1.Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans.
Longbing YANG ; Zhuqing TIAN ; Luoxiong ZHOU ; Chaoqin SUN ; Mingjiao HUANG ; Chunren TIAN ; Jian PENG ; Guo GUO
Chinese Journal of Biotechnology 2023;39(1):304-317
Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC80 values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.
Humans
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Candida albicans
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Antimicrobial Peptides
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Proteomics
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Peptides/pharmacology*
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Transcription Factors/metabolism*
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Antifungal Agents/pharmacology*
2.Screening of peptide inhibitors of acetylcholinesterase from 12-mer random phage display peptide library.
Xing-mei ZHANG ; Yu-sheng SHI ; Chun-xia WANG
Journal of Southern Medical University 2006;26(7):1053-1054
OBJECTIVETo screen the peptide inhibitor of acetylcholinesterase (AChE) from 12-mer random phage display peptide library.
METHODSHuman AChE was used as the target to screen its binding peptides from 12-mer random phage display peptide library. The positive phage clones were isolated after three rounds of biopanning followed then by sequence analysis and their activity evaluation.
RESULTSSix positive phage clones binding to human AChE were obtained, and 4 of them sharing the conservative sequence W(S/P)HY inhibited the enzyme activity of AChE.
CONCLUSIONAcquisition of AChE inhibitor from phage display library provides clues for designing peptide inhibitors of AChE.
Acetylcholinesterase ; metabolism ; Cholinesterase Inhibitors ; metabolism ; pharmacology ; Humans ; Peptide Library ; Peptides ; metabolism ; pharmacology ; Protein Binding
3.The protection of scorpion venom derived activity peptide against the change of dynorphin in the early Parkinson's disease rats.
Dong-mei WANG ; Dan ZHAO ; Sheng-ming YIN ; Dong AN ; Wei CHEN ; De-qin YU ; Hong XU ; Jie ZHAO ; Wan-qin ZHANG ; Yu-xiang TIAN
Chinese Journal of Applied Physiology 2015;31(2):120-122
Animals
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Dynorphins
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metabolism
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Parkinson Disease
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drug therapy
;
metabolism
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Peptides
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pharmacology
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Rats
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Scorpion Venoms
;
pharmacology
4.Binding characteristics between RGD-containing cyclic peptide and rat hepatic stellate cells: an in vitro study.
Shi-lin DU ; Ji-yao WANG ; Wei-yue LU
Chinese Journal of Hepatology 2005;13(5):362-365
OBJECTIVETo investigate the binding characteristics between an artificial Arg-Gly-Asp (RGD)-containing cyclic peptide [cyclo(CGRGDSPK)] and rat hepatic stellate cells (HSC).
METHODSAn artificial RGD-containing cyclic peptide was labeled with fluorescein isothiocyanate (FITC). HSCs were isolated by collagenase in situ liver recirculating and purified by density gradient centrifugation from normal rats. The cells were cultured for 5 days of primary culture (quiescent phenotype) or for 7 days of secondary culture (activated phenotype). To access the binding and uptake, HSCs were incubated with FITC-cRGD of different concentrations at 4 degree C or 37 degree C, and then the binding and uptake were investigated by flow cytometry. The location of FITC-cRGD in HSC was investigated by fluorescent microscopy. Kd and maximal binding sites per cell were calculated by radioligand binding assay (RBA) of receptors using 3H-cRGD. In the interim, FITC-cAGA was used as a peptide control devoid of any binding site.
RESULTSThe binding between FITC-cRGD and HSC was saturable, time- and dose-dependent and could compete with overdosed unlabeled cRGD. The fluorescence was mainly distributed in cytoplasma, especially near the nuclei. Kd was 7.05 x 10(-9) mol/L and Bmax per cell was nearly 6.79 x 10(5).
CONCLUSIONSThe results demonstrate that cRGD are specifically taken up by HSC through a receptor-mediated pathway. The information is useful for understanding the ligand-receptor interaction of HSC. FITC labeled cyclic RGD-peptides meet the standards of special ligands and FITC does not change the binding activation of cyclic RGD-peptides.
Animals ; Binding Sites ; Cells, Cultured ; Hepatocytes ; cytology ; metabolism ; Oligopeptides ; pharmacology ; Peptides, Cyclic ; pharmacology ; Protein Binding ; Rats
5.Effects of humanin on elevation of intracellular calcium concentration induced by beta-amyloid peptide(31-35) in cultured cortical neurons..
Ling-Min LI ; Jian-Tian QIAO ; Ce ZHANG
Acta Physiologica Sinica 2009;61(2):127-131
The disruption of the intracellular Ca(2+) homeostasis has been reported to be one of the mechanisms of beta-amyloid (Abeta) neurotoxicity in Alzheimeros disease (AD). Abeta(31-35), a small active fragment of Abeta, is believed to possess the similar biological activities of full-length Abeta molecule. Humanin (HN) is a recently identified peptide that suppresses neuronal death initiated by AD-related insults. The present study was to investigate the effects of HN on Abeta(31-35)-induced elevation of [Ca(2+)](i) in cultured cortical neurons by real-time fluorescence imaging technique using the Ca(2+)-sensitive dye, Fura-2/AM. The elevation of [Ca(2+)](i) was observed in cultured neurons exposed to Abeta(31-35) (25 mumol/L) (F340/F380: 1 042.56+/- 83.54, compared with control group: 804.73+/- 48.230, P<0.05, n=10). Pretreatment of HN (10 mumol/L) for 10 min significantly decreased the elevation of [Ca(2+)](i) induced by Abeta(31-35) (25 mumol/L) (F340/F380: 918.788+/- 50.73, compared with Abeta(31-35) group, P<0.05, n=10). When neurons were treated with HN and Abeta(31-35) simultaneously, HN (10 mumol/L) could not change the elevation of [Ca(2+)](i) induced by Abeta(31-35) (F340/F380: 1 036.68+/- 88.96, compared with Abeta(31-35) group, P>0.05, n=10), while HN (20 mumol/L) diminished the elevation of [Ca(2+)](i) induced by Abeta(31-35) (25 mumol/L) significantly (F340/F380: 898.56+/- 76.46, compared with Abeta(31-35) group, P<0.05, n=10). The findings imply that: (1) the disruption of the calcium homeostasis induced by Abeta(31-35) is possibly the basis of the neurotoxicity of Abeta(31-35) in cultured cortical neurons; (2) HN suppresses the elevation of [Ca(2+)](i) induced by Abeta(31-35) in a dose- and time-dependent manner.
Amyloid beta-Peptides
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pharmacology
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Calcium
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metabolism
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Cell Death
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Cells, Cultured
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Homeostasis
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Humans
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Intracellular Signaling Peptides and Proteins
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pharmacology
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Neurons
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drug effects
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Peptide Fragments
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pharmacology
6.The anti-thrombotic activity of surfactins.
Jong hwan LIM ; Byung kwon PARK ; Myoung seok KIM ; Mi Hyun HWANG ; Man Hee RHEE ; Seung Chun PARK ; Hyo in YUN
Journal of Veterinary Science 2005;6(4):353-355
Platelet aggregation was inhibited and the density of platelet-rich plasma (PRP) clots was decreased by the preincubation of PRP with surfactins, an acidic lipopeptide of Bacillus subtilis complex BC1212 isolated from soybean paste, in dose-dependent manner. Our findings suggest that surfactins are able to prevent a platelet aggregation leading to an inhibition of additional fibrin clot formation, and to enhance fibrinolysis with facilitated diffusion of fibrinolytic agents.
Bacillus subtilis/metabolism
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Blood Platelets/drug effects
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Fibrinolytic Agents/*pharmacology
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Humans
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Peptides, Cyclic/isolation&purification/*pharmacology
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Platelet Aggregation Inhibitors/pharmacology
7.Effects of oncostatin M on hormone release of rat pituitary cells in primary culture.
Dong Sun KIM ; Ho Soon CHOI ; Yong Soo PARK ; Tae Wha KIM
Journal of Korean Medical Science 2000;15(3):323-326
It has become increasingly clear that cytokines play an important role in modulating neuroendocrine regulation, especially in the secretion of corticotropin (ACTH) in the pituitary. Oncostatin M (OSM), a cytokine of IL-6 family has been reported to increase ACTH secretion and pro-opiomelanocortin (POMC) transcription in murine corticotroph pituitary tumor cells (AtT20 cells). The present study was undertaken to determine the effects of OSM on hormonal release in primary culture of rat pituitary cells. Growth hormone or prolactin release was not affected by OSM. OSM (1 nM) stimulated ACTH release (35.1% increase versus control, p>0.001) in dispersed pituitary cells of rat to a lesser extent than in AtT20 cells. Corticotropin releasing hormone (CRH) (10 nM) also induced a 2.3-fold increase of ACTH secretion (p>0.001), but co-treatment of OSM and CRH did not exhibit any synergistic effect on ACTH secretion. We conclude OSM has a stimulatory effect on ACTH secretion in normal rat pituitary cell cultures, and OSM acts mainly on corticotroph, supporting the potential role of OSM to modulate immune-endocrine regulation in the pituitary.
Animal
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Cells, Cultured
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Corticotropin/secretion*
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Cytokines/pharmacology
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Cytokines/metabolism*
;
Inflammation Mediators/pharmacology
;
Inflammation Mediators/metabolism*
;
Male
;
Peptides/pharmacology
;
Peptides/metabolism*
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Pituitary Gland/metabolism*
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Pituitary Gland/drug effects
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Pituitary Gland/cytology
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Prolactin/secretion*
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Rats
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Rats, Inbred WF
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Somatotropin/secretion*
8.Effects of oncostatin M on hormone release of rat pituitary cells in primary culture.
Dong Sun KIM ; Ho Soon CHOI ; Yong Soo PARK ; Tae Wha KIM
Journal of Korean Medical Science 2000;15(3):323-326
It has become increasingly clear that cytokines play an important role in modulating neuroendocrine regulation, especially in the secretion of corticotropin (ACTH) in the pituitary. Oncostatin M (OSM), a cytokine of IL-6 family has been reported to increase ACTH secretion and pro-opiomelanocortin (POMC) transcription in murine corticotroph pituitary tumor cells (AtT20 cells). The present study was undertaken to determine the effects of OSM on hormonal release in primary culture of rat pituitary cells. Growth hormone or prolactin release was not affected by OSM. OSM (1 nM) stimulated ACTH release (35.1% increase versus control, p>0.001) in dispersed pituitary cells of rat to a lesser extent than in AtT20 cells. Corticotropin releasing hormone (CRH) (10 nM) also induced a 2.3-fold increase of ACTH secretion (p>0.001), but co-treatment of OSM and CRH did not exhibit any synergistic effect on ACTH secretion. We conclude OSM has a stimulatory effect on ACTH secretion in normal rat pituitary cell cultures, and OSM acts mainly on corticotroph, supporting the potential role of OSM to modulate immune-endocrine regulation in the pituitary.
Animal
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Cells, Cultured
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Corticotropin/secretion*
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Cytokines/pharmacology
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Cytokines/metabolism*
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Inflammation Mediators/pharmacology
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Inflammation Mediators/metabolism*
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Male
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Peptides/pharmacology
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Peptides/metabolism*
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Pituitary Gland/metabolism*
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Pituitary Gland/drug effects
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Pituitary Gland/cytology
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Prolactin/secretion*
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Rats
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Rats, Inbred WF
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Somatotropin/secretion*
9.Protective effect of cyclophilin A against Alzheimer's amyloid beta-peptide (25-35)-induced oxidative stress in PC12 cells.
Yu-Song GE ; Wei-Yu TENG ; Chao-Dong ZHANG
Chinese Medical Journal 2009;122(6):716-724
BACKGROUNDbeta-amyloid peptide (Abeta) is considered responsible for the pathogenesis of Alzheimer's disease (AD). Possible mechanisms underlying Abeta-induced neuronal cytotoxicity include excessive production of reactive oxidative species (ROS) and apoptosis. Cyclophilin A (CypA), exhibits antioxidant properties and protects neurons against oxidative stress induced injury. This study was conducted to demonstrate whether CyPA added to cultured PC12 cells could alleviate Abeta-induced oxidative stress and protect them from apoptosis.
METHODSPC12 cells were pre-incubated for 30 minutes with recombinant human cyclophilin A (rhCyPA) in 0.1 nmol/L, 1.0 nmol/L, 10 nmol/L and 100 nmol/L and then incubated with 10 micromol/L Abeta(25-35). In every group, cell viability, apoptotic morphology, apoptotic rate, intracellular ROS accumulation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of PC12 cells and mitochondrial transmembrane potential were detected. Subsequently, the expression of the active form of caspase-3 was determined by Western blotting.
RESULTSIt was shown that cultures treated with 1.0 nmol/L, 10 nmol/L or 100 nmol/L rhCyPA + Abeta(25-35) had significantly higher cell viability and a lower rate of apoptosis compared with the cultures exposed only to Abeta(25-35). In addition, rhCyPA attenuated Abeta(25-35)-induced overproduction of intracellular ROS and Abeta(25-35)-induced a decrease in activity of the key antioxidant enzymes SOD and GSH-Px. Furthermore, rhCyPA also attenuated Abeta(25-35)-induced mitochondrial dysfunction and the activation of caspase-3.
CONCLUSIONCyPA may act as an ROS scavenger, and prevent Abeta(25-35)-induced neurotoxicity through attenuating oxidative stress induced by Abeta(25-35).
Amyloid beta-Peptides ; pharmacology ; Animals ; Caspase 3 ; metabolism ; Cyclophilin A ; pharmacology ; Glutathione Peroxidase ; metabolism ; Humans ; Oxidative Stress ; drug effects ; PC12 Cells ; Peptide Fragments ; pharmacology ; Rats ; Superoxide Dismutase ; metabolism
10.Effect of exendin-4 on monocyte chemoattractant protein-1 expression in cultured rat glomerular mesangial cells induced by tumor necrosis factor-α in vitro.
Yingjuan JIANG ; Yaoming XUE ; Qian ZHANG ; Yanfei ZHANG ; Yuan YUAN
Journal of Southern Medical University 2013;33(6):930-933
OBJECTIVETo investigate the effect of exendin-4 on the expression of monocyte chemoattractant protein-1 (MCP-1) and fibronectin (FN) in rat glomerular mesangial cells in vitro.
METHODSRat glomerular mesangial cells were divided into 5 groups, namely control group, tumor necrosis factor-α (TNF-α) group (10 ng/ml), TNF-α (10 ng/ml)+E1 (1 nmol/L exendin-4) group, TNF-α (10 ng/ml)+E5 (5 nmol/L exendin-4) group, and TNF-α (10 ng/ml)+E10 (10 nmol/L exendin-4) group. After cultured 24 h or 48 h, RNA were extracted to determine the expression of MCP-1 with real-time PCR, the supernatant were collected to determine the expression of MCP-1 and FN with ELISA.
RESULTSCompared with control group, the cells treated with TNF-α for 24 h showed significantly increase the expression of MCP-1 and FN (P<0.01), exendin-4 significantly reduced the expression of MCP-1 and FN in TNF-α+E5 group and TNF-α+E10 group (P<0.05). After 48h incubation, the expression of MCP-1 and FN increased significantly in TNF-α group (P<0.01), which was lowered by exendin-4 in TNF-α+E1,TNF-α+E5 and TNF-α+E10 groups (P<0.05).
CONCLUSIONExendin-4 has an intrinsic capability to concentration- and time-dependently inhibit TNF-α-induced expression of MCP-1 and FN in rat mesangial cells, suggesting the beneficial effect of exendin-4 in preventing and treating diabetic nephropathy.
Animals ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Diabetic Nephropathies ; metabolism ; Glomerular Mesangium ; cytology ; Mesangial Cells ; drug effects ; metabolism ; Peptides ; pharmacology ; Rats ; Tumor Necrosis Factor-alpha ; pharmacology ; Venoms ; pharmacology