Methods and Kit for Determination of Prostacyclin in Plasma

A solid-phase immunoassay for 6-keto-Prostaglandin F1α, the stable hydrolysis product of prostacyclin (Prostaglandin I2) is disclosed. Prostacyclin, a potent vasodilator with anti-platelet and anti-proliferative properties is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like syndrome. Levels of 6-keto-Prostaglandin F1α can be directly correlated with levels of prostacyclin. Therefore, 6-keto-Prostaglandin F1α has become the indicator of choice to measure prostacyclin levels. The single step immunoassay for 6-keto-Prostaglandin F1α uses the bioluminescent protein, aequorin as a label. Analyte-label conjugates were constructed by linking the carboxyl group of 6-keto-Prostaglandin F1α and lysine residues of aequorin by chemical conjugation methods. The binding properties of 6-keto-Prostaglandin F1α towards its antibody and the bioluminescent properties of aequorin are retained in the conjugate. The concentration of 6-keto-Prostaglandin F1α after extraction from plasma shows good correlation with the concentration of 6-keto-Prostaglandin F1α obtained without prior extraction of the same plasma sample. The assay allows the measurement of 6-keto-Prostaglandin F1α directly in plasma without any pre-treatment of the samples, which results in a much simpler method with a faster assay time

A Rapid Cellular FRET Assay of Polyglutamine Aggregation Identifies a Novel Inhibitor

AbstractMany neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC50 ≅ 5 μM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing

Expression and Affinity Purification of Recombinant Proteins from Plants

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, β-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system

Using Epitope–Aequorin Conjugate Recognition in Immunoassays for Complex Proteins

Recombinant techniques are used to fuse biologically important molecules or peptides to the N-terminus of the photoprotein aequorin such that the binding characteristics of the molecule and the bioluminescent activity of aequorin are retained. This work demonstrates that the peptide region of a bulky protein can be used to develop an assay for the protein. A heterogeneous competitive binding assay was first developed for HPC4 epitope, the binding region of protein C, using HPC4–apoaequorin conjugate. It was observed that the binding of HPC4 epitope to its monoclonal antibody and the bioluminescence properties of aequorin were retained in the fusion protein. The same strategy and the same fusion protein were used to develop the assay for protein C. This project could potentially be a model for large biomolecules utilizing only the binding region of the protein in the labeled analyte. Also, this assay can be used in clinical diagnostics for the quantitative detection of protein C

Responsive drug delivery system: New challenges to analytical chemists; Drug detection based on the conformational changes of calmodulin and the fluorescence of its enhanced green fluorescent protein fusion partner; Determination of prostacyclin in plasma through a bioluminescent immunoassay for 6-keto-prostaglandin F1alpha: implication of dosage in patients with primary pulmonary hypertension

This work describes a solid-phase immunoassay for 6-keto-prostaglandin F1alpha, the stable hydrolysis product of prostacyclin (prostaglandin I2). Prostacyclin, a potent vasodilator with antiplatelet and antiproliferative properties is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like syndrome. Levels of 6-keto- prostaglandin F1alpha can be directly correlated with levels of prostacyclin. Therefore, 6-keto-prostaglandin F1alpha, has become the indicator of choice to measure prostacyclin levels. The single-step immunoassay for 6-keto-prostaglandin F1alpha reported here was developed using the bioluminescent protein aequorin as a label. Analyte-label conjugates were constructed by linking the carboxyl group of 6-keto- prostaglandin F1alpha and lysine residues of aequorin by chemical conjugation methods. The binding properties of 6-keto-prostaglandin F1alpha toward its antibody and the bioluminescent properties of aequorin were retained in the conjugate, which was then used to generate a dose- response curve for the analyte in a convenient microtiter plate format. The concentration of 6-keto-prostaglandin F1alpha after extraction from plasma showed good correlation with the concentration of 6-ketoprostaglandin F1alpha obtained without prior extraction of the same plasma sample. This measurement demonstrated that the assay allows the measurement of 6-keto-prostaglandin F1alpha directly in plasma without any pretreatment of the samples, which results in a much simpler method with a faster assay time

Determination of prostacyclin in plasma through a bioluminescent immunoassay for 6-keto-prostaglandin F1alpha: implication of dosage in patients with primary pulmonary hypertension

This work describes a solid-phase immunoassay for 6-keto-prostaglandin F1alpha, the stable hydrolysis product of prostacyclin (prostaglandin I2). Prostacyclin, a potent vasodilator with antiplatelet and antiproliferative properties is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like syndrome. Levels of 6-keto-prostaglandin F1alpha can be directly correlated with levels of prostacyclin. Therefore, 6-keto-prostaglandin F1alpha, has become the indicator of choice to measure prostacyclin levels. The single-step immunoassay for 6-keto-prostaglandin F1alpha reported here was developed using the bioluminescent protein aequorin as a label. Analyte-label conjugates were constructed by linking the carboxyl group of 6-keto-prostaglandin F1alpha and lysine residues of aequorin by chemical conjugation methods. The binding properties of 6-keto-prostaglandin F1alpha toward its antibody and the bioluminescent properties of aequorin were retained in the conjugate, which was then used to generate a dose-response curve for the analyte in a convenient microtiter plate format. The concentration of 6-keto-prostaglandin F1alpha after extraction from plasma showed good correlation with the concentration of 6-ketoprostaglandin F1alpha obtained without prior extraction of the same plasma sample. This measurement demonstrated that the assay allows the measurement of 6-keto-prostaglandin F1alpha directly in plasma without any pretreatment of the samples, which results in a much simpler method with a faster assay time

Biologically active molecules that reduce polyglutamine aggregation and toxicity.

Polyglutamine expansion in certain proteins causes neurodegeneration in inherited disorders such as Huntington disease and X-linked spinobulbar muscular atrophy. Polyglutamine tracts promote protein aggregation in vitro and in vivo with a strict length-dependence that strongly implicates alternative protein folding and/or aggregation as a proximal cause of cellular toxicity and neurodegeneration. We used an intracellular polyglutamine protein aggregation assay based on fluorescence resonance energy transfer (FRET) to identify inhibitors of androgen receptor (AR) aggregation in three libraries of biologically active small molecules: the Annotated Compound Library, the NINDS Custom Collection and a kinase inhibitor collection. In the primary screen 10 compounds reduced AR aggregation. While 10/10 also reduced huntingtin (Htt) exon 1 aggregation, only 2/10 reduced aggregation of pure polyglutamine peptides. In a PC-12 model 9/10 compounds reduced aggregation. Five out of nine compounds tested in an Htt exon 1 assay of neurodegeneration in Drosophila partially rescued the phenotype. Three of the five compounds effective in flies are FDA-approved drugs. These compounds provide new leads for therapeutic development for the polyglutamine diseases based on their efficacy in mammalian cells and a Drosophila model. The high predictive value of the primary screen suggests that the FRET-based screening assay may be useful for further primary and secondary screens for genes or small molecules that inhibit polyglutamine protein aggregation