Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence.

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive.

Sodium activates human monocytes via the NADPH oxidase and isolevuglandin formation.

Aims: Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro.

Methods And Results: To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments.

Agonistic anti-CD148 monoclonal antibody attenuates diabetic nephropathy in mice.

CD148 is a transmembrane protein tyrosine phosphatase (PTP) that is expressed in the renal vasculature, including the glomerulus. Previous studies have shown that CD148 plays a role in the negative regulation of growth factor signals (including epidermal growth factor and vascular endothelial growth factor), suppressing cell proliferation and transformation. However, the role of CD148 in kidney disease remains unknown.

Arachidonic Acid Kills Staphylococcus aureus through a Lipid Peroxidation Mechanism.

infects every niche of the human host. In response to microbial infection, vertebrates have an arsenal of antimicrobial compounds that inhibit bacterial growth or kill bacterial cells. One class of antimicrobial compounds consists of polyunsaturated fatty acids, which are highly abundant in eukaryotes and encountered by at the host-pathogen interface.

Dendritic Cell Amiloride-Sensitive Channels Mediate Sodium-Induced Inflammation and Hypertension.

Sodium accumulates in the interstitium and promotes inflammation through poorly defined mechanisms. We describe a pathway by which sodium enters dendritic cells (DCs) through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel and the sodium hydrogen exchanger 1. This leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47, and association of p47 with gp91.

Comparative Kinetic Analysis of Closed-Ended and Open-Ended Porous Sensors.

Efficient mass transport through porous networks is essential for achieving rapid response times in sensing applications utilizing porous materials. In this work, we show that open-ended porous membranes can overcome diffusion challenges experienced by closed-ended porous materials in a microfluidic environment. A theoretical model including both transport and reaction kinetics is employed to study the influence of flow velocity, bulk analyte concentration, analyte diffusivity, and adsorption rate on the performance of open-ended and closed-ended porous sensors integrated with flow cells.

Determination of the CD148-Interacting Region in Thrombospondin-1.

CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148.

Accumulation of isolevuglandin-modified protein in normal and fibrotic lung.

Protein lysine modification by γ-ketoaldehyde isomers derived from arachidonic acid, termed isolevuglandins (IsoLGs), is emerging as a mechanistic link between pathogenic reactive oxygen species and disease progression. However, the questions of whether covalent modification of proteins by IsoLGs are subject to genetic regulation and the identity of IsoLG-modified proteins remain unclear. Herein we show that Nrf2 and Nox2 are key regulators of IsoLG modification in pulmonary tissue and report on the identity of proteins analyzed by LC-MS following immunoaffinity purification of IsoLG-modified proteins.

Immune activation caused by vascular oxidation promotes fibrosis and hypertension.

Vascular oxidative injury accompanies many common conditions associated with hypertension. In the present study, we employed mouse models with excessive vascular production of ROS (tg(sm/p22phox) mice, which overexpress the NADPH oxidase subunit p22(phox) in smooth muscle, and mice with vascular-specific deletion of extracellular SOD) and have shown that these animals develop vascular collagen deposition, aortic stiffening, renal dysfunction, and hypertension with age. T cells from tg(sm/p22phox) mice produced high levels of IL-17A and IFN-γ.

Renal Denervation Prevents Immune Cell Activation and Renal Inflammation in Angiotensin II-Induced Hypertension.

Rationale: Inflammation and adaptive immunity play a crucial role in the development of hypertension. Angiotensin II and probably other hypertensive stimuli activate the central nervous system and promote T-cell activation and end-organ damage in peripheral tissues.

Objective: To determine if renal sympathetic nerves mediate renal inflammation and T-cell activation in hypertension.

DC isoketal-modified proteins activate T cells and promote hypertension.

Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive γ-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs.

Porous silicon Bloch surface and sub-surface wave structure for simultaneous detection of small and large molecules.

A porous silicon (PSi) Bloch surface wave (BSW) and Bloch sub-surface wave (BSSW) composite biosensor is designed and used for the size-selective detection of both small and large molecules. The BSW/BSSW structure consists of a periodic stack of high and low refractive index PSi layers and a reduced optical thickness surface layer that gives rise to a BSW with an evanescent tail that extends above the surface to enable the detection of large surface-bound molecules. Small molecules were detected in the sensor by the BSSW, which is a large electric field intensity spatially localized to a desired region of the Bragg mirror and is generated by the implementation of a step or gradient refractive index profile within the Bragg mirror.

Biosensor design based on Marangoni flow in an evaporating drop.

Effective point-of-care diagnostics require a biomarker detection strategy that is low-cost and simple-to-use while achieving a clinically relevant limit of detection. Here we report a biosensor that uses secondary flows arising from surface Marangoni stresses in an evaporating drop to concentrate target-mediated particle aggregates in a visually detectable spot. The spot size increases with increasing target concentration within the dynamic range of the assay.

Thrombospondin-1 acts as a ligand for CD148 tyrosine phosphatase.

CD148 is a receptor-type protein tyrosine phosphatase that is expressed in several cell types, including vascular endothelial cells and duct epithelial cells. Growing evidence demonstrates a prominent role for CD148 in negative regulation of growth factor signals, suppressing cell proliferation and transformation. However, its extracellular ligand(s) remain unknown.

IQGAP1 is a novel CXCR2-interacting protein and essential component of the "chemosynapse".

Background: Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding.

Characterization of chemokine receptor CXCR2 interacting proteins using a proteomics approach to define the CXCR2 "chemosynapse".

Chemokine-receptor signaling is initiated upon ligand binding to the receptor and continues through the process of endocytic trafficking by the association of a variety of adaptor proteins with the chemokine receptor. In order to define the adaptor proteins that associate with CXCR2 before and after ligand activation, a protocol was developed using differentiated HL-60 cells transfected to express CXCR2 stimulated or not stimulated with ligand for one minute. CXCR2-associating proteins were isolated by immunoprecipitation with CXCR2 antibody and the eluted proteins were electrophoretically run into the separating gel directly without a stacking gel.

VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis.

Chemotaxis regulates the recruitment of leukocytes, which is integral for a number of biological processes and is mediated through the interaction of chemokines with seven transmembrane G-protein-coupled receptors. Several studies have indicated that chemotactic signaling pathways might be activated via G-protein-independent mechanisms, perhaps through novel receptor-interacting proteins. CXCR2 is a major chemokine receptor expressed on neutrophils.

Single chain fragment variable recombinant antibody functionalized gold nanoparticles for a highly sensitive colorimetric immunoassay.

In this report, the peptide linker connecting scFv V(H) and V(L) domains were genetically modified to contain different amino acids (i.e. cysteine (scFv-cys) or histidines (scFv-his)) to enable the scFv to adsorb or self-assemble onto the gold nanoparticles (NPs).

Engineering peptide linkers for scFv immunosensors.

Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs).

Identification of the protein targets of the reactive metabolite of teucrin A in vivo in the rat.

Covalent modification of proteins is associated with the toxicity of many electrophiles, and the identification of relevant in vivo protein targets is a desirable but challenging goal. Here, we describe a strategy for the enrichment of adducted proteins utilizing single-chain fragment variable (ScFv) antibodies selected using phage-display technology. Teucrin A is a furan-containing diterpenoid found in the herb germander that is primarily responsible for the herb's hepatotoxicity in rodents and humans following metabolic activation by cytochrome P450 enzymes.

Recombinant antibody piezoimmunosensors for the detection of cytochrome P450 1B1.

The phase I enzyme known as cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of many endogenous and exogenous compounds, including carcinogens. CYP1B1 is overexpressed in a wide variety of human diseases ranging from diabetes to malignancies, such as invasive breast cancer. Because of its microsomal location in the cell, CYP1B1 could not be measured directly by existing methods but only assessed indirectly via the determination of the catalytic products.

A monoclonal antibody against CD148, a receptor-like tyrosine phosphatase, inhibits endothelial-cell growth and angiogenesis.

Angiogenesis contributes to a wide range of neoplastic, ischemic, and inflammatory disorders. Definition of the intrinsic molecular controls in angiogenic vessel growth promises novel therapeutic approaches for angiogenesis-related diseases. CD148 (also named DEP-1/PTP eta) is a receptor-like protein tyrosine phosphatase that is abundantly expressed in vascular endothelial cells.

Engineered recombinant single-chain fragment variable antibody for immunosensors.

A recombinant single-chain fragment variable (scFv) antibody (designated A10B) was engineered to contain two histidines within the linker peptide used to join the scFv heavy and light chains. A piezoimmunosensor using the scFv was successfully developed. A10B scFv bound to the gold piezoimmunosensor surface were correctly oriented, retained antigen-binding activity, and coupled at high surface concentration.

Production and characterization of mouse ureteric bud cell-specific rat hybridoma antibodies utilizing subtractive immunization and high-throughput screening.

The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used.