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Pyrularia thionin (PT) is a basic 47 amino acid peptide isolated from the nuts of Pyrularia pubera. Its structure and properties have been studied in some detail. Its receptor site is a domain of membrane phosphatidyl serine (PS), where it binds with a relatively high specificity. A segment of its covalent structure, the nonapeptide Thr-Trp-Ala-Arg-Asn-Ser-Tyr-Asn-Val, designated serine nonapeptide (SNP), corresponds to amino acids 7-15 of the thionin, except for the position 12 (Ser), which substitutes for Cys, to give stability. This peptide represents what we consider to be the active site of the thionin, and it also binds to PS domains, but less tightly than thionin does. The peptide has an effect on the prothrombinase assay using the chromophore S2238 to measure the thrombin produced by the prothrombinase complex. It is shown that SNP stimulates the prothrombinase complex activity, instead of inhibiting it, as would be expected if it simply covered the PS sites on the membrane of erythrocyte ghosts, used in the prothrombinase assay. SNP appears to substitute for Va in the prothrombinase complex reaction, in a Ca(2+) independent manner, being even more effective in the absence than in the presence of ghosts. In the clotting system, SNP can also substitute for Factor Va.

Pyrularia thionin and snake venom cardiotoxin are strongly basic proteins which bind to and induce hemolysis of erythrocytes, cause depolarization of muscle cells, and influence the order and properties of phospholipids in cellular membranes. Earlier studies showed a competition between the thionin and cardiotoxin for a common binding site on erythrocytes, and the present study extends these studies to show a similar competition between prothrombin and both basic proteins. The competition between the thionin and prothrombin for binding sites on erythrocytes was shown by direct binding experiments using radiolabeled thionin. Whereas binding of thionin or cardiotoxin induces hemolysis as a consequence of membrane perturbation, prothrombin does not induce hemolysis. Although it binds to the same site, there is no penetration into and perturbation of the membrane. The competition between prothrombin and pyrularia thionin is not influenced by added Ca++. This indicates that membrane PS interacts in a specific and Ca++-independent manner with at least one site on prothrombin, as proposed earlier (Tendian and Lentz, 1990). The competition between thionin and prothrombin was also demonstrated for the release of radiolabeled chromate from loaded P388 cells. The competition observed with the P388 cells shows that the competition is not a unique phenomenon with erythrocytes, but occurs with other cell membranes.

Hemolysis rates of human erythrocytes induced by C2 and C8-C14 straight chain 1-alkanols, 1,2-alkanediols and the corresponding benzylidene derivatives (benzaldehyde acetals) have been studied and compared with hemolysis rates obtained by three peptide toxins. The peak of activity occurs at C12 for the alkanols and glycols and at C10 for the benzylidene derivatives. The most active compound is 1-dodecanol, followed by 1,2-dodecanediol and the C10 benzylidene acetal, which show 50% hemolysis at 15, 99 and 151 microM, respectively, at 37 degrees C. A few lysolecithins and longer chain cis-unsaturated alcohols were studied for comparison purposes, and were found to be more active than 1-dodecanol. The most active were the 16:0 lysolecithin and cis-9-tetradecene-1-ol, which gave 50% hemolysis at concentrations of 2.8 and 5.6 microM respectively. The hemolytic activities of 1-dodecanol, 1,2-dodecanediol and the C10 benzylidene acetal were compared to activities of Pyrularia thionin and melittin with cow, horse, sheep, pig and human erythrocytes. Whereas the peptide toxins showed clear specificity for human erythrocytes, no selectivity was shown by any of the other compounds tested. Addition of the thionin or Naja naja kaouthia cardiotoxin to erythrocyte ghosts caused a slight but reproducible increase in the order of the phospholipid bilayer, as measured with the fluorescent probe NBD-PC. Cardiotoxin gave a greater response than did the P thionin, and extensively iodinated P thionin gave a smaller change than did P thionin. Similar results were obtained with melittin, but this peptide gave a markedly greater response than all other peptides. Addition of dodecanol or the C10 benzylidene acetal caused a marked increase in membrane fluidity. All of these data indicate that the organic compounds interact directly with and are incorporated nonspecifically into the membrane lipid bilayer, but the peptide toxins interact specifically with some component on the surface of the membrane, either a protein or specific phospholipid domain, followed by insertion into the membrane and decreasing phospholipid movement.

— Pyrularia thionin is a strongly basic peptide of 47 amino acids isolated from Pyrularia pubera . This peptide, a member of the thionin family, is hemolytic, cytotoxic and neurotoxic. The characteristics of the hemolytic activity on human erythrocytes are as follows: (1) the peptide does not itself have any phospholipase activity in a micellar assay system with egg yolk phosphatidylcholine, as evidenced by a lack of pH change or uptake of oxygen in the presence of lipoxidase; (2) erythrocyte membranes treated with thionin, however, show a low level of oxygen uptake in the presence of lipoxidase as a consequence of fatty acid release, and this activity is synergistic with that of bee venom phospholipase A 2 ; (3) hemolysis caused by thionin is synergistic with added bee venom phospholipase A 2 ; (4) kinetic analysis of the hemolytic assay reveals that the reaction follows Michaelis-Menten kinetics, being saturable with thionin with a K m of 1.6 μM; (5) binding studies with 125 I-thionin show by Scatchard analysis a K d value of 2.1 μM; (6) although iodinated thionin is inactive in the hemolysis assay, it acts as a competitive inhibitor to native thionin in the hemolytic assay; the inhibitor constant, K i , for this reaction is 7.0 μM; and (7) Ca 2+ above 1 mM inhibits the reaction. All the data are consistent with thionin binding to a receptor, most likely a protein, on the erythrocyte membrane, leading to the release of free fatty acids, most likely by activation of phospholipase A 2 . The release of fatty acids is itself not sufficient to explain the hemolytic reaction.

—Pyrularia thionin (P. thionin) is a strongly basic peptide of 47 amino acids which is hemolytic, cytotoxic and neurotoxic. It shows the greatest hemolytic activity toward human erythrocytes. Rabbit, guinea pig and pig erythrocytes show decreasing activity in that order, and little or no activity is shown with sheep, horse, cow or mouse erythrocytes. Crotalus venoms are inactive, but the venoms from Naja naja atra, Naja naja ceylonicus and Naja naja melanoleuca and, more specifically, cardiotoxin from Naja naja kaouthia have significant hemolytic activities toward human erythrocytes. The cardiotoxin preparation used had no phospholipase activity, and was less active than P. thionin (23% compared to 35% hemolysis by P. thionin in 60 min at 10 μg/ml toxin). Since iodinated P. thionin is inactive, it was used as an inhibitor of hemolysis catalyzed by native P. thionin, N. ceylonicus venom and by cardiotoxin. Examination of the kinetics of the reactions catalyzed by N. ceylonicus venom and cardiotoxin in the absence and presence of iodinated P. thionin shows that both N. ceylonicus venom and cardiotoxin exhibit Michaelis-Menten kinetics, yielding apparent Km values of 7.4 μg/ml and 0.69 μM, respectively. These values compare to an apparent Km for P. thionin of 1.6 μM for erythrocyte hemolysis and a binding constant of 2.1 μM ( Osorio e Castro , V. R., van Kuiken , B. A. and Vernon , L. P. (1989) Action of a thionin isolated from nuts of Pyrularia pubera on human erythrocytes. Toxicon27, 501). The inhibition constants Ki for iodinated P. thionin in the reactions with N. ceylonicus venom and cardiotoxin are 3.8 and 5.3 μM, respectively. These compare to a Ki value of 7.0 μM for the reaction catalyzed by native P. thionin. These data point to similar mechanisms of action of cardiotoxin and P. thionin on cellular membranes, including a common binding site which could be a protein in the membrane.

12 Páginas.-- 6 Tablas.-- 1 Figura, Background-Epidemiological evidence suggests a cardioprotective role of α-linolenic acid (ALA), a plant-derived ω-3 fatty acid. It is unclear whether ALA is beneficial in a background of high marine ω-3 fatty acids (long-chain n-3 polyunsaturated fatty acids) intake. In persons at high cardiovascular risk from Spain, a country in which fish consumption is customarily high, we investigated whether meeting the International Society for the Study of Fatty Acids and Lipids recommendation for dietary ALA (0.7% of total energy) at baseline was related to all-cause and cardiovascular disease mortality. We also examined the effect of meeting the society's recommendation for long-chain n-3 polyunsaturated fatty acids (≥500 mg/day). Methods and Results-We longitudinally evaluated 7202 participants in the PREvención con DIeta MEDiterránea (PREDIMED) trial. Multivariable-adjusted Cox regressionmodels were fitted to estimate hazard ratios. ALA intake correlated towalnut consumption (r=0.94). During a 5.9-y follow-up, 431 deaths occurred (104 cardiovascular disease, 55 coronary heart disease, 32 sudden cardiac death, 25 stroke). The hazard ratios formeeting ALArecommendation (n=1615, 22.4%) were 0.72 (95% CI 0.56-0.92) for all-causemortality and 0.95 (95% CI 0.58-1.57) for fatal cardiovascular disease. The hazard ratios formeeting the recommendation for long-chain n-3 polyunsaturated fatty acids (n=5452, 75.7%) were 0.84 (95% CI 0.67-1.05) for all-causemortality, 0.61 (95% CI 0.39-0.96) for fatal cardiovascular disease, 0.54 (95% CI 0.29-0.99) for fatal coronary heart disease, and 0.49 (95% CI 0.22-1.01) for sudden cardiac death. The highest reduction in all-cause mortality occurred in participants meeting both recommendations (hazard ratio 0.63 [95% CI 0.45-0.87]). Conclusions-In participants without prior cardiovascular disease and high fish consumption, dietary ALA, supplied mainly by walnuts and olive oil, relates inversely to all-cause mortality, whereas protection from cardiac mortality is limited to fish-derived long-chain n-3 polyunsaturated fatty acids., This study was funded in part by Instituto de Salud Carlos III (ISCIII) (Spanish Ministry of Economy) through grants RTIC G03/140, RTIC RD 06/0045, Centro Nacional de Investigaciones Cardiovasculares CNIC 06/2007, ISCIII FIS PS09/01292, the Spanish Ministry of Science and Innovation (MICINN) AGL2010‐22319‐C03‐02 and AGL2009‐13906‐C02‐02, and an unrestricted grant from the California Walnut Commission. Sala‐Vila holds a Miguel Servet I fellowship from the Ministry of Economy and Competitiveness through the ISCIII.

Objective: To assess the efficacy of an electrically assisted application technique on dentine wettability (water contact angle measurements) and to determine the promoted surface roughness of dentine after applying 37% phosphoric acid etching, a self-etching primer or a self-etching adhesive system. Methods: Wettability was assessed on 30 caries-free extracted human third molars. Specimens were sectioned parallel to the occlusal surface to expose moderately deep dentine and ground flat (water-wet 180-grit SiC) to provide uniform flat surfaces. 37% H(3)PO(4), Clearfil SE Bond (CSEB) primer or Prompt-L-Pop (PLP) adhesive system were applied on dentine surfaces. In half of the specimens, the electric impulse-assisted application technique/ElectroBond-assisted application was followed. Contact angle measurements were performed to assess wettability using the Axisymmetric Drop Shape Analysis technique. Additional surfaces were conditioned for atomic force microscopy (AFM) analysis. Two-way ANOVA, Student's t and Student-Newman-Keuls tests were performed (P < 0.05). Results: PLP-treated dentine showed the highest intertubular roughness and the lowest dentine wettability. ElectroBond application reduced water contact angles when dentine was treated with H(3)PO(4) or Clearfil SE Bond primer, but not when dentine was treated with PLP. Conclusions: The use of electric current improved wettability of dentine surface following application of phosphoric acid and a mild self-etch primer, but not self-etch adhesive. (C) 2010 Elsevier Ltd. All rights reserved.

BACKGROUND: The National Pediatric Oncology Unit (UNOP) is the only pediatric hemato-oncology center in Guatemala. METHODS: Patients ages 1 to 17 years with acute lymphoblastic leukemia (ALL) were treated according to modified ALL Intercontinental Berlin-Frankfurt-Münster (IC-BFM) 2002 protocol. Risk classification was based on age, white blood cell count, immunophenotype, genetics (when available), and early response to therapy. RESULTS: From July 2007 to June 2014, 787 patients were treated, including 160 who had standard-risk ALL, 450 who had intermediate-risk ALL, and 177 who had high-risk ALL. The induction death rate was 6.6%, and the remission rate was 92.9%. The rates of death and treatment abandonment during first complete remission were 4.8% and 2.5%, respectively. At a median observation time of 3.6 years, and with abandonment considered an event, the 5-year event-free survival and overall survival estimates (± standard error) were 56.2% ± 2.1% and 64.1% ± 2.1%, respectively, with a 5-year cumulative incidence of relapse of 28.9% ± 2.0%. Twenty-one of 281 patients (7.5%) investigated were positive for the ets variant 6/runt-related transcription factor 1 (ETV6/RUNX1) fusion. CONCLUSIONS: A well organized center in a low-middle-income country can overcome the disadvantages of malnutrition and reduce abandonment. Outcomes remain suboptimal because of late diagnosis, early death, and a high relapse rate, which may have a partly genetic basis. Earlier diagnosis, better management of complications, and better knowledge of ALL will improve outcomes. Cancer 2017;123:436–448. © 2016 American Cancer Society.

One way of possibly improving bond strength is by changing the application mode of self-etch adhesives. The current study evaluated the resin-dentin microtensile bond strength (MTBS) promoted by two- and one-step self-etching adhesives after different bonding application procedures. Flat dentin surfaces from extracted human molars were bonded: 1) according to the manufacturers' instructions, 2) duplicating the number of adhesive coats and 3) doubling the application time of the acidic primers. Two-step (Clearfil SE Bond/SEB and Resulcin AquaPrime/RE) and one-step (Etch & Prime 3.0/EP and One-Up Bond F/OUB) self-etch adhesives were used. Resin-dentin beams were tested in tension at 0.5 mm/minute. Selected debonded beams were observed under scanning electron microscopy (SEM). MTBS data were analyzed by ANOVA and multiple comparison tests (p < 0.05). The highest MTBS was always attained with SEB, regardless of the bonding procedure. RE, EP and OUB showed similar MTBS when bonded as per the manufacturers' instructions. The MTBS of OUB increased after doubling the application time and duplicating the adhesive coats. The two-step self-etch adhesives were insensible to changes in bonding application procedures. Attempts to improve the bonding performance of water/ethanol-based self-etching systems by using different bonding application parameters were system-specific and only effectively detected in one-step adhesive systems.

The chemo-mechanical surface treatment of fiber posts increases their bonding properties. The combined use of atomic force and confocal microscopy allows for the assessment and quantification of the changes on surface roughness that justify this behavior. Quartz fiber posts were conditioned with different chemicals, as well as by sandblasting, and by an industrial silicate/silane coating. We analyzed post surfaces by atomic force microscopy, recording average roughness (Ra) measurements of fibers and resin matrix. A confocal image profiler allowed for the quantitative assessment of the average superficial roughness (Ra). Hydrofluoric acid, potassium permanganate, sodium ethoxide, and sandblasting increased post surface roughness. Modifications of the epoxy resin matrix occurred after the surface pre-treatments. Hydrofluoric acid affected the superficial texture of quartz fibers. Surface-conditioning procedures that selectively react with the epoxy-resin matrix of the fiber post enhance roughness and improve the surface area available for adhesion by creating micro-retentive spaces without affecting the post’s inner structure.

Objective. The aim is to determine if superficial or deep dentine microhardness (MH) is affected by different chemical dentine pre-treatments performed for resin bonding. Methods. Dentine discs of superficial (SD) and deep dentine (DD) were obtained by transversally sectioning the crowns of human third molars. Knoop MH was measured after different treatments: (1) polished up to 4000 grit, (2) polished and etched (37% ortophosphoric acid for 15 s), (3) resin (Single Bond -SB-) infiltrated dentine after acid etching, (4) polished, etched and treated with 5% NaOCl for 2 min, (5) resin infiltrated (SB) after etching and NaOCl treatment. For resin infiltrated surfaces care was taken in order to remove the excess adhesive layer, and obtain infiltrated dentine, by measuring thickness of the sample before resin infiltration and polishing after resin infiltration until obtaining the initial thickness of the sample. Indentations (n = 20) were performed on moist surfaces with a standard Knoop MH tester (20 g, 5 s). Data were expressed in Knoop Hardness Numbers (KHN) and analysed by ANOVA and multiple comparisons (P < 0.05). Results. Dentine KHN decreased on both SD and DD after acid etching. NaOCl treatment after acid etching did not affect MH on SD, but KHN of DD was towered. Resin infiltration increased KHN but did not recover the initial MH values in both SD and DD. Conclusions. Treating dentine with either H3PO4 or NaOCl caused marked reduction of its surface hardness and subsequent resin infiltration was not capable to restore it. (c) 2004 Elsevier Ltd. All rights reserved.