Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Sea anemones contain a variety of biological active compounds including some potent toxins. For this reason, many investigators have focused their studies in these proteins, isolated from sea anemones, such as Bunodosoma caissarum. The aim of this work was to study the mechanism of functional alterations produced by the crude extract and the PLA2 fraction of B. caissarum in the isolated rat kidney and arteriolar mesenteric bed. Isolated kidneys from Wistar rats, weighing 250-300g, were perfused with Krebs-Henseileit solution containing 6% of bovine serum albumin for 120 min. B. caissarum crude extract (BcE) and PLA2 were added to the system 30 min after the beginning of each experiment (internal control) and Tezosentan (TZN) and Indomethacin (IND) were administered in the initial period of each experiment (T=0min). The mesenteric bed, kept warmed at 37ÂC, was perfused with Krebs solution at a constant flow, (4mL/min) but with a variable perfusion pressure, measured for 80 min. mRNA expression of TNF-&#945;, IL-1&#946;, rennin and adenosine receptors (A1, A2a, A2b and A3) was evaluated by PCR using 18S mRNA as reference gene. The data were analyzed by Studentâs t-test (p<0.05). BcE altered kidney function increasing perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR) and sodium, potassium and chloride excretion, especially with the intermediary concentraÃÃo. Tezosentan inhibited these effects only partially. However, indomethacin presented a blockage more expressive that TZN. The PLA2 of B. caissarum showed a higher increase of PP at 60 minutes (Control= 104,17Â3,72; PLA2(0,1) = 130,9Â5,6*; PLA2(0,3) = 165,1Â12,6*; PLA2(1,0) = 142,3Â9,6*, *p<0,5) and increased RVR, UF, GFR, sodium, potassium and chloride excretion and urinary osmolarity, with descrease of Na+, K+ and Cl- distal transports.. The blockage of IND was more expressive on the smallest PLA2 concentraÃÃo and TZN showed a modest effect, blocking more efficiently the intermediary concentraÃÃo. Neither BcE, nor PLA2 altered the basal perfusion pressure of the isolated arteriolar mesenteric bed. PLA2 increased the gene expression of TNF-&#945; (Control = 1,00  0,00 vs. PLA2 = 1,116  0,033*, com p<0,0001)and upnregulated the expression of adenosine receptors A2a (Control = 1,0  0,01 vs. PLA2 = 1,238  0,043*, com p<0,0001). These results suggest that BcE and PLA2 exert significative actions in the isolated perfusion kidney, without vascular effects in the arteriolar mesenteric bed. COX-2 and endothelin appear to be important mediators of the effects of BcE and PLA2. In addition, adenosine is also involved in the mechanism of action of B. caissarum / AnÃmonas marinhas contÃm uma variedade de compostos biologicamente ativos incluindo potentes toxinas. Por esta razÃo, muitos investigadores tÃm centrado seus estudos nestas proteÃnas, isoladas de anÃmonas marinhas, como Bunodosoma caissarum. O objetivo deste trabalho foi estudar o mecanismo das alteraÃÃes funcionais produzidas pelo extrato bruto e da fraÃÃo PLA2 de B. caissarum em rim isolado de rato e leito vascular mesentÃrico. Rins isolados de ratos Wistar, pesando 250-300g, foram perfundidos durante 120 min com soluÃÃo de Krebs-Henseileit contendo 6% de albumina bovina. O extrato bruto de B. caissarum (BcE) e PLA2 foram adicionados ao sistema, em diferentes concentraÃÃes, 30 minutos apÃs o inÃcio de cada experimento (controle interno) e Tezosentan (TZN) e Indometacina (IND) foram administrados no perÃodo inicial de cada experimento (T = 0 min). O leito vascular mesentÃrico, mantido aquecido a 37  C, foi perfundido com soluÃÃo de Krebs em um fluxo constante (4mL/min) e a pressÃo medida por 80 min. A expressÃo de mRNA TNF-&#945;, IL-1 &#946;, renina e receptores de adenosina (A1, A2a, A2b e A3) foi avaliada por PCR utilizando como referÃncia o mRNA do gene 18S. Os dados foram analisados por teste t de Student (p <0,05). BcE alterou a funÃÃo renal elevando a pressÃo de perfusÃo (PP), a resistÃncia vascular renal (RVR), fluxo urinÃrio (UF), taxa de filtraÃÃo glomerular (TFG) e excreÃÃo de sÃdio, de potÃssio e cloreto, especialmente com a concentraÃÃo intermediÃria. Tezosentan inibiu estes efeitos apenas parcialmente. No entanto, indometacina apresentou um bloqueio mais expressivo que TZN. A PLA2 de B. caissarum elevou a pressÃo de perfusÃo (PP) jà aos 60 minutos (controle = 104,17  3,72; PLA2 (0,1) = 130,9  5,6 *; PLA2 (0,3) = 165,1  12,6 *; PLA2 (1,0) = 142,3  9,6 *, *p <0,5), revelando tambÃm elevaÃÃes de RVR , UF, TFG, excreÃÃo de eletrÃlitos e osmolaridade urinÃria, com diminuiÃÃo dos transportes distais de Na+, K+ e Cl-. O bloqueio com IND foi mais expressivo sobre a menor concentraÃÃo de PLA2, enquanto TZN mostrou um efeito discreto. BcE e PLA2 nÃo alteraram a pressÃo de perfusÃo basal do leito vascular mesentÃrico. PLA2 aumentou a expressÃo relativa de TNF&#945; (C = 1,00  0,00 vs. PLA2 = 1,116  0,033*, com p<0,0001) e do receptor de adenosina A2a (C = 1,0  0,01 vs. PLA2 = 1,238  0,043*, com p<0,0001).Estes resultados sugerem que BcE e PLA2 exercem aÃÃes significativas na perfusÃo de rim isolado, sem efeitos vasculares diretos no leito mesentÃrico. COX-2 e endotelina parecem ser importantes mediadores dos efeitos da BcE e PLA2. AlÃm disso, adenosina tambÃm parece estar envolvida no mecanismo de aÃÃo da PLA2 de Bunodosoma caissarum.

As fosfolipases A2 (PLA2s) de peçonhas de serpentes compreendem um grupo de enzimas de massas moleculares variáveis entre 14.000 e 18.000, e são responsáveis por vários efeitos tóxicos induzidos pela peçonha destes animais, tornando-se necessária a busca por inibidores naturais de PLA2¬s. O presente trabalho propôs a caracterização bioquímica, farmacológica e estrutural de duas proteínas inibitórias isoladas do plasma da serpente Bothrops jararacussu (BjussuMIPs), que neutralizam as atividades enzimáticas, tóxicas e farmacológicas de diferentes PLA2s. Estes inibidores foram isolados por cromatografia de afinidade em miotoxina-Sepharose, demonstrando que ambos são glicoproteínas com massas moleculares de 24.000 (BjussuMIP) e 23.500 (BjussuMIP) para os monômeros e de 120.000 (BjussuMIP) e 160.000 (BjussuMIP) para os oligômeros. O tratamento dos BjussuMIPs com a N-glicosidase F reduziram os seus pesos moleculares para aproximadamente 18.000, mas não afetaram suas atividades inibitórias sobre PLA2s, sugerindo que os carboidratos tem pouco ou nenhum papel na associação dos BjussuMIPs com estas enzimas. A análise do BjussuMIP por dicroísmo circular mostrou 44% de -hélice, 18% de folhas , 10% de voltas e 28% de estruturas aleatórias. O cDNA obtido por PCR a partir do fígado desta serpente revelou 432 pb (BjussuMIP) e 543 pb (BjussuMIP) que codificam para 144 e 181 resíduos de aminoácidos, respectivamente. O alinhamento da sequência de BjussuMIP com a de outros inibidores do tipo , denominados de PLIs, apresentou 73-92% de similaridade e o BjussuMIP mostrou 89-94% com inibidores do tipo PLIs. Os BjussuMIPs demonstraram ser relativamente estável a variações de pH (6-12) e temperatura, entretanto, perderam atividade inibitória quando submetido a altas temperaturas. A caracterização funcional indica que os BjussuMIPs apresentaram propriedades inibitórias sobre diferentes PLA2s isoladas de peçonhas de serpentes dos gêneros Bothrops e Crotalus. Ambos BjussuMIPs revelaram propriedades farmacológicas como a inibição das atividades fosfolipásica, anticoagulante, miotóxica, indução de edema, citotóxica, bactericida e letal. Os resultados obtidos demonstram que o BjussuMIP mostra maior afinidade sobre as PLA2s homólogas Lys49 como BthTX-I e PrTX-I, enquanto que o BjussuMIP apresenta-se mais específico para PLA2s Asp49, sugerindo uma especificidade entre os BjussuMIPs e tipos de PLA2s. Além disso, ambos os inibidores mostraram ser eficazes na suplementação do antiveneno botrópico em diferentes concentrações, resultando no aumento da capacidade do soro em neutralizar toxinas de serpentes. Os aspectos abordados neste trabalho poderão trazer informações complementares sobre possíveis mecanismos de ação, podendo resultar no melhor entendimento dos efeitos inibitórios exercidos pelos BjussuMIPs, assim como auxiliar o tratamento do envenenamento ofídico pela suplementação da soroterapia tradicional. / Phospholipases A2 (PLA2s) from snake venoms comprise a group of enzymes with molecular weights varying from 14,000 to 18,000, and are responsible for several toxic effects induced by the venom of these animals, making important the search for natural inhibitors of PLA2s. The present work proposed the biochemical, pharmacological and structural characterization of two protein inhibitors isolated from the plasma of Bothrops jararacussu snake (BjussuMIPs), which neutralize the enzymatic, toxic and pharmacological activities of different PLA2s. These inhibitors were isolated by an affinity chromatography on myotoxin-Sepharose, showing that both are glycoproteins with molecular weights of 24,000 (BjussuMIP) and 23,500 (BjussuMIP) for the monomers and 120,000 (BjussuMIP) and 160,000 (BjussuMIP) for the oligomers. The treatment of BjussuMIPs with N-glucosidase F reduced their molecular weights to about 18,000, but did not affect their inhibitory activity on PLA2s, suggesting that the carbohydrates have little or no role in the association of these BjussuMIPs with these enzymes. The analysis of BjussuMIP by circular dichroism showed 44% of -helix, 18% of sheets, 10% of turns and 28% of random structures. The cDNA obtained by PCR from the snake liver showed 432 bp for BjussuMIP and 543 bp for BjussuMIP, which encode for 144 and 181 amino acid residues, respectively. The alignment of the sequence of BjussuMIP with those from other -inhibitors (PLIs) showed 73-92% of similarity and 89-94% for the BjussuMIP compared to other PLIs. The BjussuMIPs showed to be relatively stable to changes in pH (6-12) and temperature, however lost of its activity when submitted to high temperatures. The functional characterization indicates that both BjussuMIPs presented inhibitory properties on different snake venom PLA2s from the genera Bothrops and Crotalus. Both BjussuMIPs showed pharmacological properties such as inhibition of phospholipase, anticoagulant, myotoxic, cytotoxic, bactericidal, edema-inducing and lethal activities. The results show that BjussuMIP presents higher affinity to Lys49-PLA2 homologous, such as BthTX-I and PrTX-I, while BjussuMIP is more specific to Asp49-PLA2s, suggesting specificity between BjussuMIPs and types of PLA2s. Moreover, both inhibitors proved effective in the supplementation of Bothrops antivenom at different concentrations, resulting in an increased capacity of serum in neutralizing snake toxins. The issues reported in this work could bring additional information on possible mechanisms of action and may result in better understanding of the inhibitory effects exerted by these BjussuMIPs, as well as assist the treatment of ophidian envenomations by supplementation of the traditional serum therapy.

Philosophiae Doctor - PhD / Lung cancer is the most common fatal cancer in terms of both incidence and mortality in the world. The most important cause of lung cancer is exposure to tobacco smoke through active or passive smoking. Nicotine which is a major component of tobacco could be assumed to be a tumour promoter since it had been indicated to stimulate tumour growth. Over expression of Bcl-2 in human lung cancer cells blocked the induction pathways (type I and II) of apoptosis. The increase in Bcl-2 in patients with lung cancer had also been linked to nicotine. In recent years nicotine replacement therapy has become a therapeutic method to treat smoker’s withdrawal symptoms and to advise cancer patients to stop smoking because, numerous cancer patients continue to smoke after their diagnosis. Non small cell lung carcinomas constitutes for approximately 80% of lung cancer cases. However, even with the development and improvement in conventional treatments of surgery, radiation and chemotherapy, the 5 year survival rate for these patients remains less than 15%. Chemoprevention, an approach to control cancer, is the use of specific natural or synthetic substances with the objective of delaying, reversing, suppressing or preventing carcinogenic progression to invasive cancer. A promising tool for chemoprevention against lung cancer could be prostaglandin A2 (PGA2), since it had been shown to have inhibitory effects on various cancer cell growth. The search for more effective agents, or combination therapies that could induce apoptosis in lung cancer are currently under investigation as a therapeutic target for the treatment of lung cancer. In order to elucidate the effect of nicotine and PGA2 on lung cancer cell proliferation in this study, an over view of the following was given;the cell cycle, tubulin, nucleoli, apoptosis, lung cancer, the etiology of cancer with reference to tobacco smoke and nicotine, the nutritional influence on carcinogenesis with reference to essential fatty acids and prostaglandins and chemoprevention.The supplements nicotine and PGA2 were administered to the NCI-H157 lung cancer cell line at the concentrations of 1 mM, 1 μM and 1 nM for nicotine and 5, 10 and 20 μg/ml PGA2. The effect of combinations of nicotine and PGA2 on the proliferation and survival was also tested. 5 μg/ml PGA2 was added to 1 mM, 1 μM and 1 nM nicotine respectively. This was also done for 10 and 20 μg/ml PGA2.These concentrations were administered to the cell culture and exposed for three different time exposures, namely 24, 48 and 72 hours. The objectives were: 1) To determine the effect of nicotine and PGA2 and combinations thereof on the growth(proliferation) of the NCI-H157 cells, where early results indicative of apoptosis lead to the investigation of the influence of nicotine and PGA2 on apoptosis. The effect of nicotine and PGA2 and their combinations on the morphology of interphase and dividing cells, as well as on the morphology of the dying cells were compared and quantified. 2) To study the effects of nicotine and PGA2 and their combinations on the nucleolar organizer region using silver stain. 3) To study the effects of nicotine and PGA2 or combinations thereof on the cytoskeleton (α-tubulin) of the cancer cells with aid of indirect immunofluorescence and to identify apoptotic cells using Hoechst 33342. 4) To determine the effect of nicotine and PGA2 and their combinations on cell cycle progression and apoptosis induction in the transformed cells using flow cytometry (DNA propidium iodide stain, Annexin V and caspase-3).In order to verify the effects of nicotine and PGA2 and their combinations on protein synthesis, SDS-PAGE and immunoblotting was employed.This study indicated the anti-apoptotic effects of nicotine. It maintained and stimulated cell proliferation of the NCI-H157 cell line. PGA2 demonstrated that it has a pro-apoptotic effect. The concentrations of 10 and 20 μg/ml PGA2 decreased cell proliferation and demonstrated its pro-apoptotic effects more effectively than 5 μg/ml PGA2. The combination of 10 and 20 μg/ml PGA2 and nicotine (1 mM, 1 μM and 1 nM) also showed a more pronounced induction of apoptosis than 5 μg/ml PGA2 and nicotine (1 mM, 1 μM and 1 nM). PGA2 therefore demonstrated that it blocked the mitogenic and anti-apoptotic effects of nicotine. With its pro-apoptotic effects, PGA2 could therefore be assumed to be a chemopreventive agent. However,it was evident that apoptotic induction was stimulated via both a dependent and an independent caspase-3 pathway and therefore further investigation is needed to indicate which pathway was activated. This study identified PGA2 as a chemopreventive agent for in vitro conditions; however, further studies are also needed to investigate the effect of in vivo conditions.

Abstract Phospholipids are the main components of cell membranes, lipoproteins and other membrane structures in living organisms. Properties of lipid molecules are important to the overall behaviour and interactions of membranes. Furthermore, characteristics of the biological membranes act as important regulators of membrane functions. Molecular dynamics (MD) simulations were applied in this thesis to study properties of biological membranes. A certain degree of acyl chain polyunsaturation is essential for the proper functioning of membranes, but earlier MD simulations had not addressed the effects of polyunsaturation. Therefore a solvated all-atom bilayer model consisting of diunsaturated 1-palmitoyl-2-linoleoyl-3-phosphatidylcholine (PLPC) molecules was simulated. The analysis of the simulation data was focused on the effects of double bonds on a membrane structure. Self-organising neural networks were applied to the analysis of the conformational data from the 1-ns simulation of PLPC membrane. Mapping of 1.44 million molecular conformations to a two-dimensional array of neurons revealed, without human intervention or requirement of a priori knowledge, the main conformational features. This method provides a powerful tool for gaining insight into the main molecular conformations of any simulated molecular assembly. Furthermore, an application of MD simulations in the comparative analysis of the effects of lipid hydrolysis products on the membrane structure was introduced. The hydrolysis products of the phospholipase A2 (PLA2) enzyme are known to have a role in a variety of physiological processes and the membrane itself acts as an important regulator of this enzyme. The simulations revealed differences in the bilayer properties between the original and hydrolysed phospholipid membranes. This study provides further evidence that MD simulations on biomembranes are able to provide information on the properties of biologically and biochemically important lipid systems at the molecular level.

BACKGROUND. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor for coronary artery disease (CAD) in nontransplant patients. We evaluated the association between Lp-PLA2, cardiac allograft vasculopathy (CAV) assessed by 3D intravascular ultrasound, and incidence of cardiac adverse events in heart transplant recipients. MATERIALS AND METHODS. Fasting blood samples were obtained and stored from a cross-section of 112 cardiac transplant recipients attending the Mayo cardiac transplant clinic in 2000 to 2001, mean of 4.7 years after transplant. Lp-PLA2 was measured in plasma aliquots using an enzyme-linked immunoassay. Fifty-six of these patients subsequently underwent two 3D intravascular ultrasound studies in 2004 to 2006 12 months apart. Cardiovascular (CV) events included percutaneous coronary intervention, coronary artery bypass grafting (CABG), reduction in left ventricular ejection fraction (LVEF) ≤45% secondary to CAV and CV death. RESULTS. High Lp-PLA2 level was associated with increase in plaque volume (r=0.43, P=0.0026) and percent plaque volume (r=0.45, P=0.0004). The association remained significant after adjusting for clinical and lipid variables. During follow-up of 5.1±1.6 years, 24 CV adverse events occurred in 15 of 112 (13%) heart transplant patients. Lp-PLA2 level>236 ng/mL (higher tertile) identified a subgroup of patients having a 2.4-fold increase of relative risk for combined endpoint of CV events (percutaneous coronary intervention, CABG, LVEF<45%, and CV death; 95% CI 1.16-5.19, P=0.012) compared with patients with Lp-PLA2≤236 ng/mL. CONCLUSIONS. Lp-PLA2 is independently associated with progression of CAV and predicts a higher incidence of CV events and CV death in transplant patients. This finding supports the concept that systemic inflammation is an important mediator of CAV. Lp-PLA2 may be a useful marker for risk of CAV and a therapeutic target in posttransplant patients.

No description available.

No description available.

El éxito de la fecundación humana depende, entre otros eventos moleculares, de la capacidad de los espermatozoides para llevar a cabo de forma adecuada la capacitación. Este proceso implica una serie de cambios bioquímicos en los espermatozoides para favorecer su interacción con el gameto femenino. Aunque es posible capacitar las células in vitro, el tiempo óptimo para que un espermatozoide complete la capacitación en estas condiciones sigue siendo objeto de debate debido a la falta de biomarcadores de capacitación adecuados. Los estudios en esta área, se han centrado en aquellos receptores espermáticos implicados en la interacción entre gametos. En particular, el complejo molecular formado por la proteína de choque térmico A2 (HSPA2; del inglés heat shock protein A2), la molécula de adhesión a hialuronidasa 1 (SPAM1; del inglés sperm adhesion molecule 1) y la proteína arilsulfatasa A (ARSA; del inglés arylsulfatase A), ha sido estudiado por varios grupos de investigación debido a su participación en el reconocimiento del ovocito por parte del espermatozoide. Los estudios más relevantes sobre la ubicación de este complejo se basan en la evidencia de la colocalización de estas proteínas en la región periacrosomal de la cabeza espermática. Sin embargo,Esta premisa es controvertida, ya que otros autores han encontrado una asociación entre diferentes áreas de distribución de HSPA2 en la cabeza del espermatozoide y la fertilidad. A pesar del importante papel que desempeña este complejo proteico durante la unión del espermatozoide a la zona pelúcida del ovocito (ZP), aún no se ha ilustrado el grado de dependencia del tiempo de capacitación sobre la presencia y distribución de una topografía específica en la superficie espermática de estas proteínas. Con esta premisa, en la presente tesis evaluamos la influencia del tiempo de capacitación in vitro en la localización y distribución de HSPA2 y ARSA en la cabeza de espermatozoides humanos. De esta manera, y mediante microscopía de fluorescencia, se evaluó la presencia de HSPA2 y ARSA en donantes normozoospérmicos2 tanto antes como tras la capacitación in vitro durante una y cuatro horas. Además, se utilizó la microscopía electrónica de campo de alta resolución (FE-SEM; microscopía electrónica de barrido de emisión de campo; del inglés field emission scanning electron microscopy) para cuantificar la densidad de ARSA y la localización específica de esta proteína en los diferentes dominios de la membrana espermática antes y después de la capacitación in vitro durante una y cuatro horas. Con respecto al porcentaje de células positivas para HSPA2, no se observaron diferencias significativas entre las poblaciones analizadas antes y después de una hora de capacitación. No obstante, observamos un porcentaje significativamente mayor de células marcadas con HSPA2 tras cuatro horas de capacitación in vitro. A pesar de que no se pudo determinar un patrón de distribución de HSPA2 predominante en las células que fueron positivas antes de la capacitación, el patrón de distribución mayoritario después de la capacitación fue de fluorescencia en la banda ecuatorial y el acrosoma. Al estudiar la distribución de ARSA se observó un aumento significativo en el porcentaje de células positivas para esta proteína tras la capacitación, pero sin diferencias entre una y cuatro horas de incubación. Al igual que ocurría con HSPA2, el análisis mediante microscopía de fluorescencia no mostró un patrón mayoritario de distribución de ARSA en la subpoblación espermática previa a la capacitación, mientras que, tras este proceso las células presentaron de manera predominante un marcaje intenso en la región acrosomal. Por otra parte, el análisis mediante microscopía electrónica de barrido de emisión de campo mostró una agregación de ARSA en la región periacrosomal tras la capacitación. Nuestros resultados apuntan que el complejo formado por HSPA2, ARSA y SPAM1 requiere más de una hora de capacitación in vitro para distribuirse correctamente en la cabeza espermática. Además, el presente estudio proporciona evidencias sólidas de la utilidad de HSPA2 y ARSA como biomarcadores de capacitación, sugiriendo su uso como biomarcadores suplementarios al clásico análisis seminal previo a una técnica de reproducción artificial. / Este trabajo de investigación ha sido subvencionado por la Cátedra Human Fertility de la Universidad de Alicante y los proyectos de I+D+i ViGrob-186 y UAIND17-03.

This study was prompted by a lack of information concerning influentials in certain Virginia counties, and the limited involvement of these individuals in Cooperative Extension programs. The reputational method was employed in identifying the influentials in a selected rural agricultural county in Virginia with 10,000 inhabitants. Additional information was collected with a pre-tested interview schedule. The following objectives constituted the framework for the study: 1. Identify the influentials and determine and describe their salient characteristics. 2. Determine the interrelationships and informal structure. 3. Determine the factors to which influence was attributed. 4. Ascertain the extension agents ability to identify the influentials. 5. Derive implications from findings pertinent to Cooperative Extension work. The 23 persons identified as being the most influential were all males who were between 32 and 81 years of age, and all except two were county natives. Occupationally the group was predominated by full-time farmers and retail merchants. The 12 persons attributed county-wide influence were older, had more associations with each other and had held a larger number of formal positions than the local community influentials. All were interrelated through organizations and business contacts and appeared to comprise a unitary structure similar to a "power pool." Each influential in the pool possessed several interrelated factors which contributed to his power. Past achievements was perceived to be one of the most important factors. Results of the study indicate the need for an organized identification program for extension agents and the recognition of the power structure as an important resource in conducting Extension programs. / Master of Science

Secreted phospholipase A2 (PLA2) is an interfacial enzyme that catalyzes the calcium-dependent hydrolysis of glycerophospholipids to free fatty acids and lyso-phospholipid, which are further converted to eicosanoids and platelet activating factor with broad biological activities. PLA2 is inactive in solution, but undergoes interfacial activation upon binding to biological membranes. Despite extensive studies on secreted PLA2s, the structural basis for interfacial activation and the effects of site-directed mutations remain largely uncharacterized. Two mutants of human group IIA PLA2, with tryptophans incorporated at the 3rd or 5th position in the N-terminal helix, display dramatic differences in activity compared to the wildtype enzyme. This project analyzes the distinct structural changes that occur in PLA2 and two Trp-mutants during interfacial activation, which are responsible for the observed disparities in activity. Additionally, the thermal stability of both mutants was determined in order to explore possible correlations between resistance to thermal denaturation and enzymatic activity. The V3W mutant shows enhanced activity and a higher optimal temperature compared to the wildtype, which may be promoted at least partially by the high affinity of tryptophan for the lipid-aqueous interface. Contrastingly, the FSW enzyme, which has a tryptophan within the substrate-binding pocket, displays greatly diminished activity compared to both the wild-type and V3W mutant, suggesting inefficient loading of substrate. Circular dichroism and fluorescence studies reveal that the differences in activity of the mutants result from distinct structural changes upon activation. Furthermore, thermal denaturation of V3W was partially reversible, whereas F5W showed no recovery of secondary structure following decrease of temperature. Thus, tryptophan incorporation at two close positions modulates the activity of PLA2 in strikingly different ways, which are associated with defined changes in the secondary structure and the thermal stability of the enzyme. Our results may find industrial or pharmaceutical applications, such as production of fatty acids or development of antibacterial agents.