Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs).Although thrombin is a recognized physiological activator of PAR 1 and PAR 4 , the endogenous enzymes responsible for activating PAR 2 in settings other than the gastrointestinal system, where trypsin can activate PAR 2 , are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR 1 , PAR 2 , and PAR 4 ; 2) PAR-dependent calcium signaling responses in cells expressing PAR 1 , PAR 2 , and PAR 4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR 4 -dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR 1 , PAR 2 , and PAR 4 and to disarm/inhibit PAR 1 . Although hK5 and -6 were also able to activate PAR 2 , they failed to cause PAR 4 -dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR 2 -mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR 1 , PAR 2 , and PAR 4 . Proteinase-activated receptors (PAR 1-4 )3 compose a unique family of four G-protein-coupled cell surface receptors for certain proteinases (1-9). Proteolytic cleavage within the extracellular N terminus reveals a tethered ligand that binds to the extracellular receptor domains to initiate cell signaling (5, 6, 9, 10). Proteinases that activate PARs include coagulation factors, enzymes from inflammatory cells, and proteinases from epithelial cells and neurons. These enzymes, generated and released during injury and inflammation, can cleave and activate PARs on many cell types from a variety of species (humans, rats, and mice) to regulate the critically important processes of hemostasis, inflammation, pain, and tissue repair. Other proteinases that cleave PARs downstream of the N-terminal tethered ligand sequence disable the receptors for further proteolytic activation, thus abrogating PAR signaling. It is of considerable interest to identify the proteinases that activate and/or disable PARs, in view of the emerging role that these receptors can play in diseases such as asthma, arthritis, inflammatory bowel disease, and cancer (5-7).In some systems, the proteinases that activate PARs have been established. For example, in the circulatory system, PAR 1 , PAR 3 , and PAR 4 are recognized physiological targets for thrombin (4), which does not efficiently acti...

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Natural abundance 13 C NMR spectra of biological extracts are recorded in a single scan provided that the samples are hyperpolarized by dissolution dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell extracts can also be obtained in a single scan. Hyperpolarized NMR of extracts opens many perspectives for metabolomics.Lying at the interface of chemistry and biology, metabolomics is one of the youngest sprouts of the sprawling tree of "omic" sciences. The last decade has witnessed a growing interest in mapping metabolic pathways, in identifying new biomarkers, and in modeling metabolic fluxes. 1,2 Nuclear Magnetic Resonance (NMR) spectroscopy is a major analytical technique in this field, offering unambiguous information about the identity of metabolites and their time-dependent concentrations in complex samples such as biofluids, extracts and tissues. However, proton NMR spectra have a limited range of chemical shifts and often suffer from overlapping signals. Carbon-13, which has a larger chemical shift dispersion, can benefit from sensitive detectors 3 or from isotopic enrichment, 4 albeit at the cost of an enhanced complexity of the spectra due to 13 C-13 C couplings. Heteronuclear ( 1 H→ 13 C) correlation spectroscopy can in principle offer a satisfactory dispersion. 5,6 So far, 13 C NMR is not routinely used in metabolomic studies, but hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) combined with cross polarization (CP) could boost its popularity in this field.Hyperpolarization techniques are capable of generating spin polarization levels that are about 50 000 times above Boltzmann equilibrium at room temperature. 7-9 Once prepared and transferred to a solution-state NMR spectrometer, the resulting magnetization can be exploited within a limited time-span that depends on the longitudinal relaxation time T 1 of the hyperpolarized nuclei. Among various methods for hyperpolarization, dynamic nuclear polarization (DNP) is least affected by the chemical substrate and sample preparation. 10 For solution-state NMR, particularly promising results can be obtained by dissolution DNP, where the sample to be analyzed is mixed with free radicals in a glass-forming solution, frozen to liquid helium temperatures, and hyperpolarized by irradiating in the vicinity of the electron spin resonance (ESR) of the unpaired electrons of the radicals. 11 Spins of 13 C nuclei may be polarized either directly or via cross-polarization from protons to 13 C. 12 Rapid melting and shuttling of the sample from the polarizer to a routine NMR spectrometer enables conventional solution-state NMR spectroscopy with a sensitivity that can be boosted by a factor of about 50 000. Most applications of D-DNP have been restricted to the injection of pure hyperpolarized 13 C-labeled molecules such as pyruvate into living organisms. 13 D-DNP has hardly been applied to complex mixtures of metabolites, 14 but only to a few wellchosen small molecules...

Two-dimensional nuclear magnetic resonance (2D NMR) forms a powerful tool for the quantitative analysis of complex mixtures such as samples of metabolic relevance. However, its use for quantitative purposes is far from being trivial, not only because of the associated experiment time, but also due to its subsequent high sensitivity to hardware instabilities affecting its precision. In this paper, an alternative approach is considered to measure absolute metabolite concentrations in complex mixtures with a high precision in a reasonable time. It is based on a "multi-scan single shot" (M3S) strategy, which is derived from the ultrafast 2D NMR methodology. First, the analytical performance of this methodology is compared to the one of conventional 2D NMR. 2D correlation spectroscopy (COSY) spectra are obtained in 10 min on model metabolic mixtures, with a precision in the 1-4% range (versus 5-18% for the conventional approach). The M3S approach also shows a better linearity than its conventional counterpart. It ensures that accurate quantitative results can be obtained provided that a calibration procedure is carried out. The M3S COSY approach is then applied to measure the absolute metabolite concentration in three breast cancer cell line extracts, relying on a standard addition protocol. M3S COSY spectra of such extracts are recorded in 20 min and give access to the absolute concentration of 14 major metabolites, showing significant differences between cell lines.

We tested the hypothesis that human tissue kallikreins (hKs) may regulate signal transduction by cleaving and activating proteinase-activated receptors (PARs). We found that hK5, 6 and 14 cleaved PAR N-terminal peptide sequences representing the cleavage/activation motifs of human PAR1 and PAR2 to yield receptor-activating peptides. hK5, 6 and 14 activated calcium signalling in rat PAR2-expressing (but not background) KNRK cells. Calcium signalling in HEK cells co-expressing human PAR1 and PAR2 was also triggered by hK14 (via PAR1 and PAR2) and hK6 (via PAR2). In isolated rat platelets that do not express PAR1, but signal via PAR4, hK14 also activated PAR-dependent calcium signalling responses and triggered aggregation. The aggregation response elicited by hK14 was in contrast to the lack of aggregation triggered by hK5 and 6. hK14 also caused vasorelaxation in a phenylephrine-preconstricted rat aorta ring assay and triggered oedema in an in vivo model of murine paw inflammation. We propose that, like thrombin and trypsin, the kallikreins must now be considered as important 'hormonal' regulators of tissue function, very likely acting in part via PARs.

Metabolomic studies by NMR spectroscopy are increasingly employed for a variety of biomedical applications. A very standardized 1D proton NMR protocol is generally employed for data acquisition, associated with multivariate statistical tests. Even if targeted approaches have been proposed to quantify metabolites from such experiments, quantification is often made difficult by the high degree of overlap characterizing (1) H NMR spectra of biological samples. Two-dimensional spectroscopy presents a high potential for accurately measuring concentrations in complex samples, as it offers a much higher discrimination between metabolite resonances. We have recently proposed an original approach relying on the (1) H 2D INADEQUATE pulse sequence, optimized for fast quantitative analysis of complex metabolic mixtures. Here, the first application of the quantitative (1) H 2D INADEQUATE experiment to a real metabonomic study is presented. Absolute metabolite concentrations are determined for different breast cancer cell line extracts, by a standard addition procedure. The protocol is characterized by high analytical performances (accuracy better than 1%, excellent linearity), even if it is affected by relatively long acquisition durations (15 min to 1 h per spectrum). It is applied to three different cell lines, expressing different hormonal and tyrosine kinase receptors. The absolute concentrations of 15 metabolites are determined, revealing significant differences between cell lines. The metabolite concentrations measured are in good agreement with previous studies regarding metabolic profile changes of breast cancer. While providing a high degree of discrimination, this methodology offers a powerful tool for the determination of relevant biomarkers.

Quantitative Ultrafast (UF) 2D NMR is a very promising methodology enabling the acquisition of 2D spectra in a single scan. The analytical performances of UF 2D NMR have been highly increased in the last few years, however little is known about the sensitivity of ultrafast experiments versus conventional 2D NMR. A fair and relevant comparison has to consider the Signal-to-Noise Ratio (SNR) per unit of time, in order to answer the following question: for a given experiment time, should we run a conventional 2D experiment or is it preferable to accumulate ultrafast acquisitions? To answer this question, we perform here a systematic comparison between accumulated ultrafast experiments and conventional ones, for different experiment durations. Sensitivity issues and other analytical aspects are discussed for the COSY experiment in the context of quantitative analysis. The comparison is first carried out on a model sample, and then extended to model metabolic mixtures. The results highlight the high analytical performance of the "multi-scan single shot" approach versus conventional 2D NMR acquisitions. This result is attributed to the absence of t(1) noise in spatially encoded experiments. The multi-scan single shot approach is particularly interesting for quantitative applications of 2D NMR, whose occurrence in the literature has been greatly increasing in the last few years.

In situ NMR spectroelectrochemistry is presented in this study as a useful hybrid technique for the chemical structure elucidation of unstable intermediate species. An experimental setting was designed to follow the reaction in real time during the experimental electrochemical process. The analysis of (1)H NMR spectra recorded in situ permitted us (1) to elucidate the reaction pathway of the electrochemical oxidation of phenacetin and (2) to reveal the quinone imine as a reactive intermediate species without using any trapping reaction. Phenacetin has been considered as hepatotoxic at high therapeutic amounts, which is why it was chosen as a model to prove the applicability of the analytical method. The use of 1D and 2D NMR experiments led to the elucidation of the major species produced from the oxidation process. We demonstrated that in situ NMR spectroelectrochemistry constitutes a fast way for monitoring unstable quinone imines and elucidating their chemical structures.