The synthetic peptide, His-Phe-Tyr-Leu-Pro-Met, is a chemoattractant for Jukat T cells

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+](i)) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanismclose3

Trp-Lys-Tyr-Met-Val-D-Met is a chemoattractant for human phagocytic cells

Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a synthetic peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes. The peptide binds to a unique cell surface receptor(s), Recently we had demonstrated that hunan neutrophils, monocytes, and B lymphocytes express this peptide-specific receptor and that stimulation of human leukocytes with the peptide leads to activation of the oxidative respiratory system and the bactericidal activity of neutrophils or monocytes. In this study we showed that the peptide induces chemotaxis of phagocytic leukocytes and studied the signaling pathway leading to chemotaxis in human monocytes. The peptide-induced monocyte chemotaxis is pertussis toxin (PTX)-sensitive. This fact correlates with the peptide's stimulation of PI hydrolysis and intracellular Ca2+ ([Ca2+](i)) release, which is also PTS-sensitive. We demonstrate that the peptide-specific receptor is different from receptor(s) for monocyte chemoattractant protein-1 (MCP-1). We also show that intracellular signaling of WKYMVm leading to monocyte chemotaxis is different from that of MCP-1, The peptide-mediated monocyte chemotaxis is insensitive to protein kinase C (PKC) inhibitor (GF109203X) and butan-1-ol, 1-ol, ruling out PKC and phospholipase D participation in this process. On the other hand, a tyrosine kinase inhibitor (genistein) and RhoA inhibitor (C3 transferase) curtailed the peptide-induced chemotaxis in a concentration-dependent mariner, implying the involvement of tyrosine kinase and RhoA, respectively. Treatment of human monocytes with the peptide stimulates tyrosine phosphorylation of several cellular proteins, including p125FAK and Pyk2 and translocation of RhoA from the cytosol to the membrane. We conclude that WKYMVm induces chemotaxis of human phagocytic leukocytes via unique receptors and signalingclose323

Independent functioning of cytosolic phospholipase A(2) and phospholipase D-1 in Trp-Lys-Tyr-Met-Val-D-Met-induced superoxide generation in human monocytes

Recently, a novel peptide (Trp-Lys-Tyr-Met-Val-D-Met, WKYMVm) has been shown to induce superoxide generation in human monocytes, The peptide stimulated phospholipase A(2) (PLA(2)) activity in a concentration- and time-dependent manner. Superoxide generation as well as arachidonic acid (AA) release evoked by treatment with WKYMVm could be almost completely blocked by pretreatment of the cells with cytosolic PLA(2) (cPLA(2))-specific inhibitors. The involvement of cPLA(2) in the peptide-induced AA release was further supported by translocation of cPLA(2) to the nuclear membrane of monocytes incubated with WKYMVm, WKYMVm-induced phosphatidylbutanol formation was completely abolished by pretreatment with PKC inhibitors. Immunoblot showed that monocytes express phospholipase D-1 (PLD1), but not PLD2, GF109203X as well as butan-1-ol inhibited peptide-induced superoxide generation in monocytes, Furthermore, the interrelationship between the two phospholipases, cPLA(2) and PLD1, and upstream signaling molecules involved in WKYMVm-dependent activation was investigated. The inhibition of cPLA(2) did not blunt peptide-stimulated PLD1 activation or vice versa, Intracellular Ca2+ mobilization was indispensable for the activation of PLD1 as well as cPLA(2), The WKYMVm-dependent stimulation of cPLA(2) activity was partially dependent on the activation of PKC and mitogen-activated protein kinase, while PKC activation, but not mitogen-activated protein kinase activation, was an essential prerequisite for stimulation of PLD,, Taken together, activation of the two phospholipases, which are absolutely required for superoxide generation, takes place through independent signaling pathways that diverge from a common pathway at a point downstream of Ca2+close343

Mechanistic understanding of insulin receptor modulation: Implications for the development of anti-diabetic drugs

The insulin receptor is an important regulator of metabolic processes in the body, and in particular of glucose homeostasis, including glucose uptake into peripheral tissues. Thus, insulin administration is an effective treatment for diabetes, which is characterized by chronic elevation of blood glucose. However, insulin is not only a metabolic regulator, but also functions as a growth hormone. Accordingly, studies of long-term insulin administration and of the hyperinsulinemia associated with type 2 diabetes have raised concerns about possible increases in the risks of cancer and atherosclerosis, due to excessive stimulation of cell proliferation. Interestingly, some insulin receptor ligands that have been developed based on a peptide, an antibody, and an aptamer selectively have metabolic effects exerted through the insulin receptor but do not cause significant cellular proliferation. Although these ligands therefore have potential as anti-diabetic drugs for advanced diabetes care, the mechanism whereby they specifically activate the insulin receptor is still unclear. Recently, studies of the structure of the insulin receptor have progressed considerably, and have provided further mechanistic understanding of insulin receptor activation. Based on this progress, we propose a mechanistic model of this specificity and discuss the potential for the development of novel anti-diabetic drugs that would not have the adverse effects caused by excessive mitogenic action.

The synthetic chemoattractant peptide, Trp-Lys-Tyr-Met-Val-D-Met, enhances monocyte survival via PKC-dependent Akt activation

Previously, we showed that Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) stimulates superoxide generation and chemotactic migration in monocytes and neutrophils. In this study, we examined the effect of WKYMVm on monocyte survival. Serum starvation-induced monocyte death was attenuated in the presence of WKYMVm, which was abated when the cells were preincubated with LY294002, suggesting the involvement of phosphoinositide-3-kinase (PI 3-kinase) in the peptide-induced monocyte survival. WKYMVm stimulated ERK and Akt activity via PI 3-kinase activation in monocytes. We also investigated the signaling pathway of WKYMVm-induced ERK and Akt activation. The WKYMVm-induced ERK activation was PI 3-kinase-dependent but PKC-independent. However, Akt activation by WKYMVm was dependent not only on PI 3-kinase but also on the PKC pathway. When monocytes were incubated with WKYMVm, caspase-3 activity, which is important for cell death, was inhibited. Pretreatment of the cells with LY294002, GF109203X, and Go 6976 but not PD98059 blocked WKYMVm-induced monocyte survival and caspase-3 inhibition. In summary, the novel chemoattractant WKYMVm enhances monocyte survival via Akt-mediated pathways, and in this process, PKC and PI 3-kinase act upstream of Aktclose111

Electrode asymmetry driven self-gating effect on the electrical detection of protein

Aptamer-immobilized gold nanoparticles (AuNP)/single-walled carbon nanotube (SWNT) field effect transistor (FET) sensors are fabricated for the detection of the ErbB2 protein. The 5'-thiol-modified 40-mer naphthyl DNA aptamers are immobilized on the AuNPs using well-established thiol chemistry. Carbodiimidazole-activated tween 20 (CDI-tween 20) layers are also introduced to the surface of the SWNT via hydrophobic interactions for blocking the attachment of nonspecific substances. Quantitative experiments reveal the specific binding effect of ErbB2 on the electrical measurements by comparing the electrical responses from a non-specific binding and control aptamer experiments. Under the optimized experimental conditions, the ErbB2 protein is clearly detectable at concentrations as low as 10 pM. The self-gating effect of the present device with a concentric electrode is able to provide a reliable electrical measurement scheme by circumventing the limits related to the isoelectric point of the target protein. Furthermore, the sensor fabrication, treatment and detection method have the potential to meet the following requirements: simplicity, portability, low cost and no need for bulky optical instruments. (C) 2013 Elsevier B.V. All rights reserved.11Nsciescopu