Development, implementation and evaluation of a multinational FLS mentorship programme in Latin America.

Realising the benefits of systematic secondary fracture prevention requires supporting local sites to get started and becoming effective. We here describe the development, implementation and impact of a regional fracture liaison service (FLS) mentorship programme in Latin America that led to 64 FLS getting started and coverage of 17,205 patients. INTRODUCTION: Despite treatments and service models to deliver effective secondary fracture prevention, most patients are left untreated after a fragility fracture. To improve the capability to get FLS started and more effective, we describe the development, implementation and evaluation of an international programme to develop national communities of FLS mentors as part of the Capture the Fracture Partnership in Latin America. METHODS: The IOF regional team and the University of Oxford developed the curriculum and associated resources for training mentors in setting up FLS, service improvement and mentorship. Mentors were selected during a preparatory meeting, trained using live online sessions followed by regular mentor-led post-training meetings. The programme was evaluated using a pre-training needs assessment and post-training evaluation based on Moore's outcomes. RESULTS: The mentorship programme was initiated in Mexico, Brazil, Colombia and Argentina. The mentors were multidisciplinary, including orthopaedic surgery, rehabilitation, rheumatology, endocrinology, geriatrics, gynaecology and internal medicine. There was 100% participation in training sessions and reported satisfaction with the training. Since the initiation of the training programme, 22 FLS have been set up in Mexico, 30 in Brazil, 3 in Colombia and 9 in Argentina, in comparison with two in Chile and none in any other LATAM countries that were not involved in the mentorship programme. This equates to approximately 17,025 additional patients identified from 2019 to 2021 after initiation of mentorship. The mentors have engaged with 58 FLS for service development. Post-training activities include two published national best practice guidelines and other country-specific resources for FLS in the local language. CONCLUSION: Despite the COVID pandemic, the mentorship pillar of the Capture the Fracture Partnership has developed a community of FLS mentors with measurable improvement in national FLS provision. The programme is a potentially scalable platform to develop communities of mentors in other countries.

Stimulation of prothrombinase activity by the nonapeptide Thr-Trp-Ala-Arg-Asn-Ser-Tyr-Asn-Val, a segment of a plant thionin.

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.

Binding to and hemolysis of human erythrocytes by pyrularia thionin and Naja naja kaouthia cardiotoxin: inhibition by prothrombin.

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 of erythrocytes and fluorescence polarization changes elicited by peptide toxins, aliphatic alcohols, related glycols and benzylidene derivatives.

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.

Action of a thionin isolated from nuts of Pyrularia pubera on human erythrocytes.

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 A2; (3) hemolysis caused by thionin is synergistic with added bee venom phospholipase A2; (4) kinetic analysis of the hemolytic assay reveals that the reaction follows Michaelis-Menten kinetics, being saturable with thionin with a Km of 1.6 microM; (5) binding studies with 125I-thionin show by Scatchard analysis a Kd value of 2.1 microM; (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, Ki, for this reaction is 7.0 microM; and (7) Ca2+ 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 A2. The release of fatty acids is itself not sufficient to explain the hemolytic reaction.

Hemolytic activity of thionin from Pyrularia pubera nuts and snake venom toxins of Naja naja species: Pyrularia thionin and snake venom cardiotoxin compete for the same membrane site.

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 micrograms/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 micrograms/ml and 0.69 microM, respectively. These values compare to an apparent Km for P. thionin of 1.6 microM for erythrocyte hemolysis and a binding constant of 2.1 microM (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. Toxicon 27, 501). The inhibition constants Ki for iodinated P. thionin in the reactions with N. ceylonicus venom and cardiotoxin are 3.8 and 5.3 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of glutaraldehyde with human erytrocytes.

The reaction of glutaraldehyde with human erythrocytes has been studied at pH values of 6.0, 7.2 and 8.0 at several glutaraldehyde concentrations, for different periods at 37 degrees C. Increasing pH and reagent concentration favours formation of cells resistant to osmotic and chemical (by n-butanol, saponin and sodium dodecyl sulfate (SDS)) hemolysis. Ultrasonic waves also have no effect upon these cells. Different behavior concerning the state of hemoglobin, K+ efflux, and swelling have also been observed, according to the pH of treatment. The results suggest that crosslinking involving hemoglobin molecules and components of the membrane (proteins, aminophospholipids) may be obtained in the reaction of human erythrocytes with glutaraldehyde.

Effect of X-537A- on the phosphorylated protein in sarcoplasmic reticulum vesicles.

The effect of the antibiotic X537A on the phosphorylated ATPase (E approximately P) was investigated. The results show that X-537A depresses the level of E approximately P which is dependent on the Ca2+ gradient, while the Ca2+-independent E approximately P is not affected.