Urine proteins identified by two-dimensional differential gel electrophoresis facilitate the differential diagnoses of scrapie.

The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.

Factors affecting the accuracy of urine-based biomarkers of BSE.

BACKGROUND: 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. OBJECTIVE: 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. RESULTS: 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. CONCLUSION: 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.

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.

Evidence for downregulation of calcium signaling proteins in advanced mouse adenocarcinoma.

BACKGROUND: Prostate 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. METHODS: The 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. RESULTS: In 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. CONCLUSIONS: Proteomics of TRAMP mice provide an excellent method to observe changes in protein abundance, revealing changes in pathways during cancer progression.

Reactions of substituted (methylthio)benzylidene Meldrum's acids with secondary alicyclic amines in aqueous DMSO. Evidence for rate-limiting proton transfer.

The replacement of the methylthio group of substituted methylthiobenzylidene Meldrum's acids (2-SMe-Z) by secondary alicyclic amines occurs by a three-step mechanism. The first step is a nucleophilic attachment of the amine to 2-SMe-Z to form a zwitterionic intermediate T(+/-)(A); the second step involves deprotonation of T(+/-)(A) to form T(-)(A); while the third step represents general acid-catalyzed conversion of T(-)(A) to products. At high amine and/or high KOH concentration nucleophilic attachment is rate limiting. At low amine and low KOH concentration the reaction follows a rate law that is characteristic for general base catalysis which, in principle, is consistent with either rate-limiting deprotonation of T(+/-)(A) or rate-limiting conversion of T(-)(A) to products. A detailed structure-reactivity analysis indicates that for the reactions with piperazine, 1-(2-hydroxyethyl)piperazine, and morpholine it is deprotonation of T(+/-)(A) that is rate limiting, while for the reaction with piperidine, conversion of T(-)(A) to products is rate limiting.

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.

Kinetic and thermodynamic acidities of pentacarbonyl(cyclobutenylidene)chromium complexes. Effect of antiaromaticity in the conjugate anion. An experimental and computational study.

The deprotonation of pentacarbonyl[(3-diethylamino-2,4-dimethyl)cyclobut-2-ene-1-ylidene]chromium (1d) and pentacarbonyl[(3-diethylamino-4-methyl-2-phenyl)cyclobut-2-ene-1-ylidene]chromium (1e) leads to antiaromatic conjugate anions by virtue of their being cyclobutadiene derivatives. Rate constants for the deprotonation of 1d and 1e by P2-Et and pKa values were determined in acetonitrile. Gas-phase B3LYP calculations of 1d, 1e, and their respective conjugate anions, using a generalized basis set, were also performed. Furthermore, for purposes of comparison with carbene complexes of similar structures, but having conjugate anions that are not antiaromatic, corresponding calculations were performed on pentacarbonyl[3-diethylamino-2,5-dimethyl)cyclopent-2-ene-1-ylidene]chromium (5), [dimethylamino(methyl)carbene]pentacarbonylchromium (3a), and [dimethylamino(iso-propyl)carbene]pentacarbonylchromium (3b) and their respective conjugate anions, and solution-phase pKa and kinetic measurements were carried out for 3a and 3b. Major points of interest include the effect of antiaromaticity on the kinetic and thermodynamic acidities of 1d and 1e, the large effect of the phenyl group on the gas-phase acidity of 1e, the strong attenuation of the acidities and the effect of the phenyl group in acetonitrile, and the position of the C=C double bonds in the cyclobutadiene ring of the conjugate anion of 1e.

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.

Physical organic chemistry of transition metal carbene complexes. 25. Kinetic and thermodynamic acidities of substituted (methylthiophenylcarbene)pentacarbonyl tungsten(0) and (Benzoxymethylcarbene)pentacarbonyl tungsten(0) in aqueous acetonitrile. Evidence for transition state imbalances.

A kinetic study of the reversible deprotonation of substituted (methylthiophenylcarbene)pentacarbonyltungsten(0) ((CO)(5)W=C(SC(6)H(4)Z)CH(3)) and of substituted (benzoxymethylcarbene)pentacarbonyltungsten(0) ((CO)(5)W=C(OCH(2)C(6)H(4)Z)CH(3)) by primary aliphatic and secondary alicyclic amines in 50% MeCN-50% water (v/v) at 25 degrees C is reported. From the dependence of the deprotonation rate constants on amine basicity and on carbene complex acidity (variation of Z), Brønsted beta(B) and alpha(CH) values, respectively, were obtained. The alpha(CH) values were found to be smaller than the beta(B) values. These results indicate a transition state imbalance in which the loss of the carbene complex stabilizing pi-donor effect of the OCH(2)Ar and SAr groups lags behind the proton transfer. These findings confirm a previously formulated hypothesis as to how pi-donor groups attached to the carbene carbon of carbene complexes can affect transition state imbalances and mask the experimental manifestation of such imbalances. It is also shown that the transition state structure of the reactions examined in this work is subject to changes with changing amine basicity and carbene complex acidity; these changes can be expressed by p(xy)() cross correlation coefficients, which are positive.