Biotinylated peptides substituted with D-amino acids with high stability as anti-anaphylactic agents targeting platelet-activating factor.

Platelet-activating factor (PAF) is an important lipid mediator of anaphylaxis and therefore can be an anti-anaphylactic agent target. Recently, we reported that several synthetic biotinylated peptides containing a Tyr-Lys-Asp-Gly sequence markedly inhibited the bioactivities of PAF in vitro and in vivo; it also inhibited anaphylactic reactions such as hypothermia, hypotension, and vascular permeability in vivo. Here, we report the anti-anaphylactic effects of three biotinylated heptapeptides (peptide 1: H-Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp-OH, peptide 2: H- -Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp-OH, and peptide 3: H- -Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly- -Asp-OH).

Estrogen Sulfotransferase is Highly Expressed in Vascular Endothelial Cells Overlying Atherosclerotic Plaques.

Cytosolic estrogen sulfotransferase (SULT1E1) mainly catalyzes the sulfoconjugation and deactivation of estrogens that are known to exert potent anti-atherogenic effects. However, it remains unknown about the connection between SULT1E1 and atherosclerosis. Recently, we reported that SULT1E1 is highly expressed in the aorta with plaques of high fat-fed ApoE knockout (KO) mice (mouse model of atherosclerosis), and interacts with oxidized low-density lipoprotein (Ox-LDL) known as a major component of atherosclerotic lesions.

Royal Jelly Proteins Inhibit Macrophage Proliferation: Interactions with Native- and Oxidized-Low Density Lipoprotein.

Macrophage proliferation is known to correlate with macrophage accumulation in atherosclerotic plaque, and therefore its inhibition and secondary reduction of plaque inflammation may have therapeutic beneficial effects on atherosclerosis. Recently, we reported that a peptide corresponding to positions 41-51 of royalisin (which consists of 51 amino acid residues), a potent antibacterial protein contained in royal jelly (RJ), can specifically bind to oxidized LDL (Ox-LDL), a major components of atherosclerotic lesions. Here, we investigated the interaction of RJ proteins including royalisin with LDL and Ox-LDL.

Interaction of Native- and Oxidized-Low-Density Lipoprotein with Human Estrogen Sulfotransferase.

Cytosolic estrogen sulfotransferase (SULT1E) mainly catalyzes the sulfate conjugation of estrogens, which decrease atherosclerosis progression. Recently we reported that a YKEG sequence in human SULT1E1 (hSULT1E1) corresponding to residues 61-64 can bind specifically to oxidized low-density lipoprotein (Ox-LDL), which plays a major role in the pathogenesis of atherosclerosis; its major oxidative lipid component lysophosphatidylcholine (LPC), and its structurally similar lipid, platelet-activating factor (PAF). In this study, we investigated the effect of Ox-LDL on the sulfating activity of hSULT1E1.

Human estrogen sulfotransferase and its related fluorescently labeled decapeptides specifically interact with oxidized low-density lipoprotein.

Estrogen sulfotransferase (SULT1E) mainly catalyzes the sulfation of estrogens, which are known to prevent the pathogenesis of atherosclerosis. Recently, we found that peptides with a YKDG sequence specifically bind to oxidized low-density lipoprotein (Ox-LDL), which plays a major role in the pathogenesis of atherosclerosis. Here, we investigated the interaction between human SULT1E1 (hSULT1E1), which has a YKEG sequence (residues 61-64) unlike other human SULTs, and Ox-LDL.

A biotinylated peptide, BP21, alleviates hypotension in anaphylactic mice.

Platelet-activating factor (PAF) is known as an important mediator of anaphylaxis and, therefore, may possibly serve as a direct target for anti-anaphylactic drugs. We recently reported that a synthetic N-terminally biotinylated peptide, BP21, alleviates hypothermia and vascular hyperpermeability during anaphylactic reactions in a mouse model of anaphylaxis via the direct binding of a Tyr-Lys-Asp-Gly sequence in the peptide to PAF. In this study, we investigated the effect of BP21 on in vivo anaphylactic hypotension.

The interaction of β-glycoprotein I with lysophosphatidic acid in platelet aggregation and blood clotting.

β-Glycoprotein I (β-GPI) is a plasma protein that binds to oxidized low-density lipoprotein (LDL) and negatively charged substances, and inhibits platelet activation and blood coagulation. In this study, we investigated the interaction of β-GPI with a negatively charged lysophosphatidic acid (LPA) in platelet aggregation and blood clotting. Two negatively charged lysophospholipids, LPA and lysophosphatidylserine, specifically inhibited the binding of β-GPI to oxidized LDL in a concentration-dependent manner.

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee-royalisin-for specific detection of oxidized low-density lipoprotein.

The probes for detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox-LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox-LDL, we investigated the interaction (with ox-LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N-terminus to FITC in the presence of 6-amino-n-caproic acid (AC) linker, (FITC-AC)-royalisin P11, which contains both sequences, Phe-Lys-Asp and Asp-Lys-Tyr, similar to Tyr-Lys-Asp in (FITC)KP6.

A Method for In Vitro Measurement of Oxidized Low-Density Lipoprotein in Blood, Using Its Antibody, Fluorescence-Labeled Heptapeptide and Polyethylene Glycol.

Two oxidized forms of low-density lipoprotein (LDL), oxidized (Ox-LDL) and minimally modified (MM-LDL), and the immune complexes (LDL-ICs) that they form with their corresponding antibodies, play a major role in the pathogenesis of atherosclerosis. Recently, we reported that the heptapeptide KP6 (Lys-Trp-Tyr-Lys-Asp-Gly-Asp) coupled through its ε-amino group present on the N-terminal Lys to fluorescein isothiocyanate (FITC)- (FITC)KP6- binds specifically to Ox-LDL and MM-LDL, but not to native LDL. Here, to develop a novel method for measuring the levels of oxidatively modified LDL in blood, using (FITC)KP6, we analyzed the latter's binding with MM-LDL-IC and Ox-LDL-IC.

A biotinylated peptide, BP21, as a novel potent anti-anaphylactic agent targeting platelet-activating factor.

Platelet-activating factor (PAF) is an important mediator of anaphylaxis and is therefore an anti-anaphylactic drug target. We recently reported that synthetic N-terminally biotinylated peptides (BP4-BP29) inhibit PAF by directly interacting with PAF and its metabolite/precursor lyso-PAF. In this study, we investigated whether the biotinylated peptides can inhibit anaphylactic reactions in vivo.

Angiotensin II induces the aggregation of native and oxidized low-density lipoprotein.

Modifications of low-density lipoprotein (LDL), such as oxidation and aggregation, and angiotensin (Ang) peptides are involved in the pathogenesis of atherosclerosis. Here, we investigated the relationship between one of the Ang peptides, AngII, and two LDL modifications, oxidation and aggregation. Using polyacrylamide gel electrophoresis and aggregation assays, we noted that AngII markedly induced the aggregation of LDL and oxidized LDL (Ox-LDL), and bound to both the aggregated and non-aggregated forms.

A Fluorescence-Labeled Heptapeptide, (FITC)KP6, as an Efficient Probe for the Specific Detection of Oxidized and Minimally Modified Low-Density Lipoprotein.

Two oxidized forms of low-density lipoprotein (LDL), oxidized LDL (ox-LDL) and minimally modified LDL (MM-LDL), are believed to play a major role in the pathogenesis of atherosclerosis. Recently, we reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminus Lys to fluorescein isothiocyanate, (FITC)KP6, bound to ox-LDL but not to LDL. In the present study, we investigated whether (FITC)KP6 could be used as a fluorescent probe for the specific detection of MM-LDL and ox-LDL.

Angiotensin peptides attenuate platelet-activating factor-induced inflammatory activity in rats.

Angiotensin (Ang)--a peptide that is part of the renin-angiotensin system-induces vasoconstriction and a subsequent increase in blood pressure; Ang peptides, especially AngII, can also act as potent pro-inflammatory mediators. Platelet-activating factor (PAF) is a potent phospholipid mediator that is implicated in many inflammatory diseases. In this study, we investigated the effects of Ang peptides (AngII, AngIII, and AngIV) on PAF-induced inflammatory activity.

Biotinylated heptapeptides substituted with a D-amino acid as platelet-activating factor inhibitors.

Platelet-activating factor (PAF), a potent lipid mediator, is implicated in many inflammatory diseases, and therefore may serve as a direct target for anti-inflammatory drugs. We previously reported that synthetic biotinylated peptides having a Tyr-Lys-Asp-Gly sequence markedly inhibit PAF-induced inflammation by direct binding, and that two synthetic fluorescence-labelled heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp and D-Lys-Trp-Tyr-Lys-Asp-Gly-Asp) with high stability in plasma specifically bind to PAF-like lipids (oxidized- and lyso-phosphatidylchoine). In this study, synthetic heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp) coupled to a biotin molecule through the N-terminal amino group and ε-amino group of N-terminus Lys, (Btn)KP6 and K(Btn)P6, respectively, and their biotinylated peptides substituted with D-Lys at the N-terminus, (Btn)dKP6 and dK(Btn)P6, respectively, were investigated for their effects on PAF-induced inflammation.

C-reactive protein specifically enhances platelet-activating factor-induced inflammatory activity in vivo.

Platelet-activating factor (PAF) is a potent lipid mediator that is implicated in numerous inflammatory diseases. C-reactive protein (CRP) is an acute-phase plasma protein that increases rapidly and dramatically in response to inflammation. In this study, we investigated the effect of the interaction between CRP and PAF on inflammatory responses in vivo.

Highly stable, fluorescence-labeled heptapeptides substituted with a D-amino acid for the specific detection of oxidized low-density lipoprotein in plasma.

Probes that can detect oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques can be useful for the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that two heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled to fluorescein isothiocyanate (FITC) through the ε-amino group of N-terminus Lys in the absence/presence of 6-amino-n-caproic acid (AC) linker to FITC-(FITC)KP6 and (FITC-AC)KP6-can be useful as fluorescent probes for the specific detection of ox-LDL. In this study, to develop the fluorescent peptides with high plasma stability for the specific detection of ox-LDL, we investigated the interaction of (FITC)KP6 and (FITC-AC)KP6 substituted with D-Lys at the N-terminus-(FITC)dKP6 and (FITC-AC)dKP6-with ox-LDL, and the in vitro stability of these peptides in mouse plasma.

A synthetic biotinylated peptide, BP21, inhibits the induction of mRNA expression of inflammatory substances by oxidized- and lyso-phosphatidylcholine.

Preclinical Research Oxidized low-density lipoprotein (ox-LDL) is implicated in many inflammatory diseases, e.g., type 2 diabetes, obesity, atherosclerosis, and metabolic syndrome.

Novel fluorescently labeled peptide compounds for detection of oxidized low-density lipoprotein at high specificity.

The probes for specific detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. In this study, to develop a fluorescent peptide probe for specific detection of ox-LDL, we investigated the interaction of fluorescein isothiocyanate (FITC)-labeled peptides with ox-LDL using polyacrylamide gel electrophoresis. Two heptapeptides (KWYKDGD and KP6) coupled through the ε-amino group of K at the N-terminus to FITC in the presence/absence of 6-amino-n-caproic acid (AC) linker to FITC--(FITC-AC)KP6 and (FITC)KP6--both bound with high specificity to ox-LDL in a dose-dependent manner.

Common mechanism in endothelin-3 and PAF receptor function for anti-inflammatory responses.

Platelet-activating factor (PAF) is a potent lipid mediator that is implicated in numerous inflammatory diseases. Under inflammatory conditions, PAF is biosynthesized through the remodelling pathway and elicits many inflammatory responses through binding to its specific PAF receptor. Endogenous bioactive endothelins (ETs: ET-1, -2, and -3) are also considered potent inflammatory mediators that play a critical role in many inflammatory diseases.

An endothelin-3-related synthetic biotinylated pentapeptide as a novel inhibitor of platelet-activating factor.

Platelet-activating factor (PAF), a potent proinflammatory mediator, is involved in many inflammatory diseases. We recently reported that synthetic biotinylated peptides having a Tyr-Lys-Asp-Gly sequence inhibit PAF-induced inflammation by directly binding to PAF. In this study, we investigated the effect of two synthetic biotinylated peptides, both of which have a sequence similar to Tyr-Lys-Asp-Gly-an endothelin-3 (ET-3)-related biotinylated pentapeptide (Tyr-Lys-Asp-Lys-Glu, BPET3) and a scavenger receptor CD36-related biotinylated tetrapeptide (Tyr-Lys-Gly-Lys, BPCD36)-on PAF-induced inflammation by using a rat model of hind paw oedema.

Endothelins specifically recognize lysophosphatidylcholine micelles.

Lysophosphatidylcholine (LPC), a major phospholipid component of oxidized low-density lipoprotein (ox-LDL), is implicated in numerous inflammatory diseases, including atherosclerosis. Here, to clarify the relationship between bioactive endothelins (ETs) (which are considered to be potent proinflammatory mediators) and LPC/ox-LDL, we investigated the interaction between ETs and LPC/ox-LDL by fluorescence spectroscopy and western blotting. Tryptophan fluorescence measurements revealed ETs specifically interacted with LPC at concentrations that exceeded the critical micelle concentration (CMC).

Endothelin-3 at low concentrations attenuates inflammatory responses via the endothelin B2 receptor.

Objective: The aim of this study was to clarify a role of endothelins (ETs: ET-1, -2, and -3) and their receptors (ETA, ETB1, and ETB2) in inflammatory responses.

Methods: Male Wistar rats (180-220 g) were used. The effects of ETs in the absence or presence of the ETA antagonist BQ-123/the selective ETB2 antagonist BQ-788, and the effect of the selective ETB1 agonist IRL-1620 and the nonselective ETB agonist BQ-3020, on rat hind paw oedema induced by several proinflammatory substances were examined.

Synthetic biotinylated peptide compound, BP21, specifically recognizes lysophosphatidylcholine micelles.

Lysophosphatidylcholine, a major phospholipid component of oxidized low-density lipoprotein, is implicated in many inflammatory diseases, including atherosclerosis. We previously reported that Asp-hemolysin-related synthetic peptide (P21) composed of 21 amino acid residues markedly inhibits the bioactivities of oxidized low-density lipoprotein and lysophosphatidylcholine, by directly binding to oxidized low-density lipoprotein and lysophosphatidylcholine. Here, to clarify whether P21 specifically binds to lysophosphatidylcholine and what forms of lysophosphatidylcholine with which P21 interact, we investigated the interaction between P21 containing two tryptophan residues and lysophosphatidylcholine by using fluorescence spectroscopy, polyacrylamide gel electrophoresis, and surface plasmon resonance.

Synthetic biotinylated peptide compounds derived from Asp-hemolysin: novel potent inhibitors of platelet-activating factor.

Platelet-activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a potent inflammatory mediator, is implicated in many inflammatory diseases and may possibly serve as a direct target for anti-inflammatory drugs. We have previously reported that Asp-hemolysin-related synthetic peptides (P4-P29) inhibit the bioactivities of oxidized low-density lipoprotein (ox-LDL) containing PAF-like lipids by direct binding to ox-LDL, which plays a key role in the atherosclerotic inflammatory process. In this study, we investigated whether these peptides inhibit the bioactivities of PAF by binding to PAF and its metabolite/precursor lyso-PAF.