Gibberellin-regulated proteins: Emergent allergens.

About 10 years ago, a protein family was shown for the first time to contain allergenic members, gibberellin-regulated protein (GRP). The first reported member was from peach, Pru p 7. One can hypothesize that it was not detected before because its physicochemical characteristics overlap with those of lipid transfer protein (LTP), a well-known allergen, or because the exposure to GRP increased due to an increase in the gibberellin phythormone level in plant food, either exogenous or endogenous. Like LTPs, GRPs are small cationic proteins with disulfide bridges, are resistant to heat and proteolytic cleavage, and are involved in the defense of the plant. Besides peach, GRP allergens have been described in Japanese apricot (Pru m 7), sweet cherry (Pru av 7), orange (Cit s 7), pomegranate (Pun g 7), bell pepper (Cap a 7), strawberry (Fra a GRP), and also in pollen with a restriction to Cupressaceae tree family (Cup s 7, Cry j 7, and Jun a 7). IgE cross-reactivities were described between GRPs, and the reported peach/cypress and citrus/cypress syndromes may therefore be explained because of these GRP cross-reactivities. GRPs are clinically relevant, and severe adverse reactions may sometimes occur in association with cofactors. More than 60% and up to 95% sequence identities are calculated between various allergenic GRPs, and three-dimensional models show a cleft in the molecule and predict at least three epitopic regions. The structure of the protein and its properties and the matrix effect in the original allergenic source should be unraveled to understand why, despite the ubiquity of the protein family in plants, only a few members are able to sensitize patients.

[Present possibilities and future development of clinical proteomics]. / Présent et futur de la Protéomique clinique.

This review focuses on "clinical proteomics" which represents an emerging discipline in biomedical research. "Clinical proteomics" relies on the analysis of the proteome, i.e. the entire set of peptides and proteins present in a biological sample, to provide relevant data for diagnosis, prognosis or therapeutic strategies of human pathologies. This new type of approach has tremendous potential for the diagnosis of complex pathologies or for the early detection of cancers. This article reports the conclusions of a workgroup of the French Society for Clinical Biology (SFBC) 2004-2006 which evaluated the status, the impact and the future development of proteomics in the clinical field. It provides therefore a broad view going from the methods already present in the clinical laboratories (multiplex technologies...), to the tools for clinical and basis research including bioinformatics.

[Angiotensin I-converting enzyme in cerebrospinal fluid and neurosarcoidosis]. / Enzyme de conversion de l'angiotensine I dans le liquide céphalorachidien et neurosarcoïdose.

Sarcoidosis is a disease of unknown aetiology. This granulomatous disease is essentially localized in lung and skin, but many other localizations are possible, such as in nervous system. Sometimes the neurological involvement is alone leading to a differential diagnosis from other neurological diseases. Angiotensin I-converting enzyme (ACE) is synthesized by sarcoidotic granulomas and diffuses in various biological fluids. The determination of ACE activity in cerebrospinal fluid (CSF) can help for the diagnosis of neurosarcoidosis, associated or not to its determination in serum. We developed a radiometric assay for the determination of ACE activity in CSF since the methods for serum cannot be used because ACE activity is low in CSF, as well as in pathological situations. At the analytical point of view this assay is sensitive, specific and reproducible. We established a normal range and yielded recommendations to give the results, particularly in function of the aspect of the CSF and the proteinorrachia. But increased level of ACE in CSF is not specific of neurosarcoidosis since elevations were also shown in meningitis. We can claim for the routine use of ACE assay in CSF for differential diagnosis by eliminating neurosarcoidosis, as well as for positive diagnosis of this disease, but in both cases with the confrontation to other parameters.

Cardiac troponin-I on diagnosis predicts early death and refractoriness in acquired thrombotic thrombocytopenic purpura. Experience of the French Thrombotic Microangiopathies Reference Center.

BACKGROUND: Cardiac involvement is a major cause of mortality in patients with thrombotic thrombocytopenic purpura (TTP). However, diagnosis remains underestimated and delayed, owing to subclinical injuries. Cardiac troponin-I measurement (cTnI) on admission could improve the early diagnosis of cardiac involvement and have prognostic value. OBJECTIVES: To assess the predictive value of cTnI in patients with TTP for death or refractoriness. PATIENTS/METHODS: The study involved a prospective cohort of adult TTP patients with acquired severe ADAMTS-13 deficiency (< 10%) and included in the registry of the French Reference Center for Thrombotic Microangiopathies. Centralized cTnI measurements were performed on frozen serum on admission. RESULTS: Between January 2003 and December 2011, 133 patients with TTP (mean age, 48 ± 17 years) had available cTnI measurements on admission. Thirty-two patients (24%) had clinical and/or electrocardiogram features. Nineteen (14.3%) had cardiac symptoms, mainly congestive heart failure and myocardial infarction. Electrocardiogram changes, mainly repolarization disorders, were present in 13 cases. An increased cTnI level (> 0.1 µg L(-1) ) was present in 78 patients (59%), of whom 46 (59%) had no clinical cardiac involvement. The main outcomes were death (25%) and refractoriness (17%). Age (P = 0.02) and cTnI level (P = 0.002) showed the greatest impact on survival. A cTnI level of > 0.25 µg L(-1) was the only independent factor in predicting death (odds ratio [OR] 2.87; 95% confidence interval [CI] 1.13-7.22; P = 0.024) and/or refractoriness (OR 3.03; 95% CI 1.27-7.3; P = 0.01). CONCLUSIONS: A CTnI level of > 0.25 µg L(-1) at presentation in patients with TTP appears to be an independent factor associated with a three-fold increase in the risk of death or refractoriness. Therefore, cTnI level should be considered as a prognostic indicator in patients diagnosed with TTP.

Anticancer drugs induce necrosis of human endothelial cells involving both oncosis and apoptosis.

The endothelium is the first physiological barrier between blood and tissues and can be injured by physical or chemical stress, particularly by the drugs used in cancer therapy. We found that four anticancer agents: etoposide, doxorubicin, bleomycin and paclitaxel induced apoptosis in human umbilical vein endothelial cells (HUVECs) (as judged by DNA fragmentation) with a time- and concentration-dependent decrease in bcl-2 protein but without the involvement of p53. As revealed by immunoblotting, bax protein was expressed in HUVECs treated with 1 mg/ml etoposide whereas bcl-2 protein disappeared. Oncosis occurred parallel to apoptosis with the release of lactate dehydrogenase into the supernatant, and, for doxorubicin and etoposide with the inversion of the distribution of angiotensin I-converting enzyme between supernatant and cells. Among the four tested anticancer drugs, only doxorubicin induced an oxidative stress, with significative malondialdehyde production. Thus, human endothelial cells in confluent cultures seem to be in an equilibrium of resistance to apoptosis related to bcl-2 expression; this equilibrium can be disrupted by a chemical stress, such as the antiproliferative drugs known as pro-apoptotic for tumour cells. For doxorubicin and bleomycin, this cellular toxicity can be related to their unwanted effects in human cancer therapy. Low doses of doxorubicin, paclitaxel or etoposide, however, could induce apoptosis of endothelial cells of new vessels surrounding the tumour, thus leading to specific vessel regression with minimal toxic effects for the endothelium of the other vessels. These findings provide evidence of relationships between endothelial toxicity of anticancer drugs and the key role of bcl-2 for resistance of endothelium cells toward apoptosis; moreover lack of p53 and bax in quiescent cells contributes to resistance of endothelial cells to DNA-damaging agents.

Vascular origin determines angiotensin I-converting enzyme expression in endothelial cells.

Previous observations on the heterogeneous distribution of von Willebrand factor in the vascular endothelium led us to examine the expression of angiotensin I-converting enzyme (ACE) in function of the vascular origin of endothelial cells (EC). EC from pig thoracic aorta, pulmonary artery, inferior vena cava and brain capillaries were cultured and assayed for ACE by enzymatic radiochemical determination and by western-blot and immunofluorescence using an antiACE polyclonal antibody. EC from the various vascular levels secreted ACE in the culture medium; western-blot analysis showed its presence at cellular level and immunofluorescence confirmed its location on the plasma membrane. But quantification revealed that EC from pulmonary artery contain more ACE than EC from the other vessels, especially from brain capillaries; immunofluorescence correlated well with the functional data. In contrast, secretion of ACE by brain capillaries EC was faster than that of arteries and of vena cava, the latter being the less effective. This differential ACE expression along the vascular tree could have a pharmacological implication since ACE inhibitors, used in the treatment of arterial hypertension, may act more at the vascular level than on the plasma renin-angiotensin system. On the other hand, endothelial distribution of ACE was different from that of von Willebrand factor; in particular we showed that EC cultured from vessels of pigs homozygous for the von Willebrand disease, in which von Willebrand factor synthesis was completely abolished, normally express ACE.

Liver veno-occlusive disease after bone marrow transplantation changes in coagulation parameters and endothelial markers.

Veno occlusive disease (VOD) is a frequent complication of allogenic bone marrow transplantation (BMT) for which no predictive blood markers are available. 39 patients grafted for severe aplastic anemia (18), and leukemia (21) were prospectively studied. Of the 39 patients, 5 leukemic patients, but no aplastic patients developed VOD. In all the 5 patients with VOD complications we demonstrated a decrease in factor VII and in protein C before the clinical onset of the disease and before any changes in hepatic enzymes were observed. This decrease is the earliest sign of hepatic involvement by the VOD suggesting that the determination of Factor VII and protein C can be used as a prediction test to identify the patients who are at risk of developing VOD after transplantation. In addition, a toxicity of the endothelial cells was suggested by the observed increase in von Willebrand factor and in Serum Angiotensin Converting Enzyme. Signs of endothelial toxicity was more pronounced in leukemic than in aplastic patients.

Plasma fibronectin and angiotensin-converting enzyme: markers of primary pulmonary injury in burn patients.

Plasma fibronectin (FBN) and angiotensin I-converting enzyme (ACE) were prospectively measured in 50 burn patients from the day of admission to day 28 after the trauma with the aim of finding biochemical markers of pulmonary injury by smoke inhalation. Patients were divided into three groups on the basis of fiberoptic bronchoscopy results (group I: healthy lungs; group II: burned lungs; group III: infected lungs). A decrease in FBN concentrations was observed in the three groups but was larger in group II than in the other groups, especially at day 2 (P < 0.05). A similar profile was observed for plasma ACE activity. Factors other than lung injury may influence plasma FBN and ACE levels, in particular the burned body surface area, an acute event such as septicemia, or outcome. However, repeated measurements of both markers could help in the assessment of lung injury in the follow-up of burn patients.

Physiochemical and immunological comparisons between angiotensin I-converting enzymes purified from different mammalian species.

Angiotensin I-converting enzyme (ACE) was purified from lungs of pig, rat, monkey and human for comparison of its physicochemical, enzymatic and immunological properties. The protocol involved three chromatographic steps after detergent extraction, i.e. DEAE-Sphérodex ion exchange, lisinopril-Sepharose affinity and Superose 12 HPLC, plus Mono-Q HPLC for monkey ACE. Purified ACE's presented numerous homologies: in particular, closely similar specific activities, catalytic efficiencies, Km's, optimal pH and chloride activations; the molecular weights were about 170 kDa by SDS-PAGE and 320 kDa by gel-filtration on Superose 12; the isoelectric points were about 4.5-4.7. Specific polyclonal antibodies recognized the antigen (porcine ACE) as well as rat, monkey and human ACEs. In contrast, three monoclonal antibodies (F02.4.1, F01.1.3 and F03) produced against porcine ACE showed some differences: they only reacted with pig enzyme and only one (F0.2.4.1) was anticatalytic. Moreover, the cross-reactivity judged on ELISA with porcine ACE characterized different epitopes specific for the porcine enzyme. In particular, the binding of F02.4.1 was not diminished by previous treatment with saturating concentrations of synthetic competitive ACE inhibitors. Thus, the extrapolation to human of data obtained on animal models should be possible at least for pharmacological and medical trials.

[Study of the intronic polymorphism of the angiotensin 1 converting enzyme among coronary Tunisians]. / Étude du polymorphisme intronique de l'enzyme de conversion de l'angiotensine I chez des coronariens tunisiens.

BACKGROUND: The acute coronary syndromes (ACS) are classified among the major causes of mortality in the industrialized countries. The increased angiotensin I converting enzyme (ACEI) activity related to a genetic polymorphism constitutes a hereditary predisposition to these syndromes. AIM: Evaluate the ACEI activity in Tunisian patients with coronary heart disease, and investigate the association between this activity and an intronic deletion of 287 pb on the intron 16 of the ACEI gene. PATIENTS AND METHODS: Seventy-two coronary patients and 34 control subjects are recruited for our study. ACEI activity was measured by kinetic method. The intronic deletion was identified by PCR technique. RESULTS: An increased activity of ACEI was observed in patients compared with control subjects (84.38 ± 33.83 UI/L vs 59.06 ± 18.2 UI/L, P=10(-5)). The molecular study showed a raised relative frequency of D/D genotype (51.4%) among patients, whereas among the witnesses, I/I genotype prevailed (62%). D/D genotype is always associated with highest ACEI activity for the patients and the control subjects. CONCLUSION: The molecular studies and the biochemical investigations of the various parameters of cardiovascular risk (including the ACEI) direct towards a better treatment.

[Angiotensin I-converting enzyme (ACE) for sarcoidosis diagnosis]. / L'enzyme de conversion de l'angiotensine I (ECA) dans le diagnostic de la sarcoïdose.

Angiotensin I-converting enzyme (ACE) is synthesized by sarcoidotic granulomas defining this enzyme as a diagnosis and prognosis marker of sarcoidosis. This granulomatous disease, a frequent disease with unknown aetiology, is essentially localized in lung and skin, but many other localizations are possible, as well as in nervous system. The diagnosis is based on a panel of clinical, biological and radiological arguments. Serum ACE has a particular interest although its sensitivity and specificity for sarcoidosis are not perfect; ACE can also help to follow this disease either spontaneously or after corticoid therapy. ACE can be measured in bronchoalveolar lavage fluid (BAL) where it better signs the activity of the pulmonary disease than in serum. ACE in cerebrospinal fluid (CSF), and eventually in other biological fluids, allows the diagnosis of sarcoidosis with extra-pulmonary locations. The methods for ACE activity determination are essentially based on the use of polypeptidic synthetic substrates. By varying their nature and the conditions for use various assays were developed for serum, plasma, BAL, CSF and other fluids where ACE can help. The data must be given in a critical manner in function of the actual knowledge on ACE gene, the enzymatic properties of the protein and the limits of sensitivity and specificity of the assays sometimes reserved to peculiar biological fluids.

Angiotensin I-converting enzyme gene polymorphism and drug response.

An insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene has been described in chromosome 17q23 of the human genome. Subjects with the genotype DD have markedly higher plasma ACE levels than those with genotype II; although ACE concentration in plasma is not rate-limiting for the production of angiotensin II, it has been suggested that the renin-angiotensin system may have an enhanced role in cardiovascular homeostasis in subjects with DD genotype or D allele. Meta-analysis confirmed the association of the D allele with an increased risk of myocardial infarction and stroke, but these relations are still uncertain with longevity and renal deterioration. Otherwise, I allele seems to be related with an improved response to physical training. The I/D polymorphism of the ACE gene is not a marker for any form of hypertension, though some conflicting results have been described. Nevertheless this polymorphism may have an influence on the antihypertensive response, particularly when using ACE inhibitors (ACEI). For example, blood pressure normalization with captopril in patients suffering from cardiac failure would be more effective in II genotype; conversely, both regression in left ventricular hypertrophy and improvement in diastolic filling would be greater after long-term treatment with enalapril in patients with essential hypertension and DD genotype. Conflicting results were also described using ACEI as a renoprotective therapy. This review therefore supports the justification for further evaluation in appropriately powered studies.

Carboxypeptidase A hydrolyses benzoylglycyl-histidyl-leucine but not furylacryloyl-phenylalanyl-glycyl-glycine, two usual substrates for angiotensin I-converting enzyme.

We compared angiotensin I-converting enzyme (ACE) and carboxypeptidase A (CPA), two zinc metallopeptidases, for the hydrolysis of the usual ACE synthetic substrate benzoylglycyl-histidyl-leucine (HHL) investigating the possible interference by CPA in the determination of ACE activity in biological fluids. Both purified enzymes hydrolyse HHL in a radiochemical assay with the same optimal pH, a characteristic divalent metal requirement, a close similar behavior against inhibitors of other metallopeptidases, such as enkephalinase and kininase I, and the involvement of arginine and lysine residues in their active site. Conversely, CPA does not show the other catalytic properties of ACe, i.e. chloride dependence, low Km for HHL, inhibition by specific synthetic ACE inhibitors and antibody, also hydrolysis of the other ACE substrate furylacryloylphenylalanyl-glycyl-glycine (FAPGG). We advise the use of ACE inhibitors to validate ACE measurement with HHL or, alternatively, FAPGG, which is a more specific substrate for ACE, must be preferred, although the poor sensitivity of the spectrophotometric assay with this substrate limits its use to blood samples.

In vitro interactions between ramiprilat and angiotensin I-converting enzyme in endothelial cells.

Ramiprilat is an angiotensin I-converting enzyme (ACE) inhibitor whose particular lipophilicity may modify its inhibitory activity on the cellular form of this enzyme in comparison to ACE inhibitors that are more hydrophilic. The inhibitory activity of ramiprilat on cellular ACE and its binding to plasma membrane ACE have been studied in cultures of pig vascular endothelial cells. ACE activity in pig pulmonary artery endothelial cells is completely inhibited by 100 nM ramiprilat; the IC50 is 2 nM, whatever the form of ACE: soluble ACE released into the culture medium, cellular ACE studied in a cell monolayer homogenate, or tissue ACE purified from pig lung tissue. When added directly to whole living cells in their culture medium, 100 nM ramiprilat inhibits less than 80% of ACE activity in the supernatant while this dose inhibits the cellular form of the enzyme completely, suggesting that ramiprilat reaches and more specifically inhibits membrane-bound ACE rather than ACE secreted by the monolayer of endothelial cells. [3H]ramiprilat binds specifically to the membrane of cultured endothelial cells, in a time and dose-dependent manner (Kd = 6 nM; Bmax = 1,600 fmol/mg of protein); specificity is confirmed by the fact that an anti-ACE antibody prevents binding of [3H]ramiprilat and that both cold ramiprilat as well as other synthetic inhibitor compounds (captopril and enalaprilat) displace [3H]ramiprilat in a dose-dependent manner. Angiotensin I and bradykinin, natural substrates of ACE, do not induce displacement of [3H]ramiprilat bound to the luminal plasma membrane of endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin-converting enzyme: clinical applications and laboratory investigations on serum and other biological fluids.

Angiotensin I-converting enzyme (ACE) is a peptidyldipeptide hydrolase that is located mainly on the luminal surface of vascular endothelial cells but also in cells derived from the monocyte-macrophage system. Physiologically, ACE is a key enzyme in the renin-angiotensin system, converting angiotensin I into the potent vasopressor angiotensin II and also inactivating the vasodilator bradykinin. Increased serum ACE activity (SACE) has been reported in pathologies involving a stimulation of the monocytic cell line, primarily granulomatous diseases. Sarcoidosis is the most frequent and the better studied of these diseases; high SACE is not only a well-established marker for the diagnosis but is also a useful tool for following its course and evaluating the effect of therapy. SACE can also be increased in nonsarcoidotic pulmonary granulomatous diseases such as silicosis and asbestosis, in extrathoracic granulomatous pathologies such as Gauchers disease and leprosis, and, to a lesser extent, in nongranulomatous disorders such as hyperthyroidism or cholestasis. On the other hand, monitoring sarcoidosis obviates the measurement of ACE activity in other biological fluids, e.g., broncho-alveolar and cerebrospinal fluids, in the search of a locoregional dissemination or dis-simulation of the disease. Decreased SACE has been reported in vascular pathologies involving an endothelial abnormality, e.g., deep vein thrombosis, and in endothelium dysfunctions related to the toxicity of chemo- and radiotherapy used in cancers, leukemias, and hematopoietic or organ transplantations. SACE is also of interest for monitoring arterial hypertension treated with specific synthetic ACE inhibitors. These various reasons for determining ACE activity have led to the development of numerous methods. The most widely used is the spectrophotometric assay using hippuryl-histidyl-leucine as substrate. Fluorimetric and radiochemical assays using both classic and novel substrates have been proposed, but they are time consuming, require special apparatus, and are not suited to automation. Kinetic spectrophotometry of furylacryloyl-phenylalanyl-glycyl-glycine hydrolysis is now used extensively because it is easy to automatize. Efforts are now required to standardize one or more of these assays. Indeed, "normal" plasma values differ not only according to the substrate, but also to the method of determination and to sex and age.

Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing.

We developed two capillary isoelectric focusing (CIEF) assays, in narrow pH gradients, with the aim of routinely separating and quantitating normal and abnormal hemoglobins (Hbs): a one-step CIEF assay where residual electroosmotic flow mobilizes the proteins during focalization, and a two-step CIEF assay where focused Hbs are mobilized by low pressure by maintaining high-voltage. The resolution of 0.10 pH unit obtained with the one-step assay allowed the separation of the Hbs A, F, S and C; but Hb A2, which represents about 2-3% of whole Hb, could not be quantitated. The better resolution of 0.02 pH unit obtained with the two-step assay allowed the separation of some Hb variants of very close isoelectric points. The reproducibility of retention times was satisfactory (C.V.<5%). Moreover, in this configuration quantitation of Hb A2, Hb F and Hb S led to a standard deviation of less than 5%, allowing the diagnosis of thalassemias. The one-step assay could be useful only for the detection of abnormal variants, while the two-step assay could be applied to the routine analysis of Hbs, with quantitation of minor fractions and presumptive identification of variants.