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Proteinase-activated receptors, targets for kallikrein signaling

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 PAR1 and PAR4, the endogenous enzymes responsible for activating PAR2 in settings other than the gastrointestinal system, where trypsin can activate PAR2, 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 PAR1, PAR2, and PAR4; 2) PAR-dependent calcium signaling responses in cells expressing PAR1, PAR2, and PAR4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR4-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 PAR1, PAR2, and PAR4 and to disarm/inhibit PAR1. Although hK5 and -6 were also able to activate PAR2, they failed to cause PAR4-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR2-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 PAR1, PAR2, and PAR4.

Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs).

Abstract 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.

Proteinase-mediated cell signalling: targeting proteinase-activated receptors (PARs) by kallikreins and more.

Abstract Serine proteinases, like trypsin, can play a hormone-like role by triggering signal transduction pathways in target cells. In many respects these hormone-like actions of proteinases can now be understood in terms of the pharmacodynamics of the G protein-coupled ‘receptor’ responsible for the cellular actions of thrombin (proteinase-activated receptor-1, or PAR1). PAR1, like the other three members of this receptor family (PAR2, PAR3 and PAR4), has a unique mechanism of activation involving the proteolytic unmasking of an N-terminally tethered sequence that can activate the receptor. The selective activation of each PAR by short synthetic peptides representing these sequences has demonstrated that PAR1, PAR2 and PAR4 play important roles in regulating physiological responses ranging from vasoregulation and cell growth to inflammation and nociception. We hypothesise that the tissue kallikreins may regulate signal transduction via the PARs. Although PARs can account for many of their biological actions, kallikreins may also cause effects by mechanisms not involving the PARs. For instance, trypsin activates the insulin receptor and thrombin can act via a mechanism involving its non-catalytic domains. Based on the data we summarise, we propose that the kallikreins, like thrombin and trypsin, must now be considered as important ‘hormonal’ regulators of tissue function.

1H-NMR-Based Metabolic Profiling of Maternal and Umbilical Cord Blood Indicates Altered Materno-Foetal Nutrient Exchange in Preterm Infants

Background Adequate foetal growth is primarily determined by nutrient availability, which is dependent on placental nutrient transport and foetal metabolism. We have used 1H nuclear magnetic resonance (NMR) spectroscopy to probe the metabolic adaptations associated with premature birth. Methodology The metabolic profile in 1H NMR spectra of plasma taken immediately after birth from umbilical vein, umbilical artery and maternal blood were recorded for mothers delivering very-low-birth-weight (VLBW) or normo-ponderal full-term (FT) neonates. Principal Findings Clear distinctions between maternal and cord plasma of all samples were observed by principal component analysis (PCA). Levels of amino acids, glucose, and albumin-lysyl in cord plasma exceeded those in maternal plasma, whereas lipoproteins (notably low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) and lipid levels were lower in cord plasma from both VLBW and FT neonates. The metabolic signature of mothers delivering VLBW infants included decreased levels of acetate and increased levels of lipids, pyruvate, glutamine, valine and threonine. Decreased levels of lipoproteins glucose, pyruvate and albumin-lysyl and increased levels of glutamine were characteristic of cord blood (both arterial and venous) from VLBW infants, along with a decrease in levels of several amino acids in arterial cord blood. Conclusion These results show that, because of its characteristics and simple non-invasive mode of collection, cord plasma is particularly suited for metabolomic analysis even in VLBW infants and provides new insights into the materno-foetal nutrient exchange in preterm infants.

Absolute quantification of metabolites in breast cancer cell extracts by quantitative 2D 1H INADEQUATE NMR

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 1H 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 1H 2D INADEQUATE pulse sequence, optimized for fast quantitative analysis of complex metabolic mixtures. Here, the first application of the quantitative 1H 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. Copyright

Fast and precise quantitative analysis of metabolic mixtures by 2D 1H INADEQUATE NMR.

Quantitative analysis of metabolic mixtures by (1)H 1D NMR offers a limited potential for precise quantification of biomarkers, due to strong overlap between the peaks. Two-dimensional spectroscopy is a powerful tool to unambiguously and simultaneously measure a larger number of metabolite contributions. However, it is still rarely used for quantification, first because quantitative analysis by 2D NMR requires a calibration procedure due to the multi-impulsional nature of 2D NMR experiments, and above all because of the prohibitive experiment duration that is necessary to obtain such a calibration curve. In this work, we develop and evaluate a 2D (1)H INADEQUATE protocol for a fast determination of metabolite concentrations in complex mixtures. The 2D pulse sequence is carefully optimized and evaluated in terms of precision and linearity. Quantitative (1)H INADEQUATE 2D spectra of metabolic mixtures are obtained in 7 min with a repeatability better than 2% for metabolite concentrations as small as 100 μM and an excellent linearity. The method described in this work allows a fast and precise quantification of metabolic mixtures, and it forms a promising tool for metabonomic studies.

Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells

Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using 1H nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.

δ 15 N and δ 13 C in hair from newborn infants and their mothers: a cohort study

Introduction:Protein intake in fetal life or infancy may play a key role in determining early growth rate, a determinant of later health and disease. Previous work has indicated that hair isotopic composition is influenced by diet and protein intake.Methods:This study analyzes the isotopic composition of hair obtained from 239 mother/newborn pairs randomly selected within a larger cohort enrolled in a study of pre- and postnatal determinants of the child’s development and health. The isotopic compositions in nitrogen (δ15N) and in carbon (δ13C) were determined by isotope ratio mass spectrometry.Results:Mother and newborn hair δ15N were tightly correlated (Pearson r = 0.88). The mean δ15N and δ13C values of hair from newborn infants were significantly higher than those for the mothers: 9.7 ± 0.7 vs. 8.8 ± 0.6‰ (P < 0.0001) for δ15N and −20.0 ± 0.4 vs. −20.4 ± 0.4‰ (P < 0.0001) for δ13C. Maternal hair δ15N at parturition was slightly and positively correlated with estimates of protein intake (r = 0.14, P = 0.04).Discussion:Hair δ15N of the fetus is both highly dependent on and systematically higher than that of the mother. Whether quantitative and qualitative protein intake, disease, or hormonal status alter hair δ15N at birth remains to be determined.

Effect of Foliar Sulfur and Nitrogen Fertilization on Wheat Storage Protein Composition and Dough Mixing Properties

ABSTRACT Nitrogen (N) and sulfur (S) supplies have a strong influence on the quality and quantity of wheat storage proteins, which play an important role in the breadmaking process. Nitrogen derived from urea, S from micronized elemental sulfur, and a mixture of both (N+S) were applied at anthesis stage on wheat by foliar spray. To relate N and S incorporation in storage proteins to the quality of dough, their incorporation into each storage protein fraction was measured: monomers, low molecular weight glutenin subunits (LMW-GS), and high molecular weight glutenin subunits (HMW-GS). Then protein fraction quantities, molecular weight distribution (MWD), polymerization index (PI), and molecular dimensions of unextractable polymeric protein (UPP), as well as dough mixing properties were determined. Fertilizers N and S were differentially incorporated into each storage protein fraction, influencing protein synthesis. Moreover, after the N+S fertilization, the increase of the polymeric proteins induced an incr...

Maternal and cord blood LC-HRMS metabolomics reveal alterations in energy and polyamine metabolism, and oxidative stress in very-low birth weight infants.

To assess the global effect of preterm birth on fetal metabolism and maternal-fetal nutrient transfer, we used a mass spectrometric-based chemical phenotyping approach on cord blood obtained at the time of birth. We sampled umbilical venous, umbilical arterial, and maternal blood from mothers delivering very-low birth weight (VLBW, with a median gestational age and weight of 29 weeks, and 1210 g, respectively) premature or full-term (FT) neonates. In VLBW group, we observed a significant elevation in the levels and maternal-fetal gradients of butyryl-, isovaleryl-, hexanoyl- and octanoyl-carnitines, suggesting enhanced short- and medium chain fatty acid β-oxidation in human preterm feto-placental unit. The significant decrease in glutamine-glutamate in preterm arterial cord blood beside lower levels of amino acid precursors of Krebs cycle suggest increased glutamine utilization in the fast growing tissues of preterm fetus with a deregulation in placental glutamate-glutamine shuttling. Enhanced glutathione utilization is likely to account for the decrease in precursor amino acids (serine, betaine, glutamate and methionine) in arterial cord blood. An increase in both the circulating levels and maternal-fetal gradients of several polyamines in their acetylated form (diacetylspermine and acetylputrescine) suggests an enhanced polyamine metabolic cycling in extreme prematurity. Our metabolomics study allowed the identification of alterations in fetal energy, antioxidant defense, and polyamines and purines flux as a signature of premature birth.