Factors affecting the accuracy of urine-based biomarkers of BSE

<p>Abstract</p> <p>Background</p> <p>Transmissible spongiform encephalopathy diseases are untreatable, uniformly fatal degenerative syndromes of the central nervous system that can be transmitted both within as well as between species. The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob disease (vCJD), have profoundly influenced beef production processes as well as blood donation and surgical procedures. Simple, robust and cost effective diagnostic screening and surveillance tools are needed for both the preclinical and clinical stages of TSE disease in order to minimize both the economic costs and zoonotic risk of BSE and to further reduce the risk of secondary vCJD.</p> <p>Objective</p> <p>Urine is well suited as the matrix for an ante-mortem test for TSE diseases because it would permit non-invasive and repeated sampling. In this study urine samples collected from BSE infected and age matched control cattle were screened for the presence of individual proteins that exhibited disease specific changes in abundance in response to BSE infection that might form the basis of such an ante-mortem test.</p> <p>Results</p> <p>Two-dimensional differential gel electrophoresis (2D-DIGE) was used to identify proteins exhibiting differential abundance in two sets of cattle. The known set consisted of BSE infected steers and age matched controls throughout the course of the disease. The blinded unknown set was composed of BSE infected and control samples of both genders, a wide range of ages and two different breeds. Multivariate analyses of individual protein abundance data generated classifiers comprised of the proteins best able to discriminate between the samples based on disease state, breed, age and gender.</p> <p>Conclusion</p> <p>Despite the presence of confounding factors, the disease specific changes in abundance exhibited by a panel of urine proteins permitted the creation of classifiers able to discriminate between control and infected cattle with a high degree of accuracy.</p

Stopped-Flow Kinetic Investigations of the Activation of Soybean Lipoxygenase-1 and the Influence of Inhibitors on the Allosteric Site †

Herein, we report on the role of the allosteric site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic site and a K(i)' of 140 +/- 9 microM for the allosteric site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an allosteric site that is separate from the catalytic iron site. We further observed that the allosteric site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS

Evidence for downregulation of calcium signaling proteins in advanced mouse adenocarcinoma

BackgroundProstate cancer (PCa) is the leading cancer related death in America. Gleason grading is currently the predominant method for prediction, with only few biomarkers available. More biomarkers, especially as they relate to cancer progression are desirable.MethodsThe abundance of several important proteins in prostate tissue was compared between wild-type mouse dorsal prostate and well-differentiated transgenic adenocarcinoma mouse prostate (TRAMP) mouse dorsal prostates, and between wild-type mouse dorsal prostate and poorly-differentiated TRAMP mouse tumor tissue. 2DIGE method in conjunction with MALDI-ToF and Western blots was used to determine differential expression.ResultsIn TRAMP dorsal prostates with well-differentiated adenocarcinoma, there were few significant changes in the protein abundances compared to wild-type dorsal prostates, with the exception of increases in proliferating cell nuclear antigen (PCNA) and beta tubulin, two proteins implicated in cell proliferation, and a more than 2-fold increase in Hsp60, a protein involved in the suppression of apoptosis. In the poorly-differentiated tumors, the changes in protein abundance were substantial. While some of those changes could be related to the disappearance of stromal tissue or the appearance of epithelial tissue, other changes in protein abundance were more significant to the cancer development itself. Most notable was the overall decrease in calcium homeostasis proteins with a 10-fold decrease in calreticulin and Hsp70 and a 40-fold decrease in creatine kinase bb in the cancerous tissue.ConclusionsProteomics of TRAMP mice provide an excellent method to observe changes in protein abundance, revealing changes in pathways during cancer progression

Kinetic, Spectroscopic, and Structural Investigations of the Soybean Lipoxygenase-1 First-Coordination Sphere Mutant, Asn694Gly † , ‡

In wild-type soybean LO-1 (WT sLO-1), Asn694 is a weak sixth ligand that is thought to be critical for enzymatic catalysis. In this investigation, N694G sLO-1 was studied to probe its contribution at this sixth ligand position to the kinetic and spectroscopic properties. The k(cat) value of N694G is approximately 230 times lower than that of WT sLO-1 at 25 degrees C, which can be partially explained by a lowered reduction potential of the iron as seen as a shift in the visible ligand-to-metal charge-transfer band (lambda(max) = 410 nm for N694G and lambda(max) = 425 nm for WT sLO-1). This conclusion was supported by a faster rate of oxidation of N694G by the product than that of WT sLO-1 (k(2) = 606 s(-)(1) for N694G and k(2) = 349 s(-)(1) for WT sLO-1). These results suggest a stronger ligand at the active site iron than the native Asn694, which is confirmed to be a water bound to the Fe(II) in the crystal structure. This produces a six-coordinate circular dichroism/magnetic circular dichroism (CD/MCD) spectra for ferrous N694G and an intermediate rhombic electron paramagnetic resonance (EPR) signal for ferric N694G. The EPR spectrum and its pH dependence suggest that the coordination environment of ferric N694G contains one hydroxide and one water. On the basis of both kinetic and structural factors, we propose that the Asn694 water-derived ligand would likely be a hydroxide and the active site, water-derived ligand a water in the ferric state, hence lowering the reaction rate of N694G more than would be expected from the lowered reduction potential alone

Tryptophan 500 and Arginine 707 Define Product and Substrate Active Site Binding in Soybean Lipoxygenase-1 †

There is much debate whether the fatty acid substrate of lipoxygenase binds "carboxylate-end first" or "methyl-end first" in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steady-state and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly pi-pi interaction) and with the positively charged amino acid residue Arg707 (charge-charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind "carboxylate-end first" with sLO-1 and not "methyl-end first" as has been proposed for human 15-lipoxygenase

Tryptophan 500 and Arginine 707 Define Product and Substrate Active Site Binding in Soybean Lipoxygenase-1 †

ABSTRACT: There is much debate whether the fatty acid substrate of lipoxygenase binds &quot;carboxylate-end first&quot; or &quot;methyl-end first&quot; in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steadystate and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly π-π interaction) and with the positively charged amino acid residue Arg707 (charge-charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind &quot;carboxylate-end first&quot; with sLO-1 and not &quot;methylend first&quot; as has been proposed for human 15-lipoxygenase. Lipoxygenases (LO) 1 catalyze the peroxidation of dienecontaining fatty acids and belong to a class of non-heme iron metalloenzymes found in both plants and mammals (1-3). Mammalian lipoxygenases serve vital roles in the biosynthesis of lipoxins and leukotrienes, which are critical signaling molecules (4, 5). There are three major mammalian isozymes, 5-LO, 12-LO, and 15-LO, which oxygenate arachidonic acid (AA) at specific carbon centers (C5, C12, and C15, respectively) (6). These isozymes of human lipoxygenase have been shown to be involved in several human diseases: asthma (7) and prostate cancer (8) for human 5-LO (5-hLO), immune disorders (9) and breast cancer (10, 11) for human 12-LO (12-hLO), and atherosclerosis (12) and colorectal cancer (13) for human 15-LO (15-hLO). To develop effective therapeutic agents against these diseases, an intimate knowledge of their active sites is needed so that specific inhibitors of a particular lipoxygenase isozyme can be designed. Sloane and co-workers made significant progress in this regard when they converted reticulocyte 15-hLO-1 into a &quot;12-hLO&quot; by increasing the active site volume; they proposed that the substrate sat deeper in the active site, with the hydrophobic methyl end inserted first (14). This hypothesis was supported by site-directed mutagenesis investigations of Gan et al., which suggested that Phe414 of 15-hLO-1 π-π-stacked with the C11-C12 double bond of the substrate and that the positively charged residue Arg402 interacted with the negatively charged carboxylate of the substrate (15). Because Arg402 is located close to the surface of 15-hLO-1, this study supported a &quot;methyl-end first&quot; binding of the substrate. In addition, mutagenesis experiments on Phe353 and Ile593 of rabbit 15-LO (15-rLO) also supported the hypothesis that the size and shape of the active site defined the specificity and were consistent with a &quot;methyl-end first&quot; binding for mammalian Nevertheless, when the rabbit 15-LO (15-rLO) structure was compared with soybean lipoxygenase-1 (sLO-1), a debate concerning the manner in which the substrate bound to the active site developed. Amzel and co-workers proposed that, to obtain the known stereochemistry of the product, only a &quot;carboxylate-end first&quot; insertion of linoleic acid (LA) into the active site was plausible for sLO-

Principle component analysis of 7 scrapie infected samples (red) at 15 and 17 weeks of age, 11 Alzheimer's disease samples (green) at 32, 36 and 40 weeks of age, and the 20 corresponding control samples (blue) when analyzed by the 20 protein Disease Discriminant Classifier.

<p><b>Samples were produced by 4 scrapie infected mice, 4 Alzheimer diseased mice and the 8 corresponding control mice.</b> Thirty seven of the 38 samples were correctly classified. The 32 week old control sample, misclassified as having Alzheimer's disease, is identified by an arrow.</p