Adsorption as a Contributor for Inflammatory Mediators Removal by Different Hemofiltration Membranes: A Pilot Study.

Atherosclerosis is an important predictor of mortality in patients with chronic kidney disease (CKD) and is associated with a wide inflammatory response. The aim of this study is to evaluate in vitro how different membranes can remove mediators associated with this pathology in a closed loop dialysis model. We performed experimental hemofiltration in vitro using three different membrane materials. Human plasma was preliminarily incubated with various inflammatory mediators and filtered in a closed loop circulation model for 240 min. Respective concentrations of 17 different mediators were measured over time to study the removal mechanisms of each membrane, including associated removal time course. The experiment was repeated three times for the assay of tumor necrosis factor (TNF)-α to document the model variability. Means were compared using Mann-Whitney test. Most of the investigated mediators were effectively removed with the different dialysis membranes. Adsorption mechanism was mainly at the origin of the decrease in mediators circulating concentrations and was maximized in the region 10 000-20 000 Da. Especially, the HeprAN membrane showed fast removal capacities of mediators with elevated isoelectric point including complement factors and chemokines or having basic groups located in the protein periphery, plasminogen activator inhibitor (PAI-1), and TNF-α-like. The latter was further significantly removed with HeprAN and polymethylmethacrylate (PMMA) compared to polyethersulfone (PES) material (P < 0.01). We concluded that dialysis using ionic adsorptive membrane could have a beneficial impact for CKD patients with atherosclerosis and would deserve further clinical investigations.

Biomarcadores de cáncer oral en saliva / Salivary analysis of oral cancer biomarkers

Esta revisión muestra los principales biomarcadores de cáncer oral en saliva. El aspecto clínico y el grado de displasia de las lesiones precancerosas de la cavidad bucal sugieren su capacidad de malignidad; sin embargo, éstas generalmente son diagnosticadas en estadios avanzados, disminuyendo la probabilidad de supervivencia, lo que justifica el diseño de nuevas pruebas diagnósticas que determinen el grado de alteración celular, permitan comprender el proceso degenerativo en el cáncer y establezcan diagnósticos precoz. Esta búsqueda para mejorar los métodos diagnósticos, apunta a que sean sensibles, específicos y menos invasivos, por lo cual el estudio de diferentes biomarcadores en saliva que desde una perspectiva molecular proporcionan información adicional al examen clínico e histopatológico, es considerada como una alternativa eficaz y más cómoda con respecto a los ensayos en sangre. Los biomarcadores que se han descrito en saliva algunos mostrando mayor relación con la carcinogénesis oral son: Ciclina D1, cyfra 21-1, endotelina-1, galectinas 1, 3 y 7, Ki67, lactato deshidrogenasa, metaloproteinasas 2 y 9, proteína p53, proteína de unión a calcio (S100P) y telomerasa (AU)

This review shows the main oral cancer biomarkers in saliva. The clinical appearance and the degree of dysplasia, precancerous lesions of the oral cavity suggests its ability to malignancy, but these are usually diagnosed in advanced stages, decreasing the likelihood of survival, justifying the design of new diagnostic tests to determine the degree of cell alteration as to understand the degenerative process in cancer diagnosis and establish early. This search for improved diagnostic methods, aims to be sensitive, specific and less invasive, so the study of biomarkers in saliva from a molecular perspective provide additional information to clinical and histopathological examination is considered as a more comfortable and effective to establish a diagnosis. Biomarkers that have been described in saliva some showing more related to oral carcinogenesis are cyclin D1, Cyfra 21-1, Endothelin-1, Galectins 1, 3 and 7, Ki67, Lactate dehydrogenase, Metalloproteinases 2 and 9, p53 protein, protein calcium-binding (S100P) and Telomerase (AU)

High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies.

Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective expression system in Escherichia coli for the production of ET-1 enriched with (15)N and (13)C isotopes. Employing thioredoxin as carrier protein, specific and nearly quantitative cleavage of ET-1 from the fusion was mediated by Factor Xa, and purification to homogeneity (final purity of >95%) was achieved by RP-HPLC. Purified recombinant ET-1 was found to be indistinguishable from the synthetic counterpart as determined by mass spectrometry and NMR spectroscopy. Our expression strategy offers the potential for production of isotopically labeled ET-1 in large (mg) quantities for the purpose of heteronuclear NMR experiments. Moreover, the method devised should be applicable for recombinant expression of small peptides in general.

Evaluation of luminal endothelin-converting enzyme activity in the pulmonary and coronary circulations.

The endothelin-converting enzymes are distributed on both the surface of the endothelium and intracellularly. Whether circulating big-endothelin-1 can be hydrolyzed in plasma by lumen-bound endothelin-converting enzymes is unknown. The lung is the major site for hydrolysis of angiotensin-I to angiotensin-II by the angiotensin-converting enzyme; because of its high content in endothelin-converting enzymes, we hypothesized that the lung could similarly hydrolyze circulating big-endothelin-1. Since big-endothelin-1 produced by the lung can modulate coronary vascular tone, the heart may also have the capacity to hydrolyze circulating big-endothelin-1. Isolated lungs and hearts from Sprague-Dawley rats were perfused at 10 mL/min. Clearance of trace doses of human I125big-endothelin-1 was quantified using the indicator-dilution curves technique with labeled albumin as a vascular reference. Single-pass hydrolysis was assessed by bolus injection of human big-endothelin-1 (24 fmol) followed by serial ELISA determinations of big-endothelin-1 and endothelin-1 levels in effluent samples. To exclude possible uptake of produced endothelin-1, 10(-6) M BQ788 was added to the perfusate. The injections had no effect on perfusion pressures. There was no detectable clearance of I125big-endothelin-1 in the lung; however the heart extracted 14 +/- 1% of the injected tracer. There was no detectable big-endothelin-1 hydrolysis in the pulmonary as well as in the coronary circulations. The pulmonary circulation does not clear or hydrolyze circulating big-endothelin-1 suggesting that endothelin-converting enzymes are predominantly used for intracellular and/or abluminal conversion of locally produced big-endothelin-1. Mild coronary uptake of big-endothelin-1 suggests that this circulating peptide could modulate coronary vascular tone.

Characterization and cardiovascular actions of endothelin-1 and endothelin-3 from the American alligator.

The structures and biological activities of the isoforms of endothelin (ET) in a reptile are unknown. ET-3, whose primary structure is identical to human ET-3 except for the substitution Phe4 --> Tyr, and a peptide identical to human ET-1 were isolated from an extract of the lung of the alligator, Alligator mississipiensis. Bolus intravenous injections of alligator ET-3 (10, 30, and 100 pmol/kg) into anesthetized alligators produced dose-dependent decreases in systemic blood pressure (P(sys)) and systemic vascular resistance (R(sys)) without change in heart rate (HR), systemic blood flow (Q(sys)), pulmonary pressure (P(pul)), pulmonary vascular resistance (R(pul)), or pulmonary blood flow (Q(pul)). At a dose of 300 pmol/kg, the initial vasodilatation was followed by an increase in R(sys) and decreases in Q(sys) and P(pul). The response to intravenous human/alligator ET-1 (10, 30, 100, and 300 pmol/kg) was biphasic at all doses with initial decreases in P(sys) and R(sys) being followed by sustained increases in these parameters. In the pulmonary circulation, ET-1 produced a dose-dependent decrease in Q(pul) and an increase in R(pul) during the first phase of the response but no significant change during the second phase. There was no change in HR in response to ET-1. The vasodilatator action of arginine, but not ET-1, was attenuated by N(omega)-nitro-L-arginine methyl ester, indicating that the effect of the peptide is probably not mediated through increased synthesis of nitric oxide. The data demonstrate that the structure of the ET isoforms has been strongly conserved during the evolution of vertebrates but that cardiovascular actions differ significantly between the alligator and mammals, especially in the magnitude and duration of the hypotensive response.

Structural characterization and effects on corticosteroid secretion of endothelin-1 and endothelin-3 from the frog Rana ridibunda.

ABSTRACT Despite the intensive study of endothelin (ET) in mammals, the primary structure and biological activity of the peptide is not known for any species of non-mammalian tetrapod. Extracts of the stomach and the liver of the European green frog Rana ridibunda contained ET-like immunoreactivity measured by RIA using an antiserum raised against human ET-1. The amino acid sequence of the peptide that was isolated in pure form from the stomach extract was identical to that of human ET-1 and the peptide purified from the liver extract was identical to human ET-3 except for a single amino acid substitution (Phe(4)-->Tyr). These observations demonstrate that the amino acid sequences of ET family peptides have been very strongly conserved during evolution of tetrapods and suggest that the pathway of post-translational processing of preproendothelin in the frog is similar to that in mammals. Both frog/human ET-1, frog ET-3 and human ET-3 produced a concentration-dependent increase in the production of corticosteroids from perifused slices of the frog interrenal gland. The maximum responses produced by the peptides (approximately 2-fold increase over basal levels for both corticosterone and aldosterone production) were not significantly different. The potency of ET-1 (-log EC(50)=9.81+/-0.01 (s.e.m.) for corticosterone and 9.52+/-0.29 for aldosterone production) was significantly (P<0.01) greater than that of frog ET-3 (-log EC(50)=8.13+/-1.6 for corticosterone and 8.15+/-0.33 for aldosterone production) but the potencies of frog ET-3 and human ET-3 (-log EC(50)=8.29+/-0.34 and 7.87+/-0.18) were not significantly different.

Simultaneous separation of angiotensin and endothelin peptide families by high-performance liquid chromatography: application to the specific radioimmunoassay measurement of angiotensin II or endothelin-1 from tissue.

Currently available measurements of endogenous angiotensin II (ANG II) and endothelin-1 (ET-1) concentrations by radioimmunoassay (RIA) lack specificity to ANG II or ET-1. ANG II and ET-1 antibodies cross-react with immuno-reactive angiotensin and endothelin family members, respectively. We have therefore developed an ion-pair reversed-phase high-performance liquid chromatography (HPLC) for simultaneously separating angiotensin and endothelin peptides and enhancing RIA specificity in the measurement of ANG II and ET-1. The developed HPLC separation was applied to canine myocardium extracts; ANG II or ET-1 fractions were collected and quantified by RIA. Elution times for both peptide families, ANG I, ANG II, ANG III, ANG IV, ANG II(4-8), bET-1, ET-1, ET-2 and ET-3 were within 25 min. In normal canine myocardium from the right atrium, right ventricle, left atrium and left ventricle, ANG II concentrations were 39+/-11, 28+/-21, 31+/-11 and 21+/-8 fmol/g and ET-1 concentrations were 43+/-16, 42+/-19, 55+/-21 and 57+/-34 fmol/g (mean+/-SD, N=7), respectively. The combination of HPLC with RIA renders the measurement of ANG II or ET-1 specific and convenient, and saves time. This HPLC separation may be applied to the specific measurement of other immuno-reactive angiotensin and endothelin peptides.