 deficiency inhibited the AS progression and regulated glucose metabolism, lipid metabolism, amino acid and phospholipid metabolism in LDLR-/- mice.

Phosphoinositide 3-Kinase (PI3K) Subunit p110δ Is Essential for Trophoblast Cell Differentiation and Placental Development in Mouse.

Maternal PI3K p110δ has been implicated in smaller litter sizes in mice, but its underlying mechanism remains unclear. The placenta is an indispensable chimeric organ that supports mammalian embryonic development. Using a mouse model of genetic inactivation of PI3K p110δ (p110δ(D910A/D910A)), we show that fetuses carried by p110δ(D910A/D910A) females were growth retarded and showed increased mortality in utero mainly during placentation. The placentas in p110δ(D910A/D910A) females were anomalously anemic, exhibited thinner spongiotrophoblast layer and looser labyrinth zone, which indicate defective placental vasculogenesis. In addition, p110δ was detected in primary trophoblast giant cells (P-TGC) at early placentation. Maternal PI3K p110δ inactivation affected normal TGCs generation and expansion, impeded the branching of chorioallantoic placenta but enhanced the expression of matrix metalloproteinases (MMP-2, MMP-12). Poor vasculature support for the developing fetoplacental unit resulted in fetal death or gross growth retardation. These data, taken together, provide the first in vivo evidence that p110δ may play an important role in placental vascularization through manipulating trophoblast giant cell.

Activation of Slit2-Robo1 signaling promotes liver fibrosis.

BACKGROUND & AIMS: The secretory protein Slit2 and its receptor Robo1 are believed to regulate cell growth and migration. Here, we aimed to determine whether Slit2-Robo1 signaling mediates the pathogenesis of liver fibrosis. METHODS: Serum levels of Slit2 in patients with liver fibrosis were determined by ELISA. Liver fibrosis was induced in wild-type (WT), Slit2 transgenic (Slit2-Tg) and Robo1(+/-)Robo2(+/-) double heterozygotes (Robo1/2(+/-)) mice by carbon tetrachloride (CCl4). The functional contributions of Slit2-Robo1 signaling in liver fibrosis and activation of hepatic stellate cells (HSCs) were investigated using primary mouse HSCs and human HSC cell line LX-2. RESULTS: Significantly increased serum Slit2 levels and hepatic expression of Slit2 and Robo1 were observed in patients with liver fibrosis. Compared to WT mice, Slit2-Tg mice were much more vulnerable to CCl4-induced liver injury and more readily develop liver fibrosis. Development of hepatic fibrosis in Slit2-Tg mice was associated with a stronger hepatic expression of collagen I and α-smooth muscle actin (α-SMA). However, liver injury and hepatic expression of collagen I and α-SMA were attenuated in CCl4-treated Robo1/2(+/-) mice in response to CCl4 exposure. In vitro, Robo1 neutralizing antibody R5 and Robo1 siRNA downregulated phosphorylation of Smad2, Smad3, PI3K, and AKT in HSCs independent of TGF-ß1. R5 and Robo1 siRNA also inhibited the expression of α-SMA by HSCs. Finally, the protective effect of R5 on the CCl4-induced liver injury and fibrosis was further verified in mice. CONCLUSIONS: Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs.

Glipizide suppresses embryonic vasculogenesis and angiogenesis through targeting natriuretic peptide receptor A.

Glipizide, a second-generation sulfonylurea, has been widely used for the treatment of type 2 diabetes. However, it is controversial whether or not glipizide would affect angiogenesis or vasculogenesis. In the present study, we used early chick embryo model to investigate the effect of glipizide on angiogenesis and vasculogenesis, which are the two major processes for embryonic vasculature formation as well as tumor neovascularization. We found that Glipizide suppressed both angiogenesis in yolk-sac membrane (YSM) and blood island formation during developmental vasculogenesis. Glipizide did not affect either the process of epithelial to mesenchymal transition (EMT) or mesoderm cell migration. In addition, it did not interfere with separation of smooth muscle cell progenitors from hemangioblasts. Moreover, natriuretic peptide receptor A (NPRA) has been identified as the putative target for glipizide׳s inhibitory effect on vasculogenesis. When NPRA was overexpressed or activated, blood island formation was reduced. NPRA signaling may play a crucial role in the effect of glipizide on vasculogenesis during early embryonic development.

¹H NMR based serum metabolic profiles associated with pathological progression of pancreatic islet ß cell tumor in Rip1-Tag2 mice.

Pancreatic islet ß cell tumor is the most common islet cell tumor. A well-characterized tumor progression in Rip1-Tag2 mice undergoes five stages, involving normal, hyperplasia, angiogenic islets, tumorigenesis and invasive carcinoma. (1)H NMR based metabonomics was applied to identify potential biomarkers for monitoring pancreatic islet ß cell tumor progression in Rip1-Tag2 mice. Multivariate analysis results showed the serum metabonome at hyperplasia stage shared the similar characteristics with the ones at normal stage as a result of slight proliferation of pancreatic islet ß cells. At angiogenic islets stage, the up-regulated glycolysis, disturbed choline and phospholipid metabolism composed the metabolic signature. In addition to the changes mentioned above, several metabolites were identified as early biomarkers for tumorigenesis, including increased methionine, citrate and choline, and reduced acetate, taurine and glucose, which suggested the activated energy and amino acid metabolism. All the changes were aggravated at invasive carcinoma stage, coupled with notable changes in alanine, glutamate and glycine. Moreover, the distinct metabolic phenotype was found associated with the implanting of SV40 large T antigen in Rip1-Tag2 mice. The combined metabolic and multivariate statistics approach provides a robust method for screening the biomarkers of disease progression and examining the association between gene and metabolism.

Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/ß-catenin pathway.

Slit2 is often overexpressed in cancers. Slit2 is a secreted protein that binds to Roundabout (Robo) receptors to regulate cell growth and migration. Here, we employed several complementary mouse models of intestinal cancers, including the Slit2 transgenic mice, the ApcMin/+ spontaneous intestinal adenoma mouse model, and the DMH/DSS-induced colorectal carcinoma model to clarify function of Slit2/Robo1 signaling in intestinal tumorigenesis. We showed that Slit2 and Robo1 are overexpressed in intestinal tumors and may contribute to tumor generation. The Slit2/Robo1 signaling can induce precancerous lesions of the intestine and tumor progression. Ectopic expression of Slit2 activated Slit2/Robo1 signaling and promoted tumorigenesis and tumor growth. This was mediated in part through activation of the Src signaling, which then down-regulated E-cadherin, thereby activating Wnt/ß-catenin signaling. Thus, Slit2/Robo1 signaling is oncogenic in intestinal tumorigenesis.

Inactivation of PI3Kδ induces vascular injury and promotes aneurysm development by upregulating the AP-1/MMP-12 pathway in macrophages.

OBJECTIVE: An aneurysm is an inflammatory vascular condition. Phosphatidylinositol 3-kinases δ is highly expressed in leukocytes, and play a key role in innate immunity. However, the link between phosphatidylinositol 3-kinases δ and aneurysm development has not yet been elucidated. APPROACH AND RESULTS: Carotid ligation unexpectedly induced characteristic aneurysm formation beneath the ligation point in p110δ(D910A/D910A) mice (n=25; P<0.001 versus wild-type). Besides, p110δ inactivation exacerbated CaCl2-induced abdominal aortic aneurysms development. A reverse transcription polymerase chain reaction microarray revealed significant extracellular matrix components degradation and matrix metalloproteinases (MMPs) upregulation in the abdominal aorta of p110δ(D910A/D910A) mice. Similarly, the expression of both collagen I and IV was significantly decreased (n=10; P<0.05 versus wild-type) in carotid artery. Western blot assay confirmed that MMP-12 was significantly upregulated in arteries of p110δ(D910A/D910A) mice (n=10; P<0.01 versus wild-type). In vitro, p110δ inactivation marked increase peritoneal macrophages recruitment and synergistically enhance tumor necrosis factor-α-induced recruitment. A specific phosphatidylinositol 3-kinases δ inhibitor (IC87114) or genetic p110δ inactivation upregulated MMP-12 expression and c-Jun phosphorylation (n=6; P<0.05 versus wild-type macrophages). IC87114 also increased activator protein-1 DNA-binding activity (n=6; P<0.001 versus control) and enhanced the effect of tumor necrosis factor-α on activator protein-1-binding activity (n=5; P<0.01 versus tumor necrosis factor-α treatment groups). Knockdown of c-Jun suppressed the effect of the IC87114 and tumor necrosis factor-α on MMP-12 mRNA expression (n=5 in each group; P<0.01 versus scrRNA treatment groups). CONCLUSIONS: Our findings demonstrate that p110δ inactivation leads to extracellular matrix degradation in vessels and promotes aneurysm development by inducing macrophages migration and upregulating the activator protein-1/MMP-12 pathway in macrophages.

Increased permeability of the blood-brain barrier and Alzheimer's disease-like alterations in slit-2 transgenic mice.

Alzheimer's disease (AD) is a progressive neurological disorder that primarily affects memory, and its prevalence is rising. Increasing evidence suggests that dysfunction of the blood-brain barrier (BBB) may be involved in AD and other neurodegenerative diseases. Herein, we report that the permeability of the BBB is increased and that AD-like alterations are present in Slit-2 overexpressing transgenic mice. We found that behavioral change and the corresponding molecular diagnostic markers of AD, such as hippocampal neuron apoptosis, amyloid-ß (Aß) protein deposition, and acetylcholinesterase expression, were increased in the Slit-2 transgenic mice. Moreover, the endothelial cells were dysfunctional, the size of the lateral ventricle cavity increased, and the permeability of the BBB increased. Additionally, there was an increased serum level of glutamate indicating that the BBB is related to AD. Finally, histopathological analysis of other organs in the Slit-2 overexpressing mice did not show any marked abnormalities. These findings demonstrate that Slit2 overexpression may be responsible for AD-like alterations and the increased BBB permeability in these mice. Our study provides a potential novel mechanism for the development of AD.

Andrographolide inhibits melanoma tumor growth by inactivating the TLR4/NF-κB signaling pathway.

The TLR4/NF-κB signaling pathway plays a critical role in tumor progression. Andrographolide (Andro) has been reported to have anticancer activity in multiple types of cancer. However, the pharmacological activities of Andro in melanoma are not completely understood. In this study, we defined the anticancer effects of Andro in melanoma and elucidated its potential mechanisms of action. Our experiments showed that Andro significantly inhibited melanoma tumor growth and metastasis by inducing cell cycle arrest and apoptosis. In addition, Andro significantly inhibited the TLR4/NF-κB signaling pathway. Furthermore, the inactivation of TLR4/NF-κB signaling inhibited the mRNA and protein expression of CXCR4 and Bcl-6, which are antitumor genes. This work provides evidence that the TLR4/NF-κB signaling pathway is a potential therapeutic target and may also be indispensable in the Andro-mediated anticancer effect in melanoma.

Inhibition of angiogenesis by a synthetic fusion protein VTF derived from vasostatin and tumstatin.

The inhibition of angiogenesis represents a potential strategy for antitumor therapy. A novel synthetic fusion protein VTF, composed of bioactive fragments from two different angiogenesis inhibitors, vasostatin and tumstatin with a (Gly-Ser-Gly)2 bridge, was generated using the pET-15b expression vector. The fusion protein VTF showed significantly enhanced efficacy in inhibiting human endothelial cell proliferation and tube formation and neovascularization on chick embryo chorioallantoic membrane. Moreover, VTF suppressed the growth of B16 melanoma and the formation of tumor blood vessels potently in vivo. These results indicated that the fusion protein containing the bioactive fragments of multiple angiogenesis inhibitors might be a promising therapeutic agent for tumor treatment.

Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library.

Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.