Detection and identification of multiple genetically modified events using DNA insert fingerprinting.

Current screening and event-specific polymerase chain reaction (PCR) assays for the detection and identification of genetically modified organisms (GMOs) in samples of unknown composition or for the detection of non-regulated GMOs have limitations, and alternative approaches are required. A transgenic DNA fingerprinting methodology using restriction enzyme digestion, adaptor ligation, and nested PCR was developed where individual GMOs are distinguished by the characteristic fingerprint pattern of the fragments generated. The inter-laboratory reproducibility of the amplified fragment sizes using different capillary electrophoresis platforms was compared, and reproducible patterns were obtained with an average difference in fragment size of 2.4 bp. DNA insert fingerprints for 12 different maize events, including two maize hybrids and one soy event, were generated that reflected the composition of the transgenic DNA constructs. Once produced, the fingerprint profiles were added to a database which can be readily exchanged and shared between laboratories. This approach should facilitate the process of GMO identification and characterization.

The effect of electronegativity and angiotensin-converting enzyme inhibition on the kinin-forming capacity of polyacrylonitrile dialysis membranes.

The combination of negatively-charged membranes and angiotensin I-converting enzyme inhibitors (ACEi) evokes hypersensitivity reactions (HSR) during hemodialysis and bradykinin (BK)-related peptides have been hypothesized as being responsible for these complications. In this study, we tested the effects of neutralizing the membrane electronegativity (zeta potential) of polyacrylonitrile AN69 membranes by coating a polyethyleneimine layer (AN69-ST membranes) over the generation of kinins induced by blood contact with synthetic membranes. We used minidialyzers with AN69 or AN69-ST membranes in an ex vivo model of plasma and we showed that plasma dialysis with AN69 membranes led to significant BK and des-Arg(9)-BK release, which was potentiated by ACEi. This kinin formation was dramatically decreased by AN69-ST membranes, even in the presence of an ACEi, and kinin recovery in the dialysates was also significantly lower with these membranes. High molecular weight kininogen and factor XII detection by immunoblotting of the protein layer coating both membranes corroborated the results: binding of these proteins and contact system activation on AN69-ST membranes were reduced. This ex vivo experimental model applied to the plasma, dialysate and dialysis membrane could be used for the characterization of the kinin-forming capacity of any biomaterial potentially used in vivo in combination with drugs which modulate the pharmacological activity of kinins.

Cardiovascular expression of inflammatory signaling molecules, the kinin B1 receptor and COX2, in the rabbit: effects of LPS, anti-inflammatory and anti-hypertensive drugs.

We first aimed to test the effect of anti-inflammatory drugs, etanercept and dexamethasone sodium phosphate (DSP), on the expression of inducible inflammatory signaling molecules (the bradykinin [BK] B(1) receptor [B(1)R], cyclooxygenase [COX]-2) in lipopolysaccharide (LPS)-treated rabbits. Preliminary experiments mostly based on a novel cellular model, rabbit dermis fibroblasts, showed that etanercept inhibited TNF-alpha-induced B(1)R expression ([(3)H]Lys-des-Arg(9)-BK binding), but that DSP also inhibited cytokine-induced B(1)R upregulation with less selectivity. LPS (100 microg/kg i.v.) induced the expression of the B(1)R (aortic contractility ex vivo, mRNA in hearts) and COX2 (immunoblots, heart extracts). However, the function of the BK B(2) receptor was unchanged (jugular vein contractility ex vivo). DSP pre-treatment profoundly reduced the induction of the B(1)R and COX2 whereas etanercept significantly inhibited only COX2 expression. The second aim was to verify whether chronic angiotensin converting enzyme (ACE) blockade in rabbits would induce B(1)R expression, as reported in other species. 14-Day enalapril oral dosing, but not treatment with the angiotensin receptor antagonist losartan, significantly increased aortic contractions mediated by B(1)Rs, however much less than LPS. Enalapril treatment did not increase COX2 expression but increased the ex vivo relaxation of the mesenteric artery mediated by endogenous prostaglandins. Chronic ACE inhibition recruits inflammatory signaling systems.

[Physiopathology of the acute adverse effects of angiotensin-converting-enzyme inhibitors]. / Physiopathologie des effets secondaires aigus des inhibiteurs de l'enzyme de conversion de l'angiotensine.

Angiotensin-I-converting-enzyme inhibitors are currently used to treat more than 40 million cardiovascular patients worldwide. These drugs have a variety of acute adverse effects, the nature of which depends on the clinical context, and which include angioedema, anaphylactoid reactions in hemodialysis patients, and severe hypotensive reactions during blood product transfusions. These adverse effects result from a combination of factors affecting the synthesis, metabolism and pharmacological activity of bradykinin and des-arginine9-bradykinin, two powerful vasodilatory and pro-inflammatory peptides. Experimental evidence obtained in our laboratory suggests that acquired, genetic and pharmacological factors can influence the risk of these rare but potentially life-threatening effects.

Role of nuclear factor-kappaB and protein kinase C signaling in the expression of the kinin B1 receptor in human vascular smooth muscle cells.

Kinin B1 receptor expression was characterized in human umbilical artery smooth muscle cells to further elucidate the function and specificity of three previously proposed pathways [nuclear factor-kappaB (NF-kappaB), protein kinase C, and agonist autoregulation] that regulate this inducible G protein-coupled receptor. Radioligand binding assays, real-time reverser transcription/polymerase chain reaction with an optional actinomycin D treatment period, and NF-kappaB immunofluorescence were primarily employed in these primary cell cultures. Various stimulatory compounds that increase receptor mRNA stability only (human and bovine sera, cycloheximide) or that stimulate NF-kappaB nuclear translocation and both mRNA concentration and stability [interleukin (IL)-1beta, phorbol 12-myristate 13-acetate (PMA)] all increased the density of binding sites for the tritiated B1 receptor agonist [3H]Lys-des-Arg9-bradykinin (without change in receptor affinity) in cell-based assays. Small interfering RNA assays indicated that NF-kappaB p65 is necessary for the effective expression of the cell surface B1 receptor under basal or IL-1beta, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1beta-, FBS-, or PMA-induced stabilization of B1 receptor mRNA was inhibited by the addition of the protein kinase C inhibitor 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF-109203x), which also diminished the Bmax under FBS or PMA treatment. Lys-des-Arg9-bradykinin had little effect on NF-kappaB activation, the Bmax, or receptor mRNA abundance or stability. Both NF-kappaB and protein kinase C signaling are required for the effective expression of the kinin B1 receptor in human umbilical artery smooth muscle cells.

Generation of kinins during preparation and storage of whole blood-derived platelet concentrates.

BACKGROUND: Leukoreduction of platelet (PLT) concentrates (PCs) may be associated with hypotension in recipients, and a role for bradykinin (BK)-related peptides has been proposed for this side effect. STUDY DESIGN AND METHODS: The concentration of BK and one of its vasoactive metabolites, des-arginine(9)-BK (des-Arg(9)-BK), was measured in a large number of PCs as a function of leukoreduction and storage duration with specific enzyme immunoassays and complementary techniques. RESULTS: On Day 0 of storage, kinins were detected in leukoreduced and unfiltered PCs at a concentration lower than 100 pg per mL. During storage, both kinin levels peaked on Day 5 of storage, with a concentration higher than 1 ng per mL in 22 percent of PCs whether filtered on Day 0 or not. Physicochemical and pharmacologic characterizations of immunoreactive kinins confirm their nature. In vitro activation of the contact system of the corresponding PLT-poor plasma showed that a high kinin concentration on Day 5 of the storage corresponded with a low kinin-forming capacity of plasma. On Day 7, BK was no longer elevated presumably due to its degradation and the depletion of kinin-forming capacity of the plasma in stored PCs. The activities of metallopeptidases that metabolize BK-related peptides in plasma from PCs were at levels similar to those recorded in the plasma of a normal reference population and were unaffected by storage. CONCLUSION: Storage of PCs contributes to the hydrolysis of high-molecular-weight kininogen and generation of pharmacologically relevant BK levels that might pose a hazard in susceptible patients.

The kallikrein-kinin system: current and future pharmacological targets.

The kallikrein-kinin system is an endogenous metabolic cascade, triggering of which results in the release of vasoactive kinins (bradykinin-related peptides). This complex system includes the precursors of kinins known as kininogens and mainly tissue and plasma kallikreins. The pharmacologically active kinins, which are often considered as either proinflammatory or cardioprotective, are implicated in many physiological and pathological processes. The interest of the various components of this multi-protein system is explained in part by the multiplicity of its pharmacological activities, mediated not only by kinins and their receptors, but also by their precursors and their activators and the metallopeptidases and the antiproteases that limit their activities. The regulation of this system by serpins and the wide distribution of the different constituents add to the complexity of this system, as well as its multiple relationships with other important metabolic pathways such as the renin-angiotensin, coagulation, or complement pathways. The purpose of this review is to summarize the main properties of this kallikrein-kinin system and to address the multiple pharmacological interventions that modulate the functions of this system, restraining its proinflammatory effects or potentiating its cardiovascular properties.

Expression of metallopeptidases and kinin receptors in swine oropharyngeal tissues: effects of angiotensin I-converting enzyme inhibition and inflammation.

Angiotensin I-converting enzyme inhibitors (ACEi) cause both chronic and acute side effects, including rare but potentially life-threatening angioedema (AE). The main hypothesis to be tested in this study was that metallopeptidases and kinin receptors are present in oropharyngeal tissues and that their expression is modulated by ACEi and inflammation. Novel real-time polymerase chain reaction analysis was developed and allowed the relative quantification of tissue's gene expression for neprilysin, membrane-bound aminopeptidase P (mAPP), and both B1 and B2 kinin receptor subtypes in tongue, parotid gland, and laryngeal tissue (areas especially involved in the gravest clinical forms of AE) and in kidney in a porcine model (single injection or 7-day ACEi oral treatments applied or lipopolysaccharide injected as a positive inflammatory control). The results provide evidence of the expression and activities of kininases in oropharyngeal tissues in the swine. ACEi treatment modulated the expression of neutral endopeptidase and mAPP mRNA, but the corresponding enzyme activities and that of angiotensin I-converting enzyme (ACE) were generally stable in tissues. The 7-day ACEi treatment up-regulated both kinin receptor mRNAs in the oropharynx and the B1 receptor mRNA in the lingual vascular endothelium (immunohistochemistry). The inhibition of ACE in plasma is responsible for an accumulation of bradykinin and des-arginine9-bradykinin generated during activation of the contact system with glass beads. The expression of critical components of the kallikrein-kinin system in the oropharyngeal tissues supports the role of kinins in ACEi-induced AE.

Interfacial enzymology of parvovirus phospholipases A2.

The capsid of parvoviruses proteins were recently shown to contain secreted phospholipase A(2) (sPLA(2))-like activity that is required during host cell entry. Parvoviral PLA(2) domains have little sequence identity with sPLA(2)s and lack disulfide bonds. In the present study, after bacterial expression and purification, the biochemical characterizations of these first PLA(2)s identified in viruses have been investigated, and a comparison has been made with other known PLA(2)s. The specific activities of three viral PLA(2)s differed by 3 orders of magnitude, with porcine parvovirus PLA(2) displaying a specific activity similar to that of the most active sPLA(2)s (e.g. human group IIA) and the human AAV2 and B19 parvoviral enzymes displaying approximately 10(3) lower specific activities (similar to human sPLA(2) groups IIE and XIIA). These differences were not caused by weaker Ca(2+) or interfacial binding. The specific activities of the viral PLA(2)s on zwitterionic or anionic phospholipid vesicles were comparable. The viral PLA(2)s did not display a preference for unsaturated versus saturated sn-2 fatty acyl chains and hydrolyzed all major classes of glycero-phospholipids except phosphatidylinositol. Incubation of mammalian cells with porcine parvovirus PLA(2) led to the release of arachidonic acid into the culture medium. Interestingly, among nine previously known sPLA(2) inhibitors, only a subset showed inhibition of the viral PLA(2)s and with weak potency, indicating that the active sites of these new enzymes are structurally distinct from those of sPLA(2)s. Based on these distinct enzymatic and structural properties, we propose to classify the parvovirus PLA(2)s within the PLA(2) superfamily as group XIII enzymes.