Sensitive high-performance liquid chromatography-tandem mass spectrometry method for quantitative analysis of mycophenolic acid and its glucuronide metabolites in human plasma and urine

A method to determine total and free mycophenolic acid (MPA) and its metabolites, the phenolic (MPAG) and acyl (AcMPAG) glucuronides, using HPLC and mass spectrometry was developed. Mean recoveries in plasma and urine samples were >85%, and the lower limits of quantification for MPA, MPAG and AcMPAG were 0.05, 0.05 and 0.01 mg/L, respectively. For plasma, the assay was linear over 0.05-50 mg/L for MPA and MPAG, and from 0.01 to 10mg/L for AcMPAG. A validation study demonstrated good inter- and intra-day precision (CV相似文献   

Targeting Toll-like receptor (TLR) signaling by Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal)-derived decoy peptides

Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal) is an adapter protein that facilitates recruitment of MyD88 to TLR4 and TLR2 signaling complexes. We previously generated a library of cell-permeating TLR4 TIR-derived decoy peptides fused to the translocating segment of the Drosophila Antennapedia homeodomain and examined each peptide for the ability to inhibit TLR4 signaling (Toshchakov, V. Y., Szmacinski, H., Couture, L. A., Lakowicz, J. R., and Vogel, S. N. (2011) J. Immunol. 186, 4819-4827). We have now expanded this study to test TIRAP decoy peptides. Five TIRAP peptides, TR3 (for TIRAP region 3), TR5, TR6, TR9, and TR11, inhibited LPS-induced cytokine mRNA expression and MAPK activation. Inhibition was confirmed at the protein level; select peptides abolished the LPS-induced cytokine production measured in cell culture 24 h after a single treatment. Two of the TLR4 inhibitory peptides, TR3 and TR6, also inhibited cytokine production induced by a TLR2/TLR1 agonist, S-(2,3-bis(palmitoyloxy)-(2R,2S)-propyl)-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH; however, a higher peptide concentration was required to achieve comparable inhibition of TLR2 versus TLR4 signaling. Two TLR4 inhibitory peptides, TR5 and TR6, were examined for the ability to inhibit TLR4-driven cytokine induction in mice. Pretreatment with either peptide significantly reduced circulating TNF-α and IL-6 in mice following LPS injection. This study has identified novel TLR inhibitory peptides that block cellular signaling at low micromolar concentrations in vitro and in vivo. Comparison of TLR4 inhibition by TLR4 and TIRAP TIR-derived peptides supports the view that structurally diverse regions mediate functional interactions of TIR domains.     

Ligand interactions in the distal heme pocket of Mycobacterium tuberculosis truncated hemoglobin N: roles of TyrB10 and GlnE11 residues

The crystallographic structure of oxygenated trHbN from Mycobacterium tuberculosis showed an extended heme distal site hydrogen-bonding network that includes Y(B10), Q(E11), and the bound O(2) (Milani, M., et al. (2001) EMBO J. 20, 3902-3909). In the present work, we analyze the effects that substitutions at the B10 and E11 positions exert on the heme and its coordinated ligands, using steady-state resonance Raman spectroscopy, absorption spectroscopy and X-ray crystallography. Our results show that (1) residues Y(B10) and Q(E11) control the binding and the ionization state of the heme-bound water molecules in ferric trHbN and are important in keeping the sixth coordination position vacant in deoxy trHbN; (2) residue Q(E11) plays a role in maintaining the integrity of the proximal Fe-His bond in deoxy trHbN; (3) in wild-type oxy-trHbN, the size and hydrogen-bonding capability of residue E11 is important to sustain proper interaction between Y(B10) and the heme-bound O(2); (4) CO-trHbN is in a conformational equilibrium, where either the Y(B10) or the Q(E11) residue interacts with the heme-bound CO; and (5) Y(B10) and Q(E11) residues control the conformation (and likely the dynamics) of the protein matrix tunnel gating residue F(E15). These findings suggest that the functional processes of ligand binding and diffusion are controlled in trHbN through the dynamic interaction of residues Y(B10), Q(E11), F(E15), and the heme ligand.     

Anti-diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait.

Incidence of type II diabetes is rapidly increasing worldwide. In order to identify complementary or alternative approaches to existing medications, we studied anti-diabetic properties of Vaccinium angustifolium Ait., a natural health product recommended for diabetes treatment in Canada. Ethanol extracts of root, stem, leaf, and fruit were tested at 12.5 μg/ml for anti-diabetic activity in peripheral tissues and pancreatic β cells using a variety of cell-based bioassays. Specifically, we assessed: (1) deoxyglucose uptake in differentiated C2C12 muscle cells and 3T3-L1 adipocytes; (2) glucose-stimulated insulin secretion (GSIS) in β TC-tet pancreatic β cells; (3) β cell proliferation in β TC-tet cells; (4) lipid accumulation in differentiating 3T3-L1 cells; (5) protection against glucose toxicity in PC12 cells. Root, stem, and leaf extracts significantly enhanced glucose transport in C2C12 cells by 15–25% in presence and absence of insulin after 20 h of incubation; no enhancement resulted from a 1 h exposure. In 3T3 cells, only the root and stem extracts enhanced uptake, and this effect was greater after 1 h than after 20 h; uptake was increased by up to 75% in absence of insulin. GSIS was potentiated by a small amount in growth-arrested β TC-tet cells incubated overnight with leaf or stem extract. However, fruit extracts were found to increase ³H-thymidine incorporation in replicating β TC-tet cells by 2.8-fold. Lipid accumulation in differentiating 3T3-L1 cells was accelerated by root, stem, and leaf extracts by as much as 6.5-fold by the end of a 6-day period. Stem, leaf, and fruit extracts reduced apoptosis by 20–33% in PC12 cells exposed to elevated glucose for 96 h. These results demonstrate that V. angustifolium contains active principles with insulin-like and glitazone-like properties, while conferring protection against glucose toxicity. Enhancement of proliferation in β cells may represent another potential anti-diabetic property. Extracts of the Canadian blueberry thus show promise for use as a complementary anti-diabetic therapy.     

Structure and heme binding properties of Escherichia coli O157:H7 ChuX

For many pathogenic microorganisms, iron acquisition from host heme sources stimulates growth, multiplication, ultimately enabling successful survival and colonization. In gram‐negative Escherichia coli O157:H7, Shigella dysenteriae and Yersinia enterocolitica the genes encoded within the heme utilization operon enable the effective uptake and utilization of heme as an iron source. While the complement of proteins responsible for heme internalization has been determined in these organisms, the fate of heme once it has reached the cytoplasm has only recently begun to be resolved. Here we report the first crystal structure of ChuX, a member of the conserved heme utilization operon from pathogenic E. coli O157:H7 determined at 2.05 Å resolution. ChuX forms a dimer which remarkably given low sequence homology, displays a very similar fold to the monomer structure of ChuS and HemS, two other heme utilization proteins. Absorption spectral analysis of heme reconstituted ChuX demonstrates that ChuX binds heme in a 1:1 manner implying that each ChuX homodimer has the potential to coordinate two heme molecules in contrast to ChuS and HemS where only one heme molecule is bound. Resonance Raman spectroscopy indicates that the heme of ferric ChuX is composed of a mixture of coordination states: 5‐coordinate and high‐spin, 6‐coordinate and low‐spin, and 6‐coordinate and high‐spin. In contrast, the reduced ferrous form displays mainly a 5‐coordinate and high‐spin state with a minor contribution from a 6‐coordinate and low‐spin state. The ν_Fe‐CO and ν_C‐O frequencies of ChuX‐bound CO fall on the correlation line expected for histidine‐coordinated hemoproteins indicating that the fifth axial ligand of the ferrous heme is the imidazole ring of a histidine residue. Based on sequence and structural comparisons, we designed a number of site‐directed mutations in ChuX to probe the heme binding sites and dimer interface. Spectral analysis of ChuX and mutants suggests involvement of H65 and H98 in heme coordination as mutations of both residues were required to abolish the formation of the hexacoordination state of heme‐bound ChuX.     

Adhesion and transcellular migration of neutrophils and B lymphocytes on fibroblasts

During tissue inflammation, infiltrated leukocytes may have physical contacts with fibroblasts. We observed that neutrophils and B lymphocytes adhered in a larger proportion than T cells on cultured fibroblasts. Microscopy showed that adhesion was also characterized by leukocyte engulfment by the fibroblasts. In migration assays, only neutrophils and B lymphocytes were selectively able to migrate through a fibroblast barrier. Adhesion and migration were increased by stimulation with tumor necrosis factor-α (TNF-α) and phorbol-12-myristate-13-acetate (PMA). Antibodies against ICAM-1/β2 integrin blocked the interaction of neutrophils to fibroblasts. For B lymphocytes the couple VCAM-1/α4 integrin was also involved in this interaction. Human skin fibroblasts presented similar adhesion characteristics as rat cardiac fibroblasts. By measuring the distance between the border of migration holes and cadherin-positive adherens junctions, more than 65% of the holes correspond to the transcellular route over the paracellular route. Furthermore, vimentin staining revealed that the migration holes were highly nested by intermediate filaments in accordance with the transcellular route. Our results demonstrated that engulfment of neutrophils and B lymphocytes by fibroblasts resulted in selective passage by a transcellular route.     

Effects of ezetimibe and simvastatin on apolipoprotein B metabolism in males with mixed hyperlipidemia

Sixteen hyperlipidemic men were enrolled in a randomized, placebo-controlled, double-blind, cross-over study to evaluate the effect of ezetimibe 10 mg and simvastatin 40 mg, coadministered and alone, on the in vivo kinetics of apolipoprotein (apo) B-48 and B-100 in humans. Subjects underwent a primed-constant infusion of a stable isotope in the fed state. The coadministration of simvastatin and ezetimibe significantly reduced plasma concentrations of cholesterol (−43.0%), LDL-C (−53.6%), and triglycerides (−44.0%). Triglyceride-rich lipoproteins (TRL) apoB-48 pool size (PS) was significantly decreased (−48.9%) following combination therapy mainly through a significant reduction in TRL apoB-48 production rate (PR) (−38.0%). The fractional catabolic rate (FCR) of VLDL and LDL apoB-100 were significantly increased with all treatment modalities compared with placebo, leading to a significant reduction in the PS of these fractions. We also observed a positive correlation between changes in TRL apoB-48 PS and changes in TRL apoB-48 PR (r = 0.85; P < 0.0001) with combination therapy. Our results indicate that treatment with simvastatin plus ezetimibe is effective in reducing plasma TRL apoB-48 levels and that this effect is most likely mediated by a reduction in the intestinal secretion of TRL apoB-48. Our study also indicated that the reduction in LDL-C concentration following combination therapy is mainly driven by an increase in FCR of apoB-100 containing lipoproteins.     

Physiological correlates of seasonal growth patterns in lake trout Salvelinus namaycush

Physiological correlates of seasonal growth patterns were measured in lake trout Salvelinus namaycush from two populations with contrasting diets (zooplankton-dominated diet in Louisa Lake; fish-dominated diet in Opeongo Lake). Fish in Opeongo Lake grew faster and were in better condition than fish in Louisa Lake. The most prominent biochemical difference between populations was higher citrate synthase (CS) and cytochrome c oxidase activity in the white muscle of fish from Opeongo Lake, indicating greater sustained swimming activity in this lake. In contrast, lactate dehydrogenase (LDH) activity in white muscle, an indicator of capacity for burst swimming, was similar between lakes. Nucleoside diphosphate kinase (NDPK) activity in white muscle, an indicator of protein synthesis, was higher in Opeongo Lake than in Louisa Lake but only in the autumn. In both lakes, protein concentration and therefore nutritional status increased as the growing season progressed from spring to summer to autumn. Biochemical indicators of growth and activity showed similar seasonal patterns in the two lakes with the spring characterized by high NDPK, high CS and high LDH activities (i.e. high levels of protein synthesis in association with high aerobic and anaerobic activities). These results suggest high foraging effort and allocation to growth early in the growing season in both lakes.     

Bradykinin receptors: towards new pathophysiological roles

In addition to being a pro-inflammatory mediator, bradykinin is now recognized as a neuromediator and regulator of several vascular and renal functions. New breakthroughs point to unusual and atypical signalling pathways for a G-protein coupled receptor that could explain the anti-proliferative and anti-fibrogenic effects of bradykinin. The availability of transgenic and knock out animal models for bradykinin receptors or bradykinin-synthesizing or -catabolic enzymes confirms these cardiac and renal protective roles for this peptide system. Bradykinin receptors are involved in the therapeutic action of angiotensin-1 converting enzyme inhibitors that are used in the treatment of arterial hypertension, heart failure and diabetes. Nevertheless, recent evidence highlights dissimilar mechanisms in the regulation and function of these receptors between the central nervous system and peripheral tissues. Therefore, the development of more specific bradykinin receptor agonists or antagonists devoid of central actions seems to evolve as a new therapeutic approach.