Catalytic properties of thimet oligopeptidase H600A mutant

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His⁶⁰⁰. In the present work, the role of His⁶⁰⁰ of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S₁ and S₁′ specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His⁶⁰⁰ residue makes important interactions with the substrate, supporting the prediction that His⁶⁰⁰ moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K_m and k_cat, showing the importance of His⁶⁰⁰ for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His⁶⁰⁰ in TOP catalysis, transferring a proton to the newly generated NH₂-terminus or helping Tyr⁶⁰⁵ and/or Tyr⁶¹² in the intermediate oxyanion stabilization.     

QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

Background

Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes.

Results

The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue.

Conclusions

A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.     

Quantitative evaluation of yeast's requirement for glycerol formation in very high ethanol performance fed-batch process

Background

Glycerol is the major by-product accounting for up to 5% of the carbon in Saccharomyces cerevisiae ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardising growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH). Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP) fed-batch process.

Results

The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The fine-tuned reduction of specific GPDH activity was achieved by replacing the native GPD1 promoter in the yeast genome by previously generated well-characterized TEF promoter mutant versions in a gpd2 Δ background. Two TEF promoter mutant versions were selected for this study, resulting in a residual GPDH activity of 55 and 6%, respectively. The corresponding strains were referred to here as TEFmut7 and TEFmut2. The genetic modifications were accompanied to a strong reduction in glycerol yield on glucose; the level of reduction compared to the wild-type was 61% in TEFmut7 and 88% in TEFmut2. The overall ethanol production yield on glucose was improved from 0.43 g g^-1 in the wild type to 0.44 g g^-1 measured in TEFmut7 and 0.45 g g^-1 in TEFmut2. Although maximal growth rate in the engineered strains was reduced by 20 and 30%, for TEFmut7 and TEFmut2 respectively, strains' ethanol stress robustness was hardly affected; i.e. values for final ethanol concentration (117 ± 4 g L^-1), growth-inhibiting ethanol concentration (87 ± 3 g L^-1) and volumetric ethanol productivity (2.1 ± 0.15 g l^-1 h^-1) measured in wild-type remained virtually unchanged in the engineered strains.

Conclusions

This work demonstrates the power of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Under the conditions used in this study (VHEP fed-batch), the two strains with "fine-tuned" GPD1 expression in a gpd2 Δ background showed slightly better ethanol yield improvement than previously achieved with the single deletion strains gpd1 Δ or gpd2 Δ. Although glycerol reduction is known to be even higher in a gpd1 Δ gpd2 Δ double deletion strain, our strains could much better cope with process stress as reflected by better growth and viability.     

Steady-state mycophenolate mofetil pharmacokinetic parameters enable prediction of systemic lupus erythematosus clinical flares: an observational cohort study

Introduction  

The aim of this study was to determine whether mycophenolate mofetil (MMF) pharmacokinetics (PK) under combined MMF and prednisone remission-maintenance therapy can predict systemic lupus erythematosus (SLE) clinical flares.     

Adrenomedullin increases fibroblast-like synoviocyte adhesion to extracellular matrix proteins by upregulating integrin activation

Introduction  

Rheumatoid arthritis (RA) is characterized by bone and cartilage invasion by fibroblast-like synoviocytes (FLSs). Adrenomedullin, a peptide with anabolic and antiapoptotic properties, is secreted by rheumatoid FLSs. Adrenomedullin also increases the expression of adhesion molecules in endothelial cells and keratinocytes. Here, we investigated whether adrenomedullin mediated FLS adhesion to extracellular matrix (ECM) proteins.     

Chips from chips: application to the study of antibody responses to methylated proteins

Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.     

Angiotensin II increases adipose angiotensinogen expression

In addition to the well-defined contribution of the liver, adipose tissue has been recognized as an important source of angiotensinogen (AGT). The purpose of this study was to define the angiotensin II (ANG II) receptors involved in regulation of adipose AGT and the relationship of this control to systemic AGT and/or angiotensin peptide concentrations. In LDL receptor-deficient (LDLR(-/-)) male mice, adipose mRNA abundance of AGT was 68% of that in liver, and adipose mRNA abundance of the angiotensin type 1a (AT(1a)) receptor (AT(1a)R) was 38% of that in liver, whereas mRNA abundance of the angiotensin type 2 (AT(2)) receptor (AT(2)R) was 57% greater in adipose tissue than in liver. AGT and angiotensin peptide concentrations were decreased in plasma of AT(1a)R-deficient (AT(1a)R(-/-)) mice and were paralleled by reductions in AGT expression in liver. In contrast, adipose AGT mRNA abundance was unaltered in AT(1a)R(-/-) mice. AT(2)R(-/-) mice exhibited elevated plasma angiotensin peptide concentrations and marked elevations in adipose AGT and AT(1a)R mRNA abundance. Increases in adipose AGT mRNA abundance in AT(2)R(-/-) mice were abolished by losartan. In contrast, liver AGT and AT(1a)R mRNA abundance were unaltered in AT(2)R(-/-) mice. Infusion of ANG II for 28 days into LDLR(-/-) mice markedly increased adipose AGT and AT(1a)R mRNA but did not alter liver AGT and AT(1a)R mRNA. These results demonstrate that differential mRNA abundance of AT(1a)/AT(2) receptors in adipose tissue vs. liver contributes to tissue-specific ANG II-mediated regulation of AGT. Chronic infusion of ANG II robustly stimulated AT(1a)R and AGT mRNA abundance in adipose tissue, suggesting that adipose tissue serves as a primary contributor to the activated systemic renin-angiotensin system.     

Autoantigen-specific TGFbeta-induced Foxp3+ regulatory T cells prevent autoimmunity by inhibiting dendritic cells from activating autoreactive T cells

Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+ Foxp3- T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFbeta. TGFbeta-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.     

Neuroprotection and stroke: time for a compromise

In April 2007, there existed a repertory of 286 trials concerned with acute ischemic stroke on the Stroke Trials Registry ( http://www.strokecenter.org/trials/ ), of which 209 trials were considered as complete (with no evidence of patient benefit unless one considers the much hard fought for and modest results of the tPA studies). Among other questions arising from such failures, one can wonder whether the plethora of pharmacological agents that exhibited neuroprotective properties in pre-clinical studies were selected for clinical trials entirely based upon their experimental efficacy. This mini-review will try to point out some of the weaknesses that could underline the failure of both researchers and clinicians involved in the field of stroke to obtain their ultimate goal – brain protection.