IPD072Aa from Pseudomonas chlororaphis Targets Midgut Epithelial Cells in Killing Western Corn Rootworm (Diabrotica virgifera virgifera).

IPD072Aa from Pseudomonas chlororaphis is a new insecticidal protein that has been shown to have high activity against western corn rootworm (WCR). IPD072 has no sequence signatures or predicted structural motifs with any known protein revealing little insight into its mode of action using bioinformatic tools. As many bacterially derived insecticidal proteins are known to act through mechanisms that lead to death of midgut cells, we evaluated whether IPD072Aa also acts by targeting the cells of WCR midgut. IPD072Aa exhibits specific binding to brush border membrane vesicles (BBMVs) prepared from WCR guts. The binding was found to occur at binding sites that are different than those recognized by Cry3A or Cry34Ab1/Cry35Ab1, proteins expressed by current maize traits that target WCR. Using fluorescence confocal microscopy, immuno-detection of IPD072Aa in longitudinal sections from whole WCR larvae that were fed IPD072Aa revealed the association of the protein with the cells that line the gut. High-resolution scanning electron microscopy of similar whole larval sections revealed the disruption of the gut lining resulting from cell death caused by IPD072Aa exposure. These data show that the insecticidal activity of IPD072Aa results from specific targeting and killing of rootworm midgut cells. IMPORTANCE Transgenic traits targeting WCR based on insecticidal proteins from Bacillus thuringiensis have proven effective in protecting maize yield in North America. High adoption has led to WCR populations that are resistant to the trait proteins. Four proteins have been developed into commercial traits, but they represent only two modes of action due to cross-resistance among three. New proteins suited for trait development are needed. IPD072Aa, identified from the bacterium Pseudomonas chlororaphis, was shown to be effective in protecting transgenic maize against WCR. To be useful, IPD072Aa must work through binding to different receptors than those utilized by current traits to reduce risk of cross-resistance and understanding its mechanism of toxicity could aid in countering resistance development. Our results show that IPD072Aa binds to receptors in WCR gut that are different than those utilized by current commercial traits and its targeted killing of midgut cells results in larval death.

Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests.

Various lepidopteran insects are responsible for major crop losses worldwide. Although crop plant varieties developed to express Bacillus thuringiensis (Bt) proteins are effective at controlling damage from key lepidopteran pests, some insect populations have evolved to be insensitive to certain Bt proteins. Here, we report the discovery of a family of homologous proteins, two of which we have designated IPD083Aa and IPD083Cb, which are from Adiantum spp. Both proteins share no known peptide domains, sequence motifs, or signatures with other proteins. Transgenic soybean or corn plants expressing either IPD083Aa or IPD083Cb, respectively, show protection from feeding damage by several key pests under field conditions. The results from comparative studies with major Bt proteins currently deployed in transgenic crops indicate that the IPD083 proteins function by binding to different target sites. These results indicate that IPD083Aa and IPD083Cb can serve as alternatives to traditional Bt-based insect control traits with potential to counter insect resistance to Bt proteins.

Disabled insecticidal proteins: A novel tool to understand differences in insect receptor utilization.

The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or "stacking" of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.

[Final results of an initiative aiming to improve the referrals from primary care to a Department of Urology]. / Resultados finales de un programa para la mejora de las derivaciones desde las unidades de atención primaria a un Servicio de Urología.

OBJECTIVES: To test two interventions aiming at improving the compliance of primary health care physicians with an agreed-on protocol of referrals to a urology department. METHODS: Joint formative meetings (every six months) were celebrated throughout a 24-month period. Also a counseling telephone line was implemented. 18.088 referrals were analyzed. The effect of both expositions was tested comparing basal data (T0) with the performance at 6, 12, 18 and 24 months later (T1, T2, T3 and T4, respectively). An additional comparison was conducted to approach the results 1 year after the study ended (T5). RESULTS: 61.7% of the referrals at baseline complied with the protocol. A significant improvement was detected at T1 (compliance 73.4%, RR with respect to T0 1.19, 95% CI 1.14- 1.23). At T2, 73.7% of referrals were adequate (RR with respect to T0 1.19, 95% CI 1.15-1.24). The percent of adequate referrals at T3 remained stable (73.4%, RR with respect to T0 1.18, 95% CI 1.15-1.23). Nevertheless, adequacy of referrals by the end of the second year (T4) significantly decreased (67.3%, RR with respect to T0, 1.09, 95% CI 1.05-1.12). Adequacy at T5 was almost identical to the basal (64.4%, RR with respect to T0, 1.04 95% CI 1.04-1.07). CONCLUSIONS: Learning activities can be effective in improving the quality of referrals from primary care to one urology department. Stopping the activities entails an immediate return to the basal standards.

Resultados finaels de un programa apra la mejora de las derivaciones desde las unidades de atención primaria a un servicio de urologia / Final results of an initiative aiming to improve the referrals from primary care to a departament of urology

OBJETIVO: Analizar el efecto de dos intervenciones formativas orientadas a mejorar la adhesión a un protocolo de derivación desde Atención Primaria (AP) a un servicio de urología. MÉTODOS: Se celebraron reuniones de trabajo cada seis meses, por espacio de 24 meses, con la participación de urólogos y médicos de AP. Además, se habilitó un sistema de información permanente para profesionales. La valoración del efecto se llevó a cabo comparando la adecuación de las derivaciones antes de la activación del programa (T0), con los datos registrados a los 6, 12, 18 y 24 meses del comienzo del estudio (T1, T2, T3 y T4, respectivamente). Adicionalmente, se analizó la adecuación un año después de cesar las exposiciones. Para este análisis se revisaron 18.088 derivaciones. RESULTADOS: En T0 se consideró adecuado el 61,7% de las derivaciones. Ya en T1 (adecuación 73,4%) se detectó una mejora significativa de la tasa de adecuación (RR con relación a T0, 1,19, IC 1,14-1,23). En T2 el 73,7% de las derivaciones fueron adecuadas (RR con relación a T0, 1,19, IC 1,15-1,24). En T3 el porcentaje de derivaciones adecuadas se mantuvo estable (adecuación 73,4%, RR con relación a T0, 1,18, IC 1,15-1,23). Sin embargo, al final del segundo año (T4), la adecuación disminuyó significativamente (adecuación 67,3%, IC 65,4-69, RR con relación a T0, 1,09, IC 1,05-1,12). Transcurrido un año de la finalización del programa (T5), el porcentaje de derivaciones adecuadas fue superponible al basal (adecuación 64,4%, RR con relación a T0, 1,04, IC 1,04-1,07). CONCLUSIONES: Las actividades educativas permiten mejorar la calidad de las derivaciones desde AP a un servicio de urología. El cese de esas actividades implica el retorno a los valores originales

OBJECTIVES: To test two interventions aiming at improving the compliance of primary health care physicians with an agreed-on protocol of referrals to a urology department. METHODS: Joint formative meetings (every six months) were celebrated throughout a 24-month period. Also a counseling telephone line was implemented. 18.088 referrals were analyzed. The effect of both expositions was tested comparing basal data (T0) with the performance at 6, 12, 18 and 24 months later (T1, T2, T3 and T4, respectively). An additional comparison was conducted to approach the results 1 year after the study ended (T5). RESULTS: 61.7% of the referrals at baseline complied with the protocol. A significant improvement was detected at T1 (compliance 73.4%, RR with respect to T0 1.19, 95% CI 1.14- 1.23). At T2, 73.7% of referrals were adequate (RR with respect to T0 1.19, 95% CI 1.15-1.24).The percent of adequate referrals at T3 remained stable (73.4%, RR with respect to T0 1.18, 95% CI 1.15-1.23). Nevertheless, adequacy of referrals by the end of the second year (T4) significantly decreased (67.3%, RR with respect to T0, 1.09, 95% CI 1.05-1.12). Adequacy at T5 was almost identical to the basal (64.4%, RR with respect to T0, 1.04 95% CI 1.04-1.07). CONCLUSIONS: Learning activities can be effective in improving the quality of referrals from primary care to one urology department. Stopping the activities entails an immediate return to the basal standards

Concordance analysis of ultrasonographic diagnoses by urology nurses.

OBJECTIVES: To check the degree of concordance in renal ultrasound examination between two nurses and one experienced urologist with the aim of testing nurses' competence performing renal ultrasound. METHODS: The echographic aspect of both kidneys (normal or abnormal) was evaluated. Every abnormal finding resulted in the automatic classification of the kidney as 'abnormal'. The agreement between observers was tested using the Kappa concordance index. RESULTS: Eventually, 75 and 45 consecutive examinations performed by the urologist and nurse MM, and the urologist and nurse NJ, respectively, were evaluable. Overall, the study tested 120 patients. Prevalence of 'abnormal' kidneys was intermediate (28-36%). The overall agreement percentage exceeded 88% (88,8-92%). Kappa coefficient was always 0.7. CONCLUSIONS: Urological ultrasound examination by qualified well-trained nurses provides records very similar to those delivered by an experienced urologist.

Analisis de concordancia de los diagnosticos ecográficos del personal de enfermeria urologica / Concordance analysis of ultrasonographic diagnoses by Urology nurses

OBJETIVO: Evaluar el grado de concordancia en el diagnóstico ecográfico entre dos enfermeras y un urólogo experimentado en esa técnica, como medio de validar la competencia de las enfermeras para la exploración ecográfica urológica. MÉTODOS: Se valoró el aspecto de los riñones (normales o anormales). Todo hallazgo fuera de la normalidad supuso la automática clasificación del órgano como ‘anormal’. Acto seguido se determinó el grado de acuerdo entre los observadores y el índice de concordancia kappa. RESULTADOS: Resultaron evaluables 75 y 45 exploraciones consecutivas llevadas a cabo conjuntamente por el urólogo y la enfermera MM, y por el urólogo y la enfermera NJ, respectivamente. En definitiva, el estudio tuvo lugar sobre 120 pacientes; todos autorizaron la doble exploración. La prevalencia de riñones ‘anormales’ resultó intermedia (28-36% de la exploraciones). El porcentaje de acuerdos fue superior al 88% (88,8-92%). En todas las cuestiones analizadas (aspecto del riñón derecho y del riñón izquierdo), el coeficiente kappa resultó igual o superior a 0,7. CONCLUSIONES: La exploración ecográfica por enfermeras bien formadas y entrenadas proporciona registros superponibles a los obtenidos por un urólogo experimentado(AU)

OBJECTIVES: To check the degree of concordance in renal ultrasound examination between two nurses and one experienced urologist with the aim of testing nurses’ competence performing renal ultrasound. METHODS: The echographic aspect of both kidneys (normal or abnormal) was evaluated. Every abnormal finding resulted in the automatic classification of the kidney as ‘abnormal’. The agreement between observers was tested using the Kappa concordance index. RESULTS: Eventually, 75 and 45 consecutive examinations performed by the urologist and nurse MM, and the urologist and nurse NJ, respectively, were evaluable. Overall, the study tested 120 patients. Prevalence of ‘abnormal’ kidneys was intermediate (28-36%). The overallagreement percentage exceeded 88% (88,8-92%). Kappa coefficient was always 0.7. CONCLUSIONS: Urological ultrasound examination by qualified well-trained nurses provides records very similar to those delivered by an experienced urologist(AU)

Quality perception in the outpatient area of an Urology department. Can we improve it?

OBJECTIVES: To test patient's satisfaction after consultation in the outpatient area of the Urology Department in a public hospital using a structured interview. METHODS: We used the Comunidad Autónoma de Madrid (CAM) standard interview form modified to include three questions related to the implementation of a 'one-visit'policy and nurses' empowerment. Patients' opinions were gathered with respect to waiting times in the waiting room, facilities, and staff kindness and professionalism. Sample size was estimated in 386 patients. The effect of every predictive factor on the overall satisfaction was tested using the chi square test. To define the effect of every variable in presence of the rest of covariates a logistic regression model was used. RESULTS: Participation reached 65.5%. Overall, 86.4% of the patients were satisfied. Irrespectively of the professional and personal style, the quality perception was homogeneous (p=ns). Multivariate analysis couldn't disclose any independent predictive variable. Only the perception in the item 'overall time available for the consultation' approached statistical significance (p=0.08), with patients scoring high in this variable getting the highest overall satisfaction scores. CONCLUSIONS: There was no personal or professional style particularly related with patient satisfaction. Nevertheless, there is a slight trend towards a higher satisfaction when patients feel enough time has been spent in their consultation. The new organizational resources (one-visit clinic and nurses' empowerment) are both welcome but are not clearly related to patient satisfaction.

Calidad percibida en el area de consultas de un servicio de urologia / Quality perception in the outpatient area of an Urology department. Can we improve it?

OBJETIVO: Establecer el grado de satisfacción de los pacientes tras su visita al área de consultas de un servicio de urología de un hospital público nacional. MÉTODOS: Se utilizó el modelo de encuesta autoaplicado de la Comunidad Autónoma de Madrid (CAM) modificado para recoger tres preguntas relacionadas con la implantación de un modelo de visita única, y con la participación directa de la enfermería en el proceso de producción. Se recogieron las opiniones de los pacientes en relación al tiempo de espera, instalaciones, amabilidad y profesionalidad de los profesionales, y tiempo dedicado a la entrevista. Para el análisis del efecto de cada uno de los factores predictivos sobre el grado de satisfacción, se empleó el test de la chi cuadrado. Para valorar el efecto de las variables predictivas en presencia del resto de covariables se utilizó un modelo de regresión logística. RESULTADOS: La participación fue del 65,5%. Globalmente, el 86,4% de los pacientes manifestaron encontrarse satisfechos con la atención recibida. Con independencia de los estilos profesionales de médicos y enfermeras, la percepción de la calidad fue homogénea (p=ns). En el análisis multivariante no fue posible identificar ninguna variable predictiva independiente. Sólo el tiempo dedicado a la entrevista con el paciente se aproximó a la significación estadística (p=0,08), de modo que, a mayor percepción en esta categoría, mayor probabilidad de satisfacción global. CONCLUSIONES: Ningún estilo profesional (sensación de profesionalidad) ni personal (sensación de amabilidad) se asocia a un mayor grado de satisfacción. Sin embargo, parece existir una tendencia hacia una mayor satisfacción cuando el paciente percibe que se le ha dedicado suficiente tiempo. Las nuevas formas de atención (visita única e implicación de la enfermería) son bienvenidas por los usuarios de un servicio de urología, pero no determinan mayor satisfacción entre los usuarios(AU)

OBJECTIVES: To test patient`s satisfaction after consultation in the outpatient area of the Urology Department in a public hospital using a structured interview. METHODS: We used the Comunidad Autónoma de Madrid (CAM) standard interview form modified to include three questions related to the implementation of ‘one-visit’ policy and nurses’ empowerment. Patients’ opinions were gathered with respect to waiting times in the waiting room, facilities, and staff kindness and professionalism. Sample size was estimated in 386 patients. The effect of every predictive factor on the overall satisfaction was tested using the chi square test. To define the effect of every variable in presence of the rest of covariates a logistic regression model was used. RESULTS: Participation reached 65.5%. Overall, 86.4% of the patients were satisfied. Irrespectively of the professional and personal style, the quality perception was homogeneous (p=ns). Multivariate analysis couldn’t disclose any independent predictive variable. Only the perception in the item ‘overall time available for the consultation’ approached statistical significance (p=0.08), with patients scoring high in this variable getting the highest overall satisfaction scores. CONCLUSIONS: There was no personal or professional style particularly related with patient satisfaction. Nevertheless, there is a slight trend towards a higher satisfaction when patients feel enough time has been spent in their consultation. The new organizational resources (one-visit clinic and nurses’ empowerment) are both welcome but are not clearly related to patient satisfaction(AU)

Correlations between Different Heavy Metals in Diverse Body Fluids: Studies of Human Semen Quality.

It has been hypothesized that exposure to heavy metals may impair male reproduction. To measure the effect produced by low doses of heavy metals on semen parameters, it is necessary to clarify in which body fluids those measurements must be performed. Sixty-one men attending infertility clinics participated in our study. Concentrations of lead, cadmium, and mercury were measured in whole blood, blood plasma, and seminal plasma using spectroanalytical and electrochemical methods. Semen analyses were performed according to World Health Organization criteria. For statistical analysis, Spearman's rank correlations, mean comparison tests, and discriminant analysis were calculated. Significant correlations between the measured concentrations of the three heavy metals in the same biological fluids were observed. However, no similar relationship was seen when comparing the concentrations in different body fluids of the same metal. According to our results and previous publications, seminal plasma might be the best body fluid for assessing impairment of human semen parameters.

The one-stop clinic as the standard of out-patient care in a hospital urology department.

PURPOSE: To evaluate the performance of a 'one-stop' clinic in terms of proportion of discharges or inclusion in surgical waiting lists. MATERIALS AND METHODS: All patients were referred from primary care facilities (population 220,646) and from different departments in the hospital. Eight senior urologists, two registered nurses and two nurse attendants participated in the experience. Prior to the start of the project, referral protocols had been agreed with the primary care physicians involved. Compliance with the protocols was periodically tested. Eventually 5537 first visits (January-December 2009) where evaluable. RESULTS: Overall, the 'one-stop' format proved feasible in 74.2% of the patients (4108/5537). Patients, who successfully used the 'one-stop' format, were significantly younger than those who required additional consultations (43 vs 50 years old, respectively, Student 's t test < 0.001). For obvious reasons the 'one-stop' format was universally possible in male sterilization and penile phimosis patients. Similarly, the 'one-stop' policy was applied in most consultations due to male sexual dysfunction (75%) and urinary tract infection (73%). Other health problems, such as haematuria (62%) and renal colic (46%), required more than one visit so that care of the patient reverted to the traditional, outpatient care model. CONCLUSION: A 'one-stop' philosophy is feasible for a number of procedures in a urological outpatient clinic. The costs to implement such an approach would be limited to managerial expenditure.

The one-stop clinic as the standard of out-patient care in a hospital urology department

PURPOSE: To evaluate the performance of a 'one-stop' clinic in terms of proportion of discharges or inclusion in surgical waiting lists. MATERIALS AND METHODS: All patients were referred from primary care facilities (population 220.646) and from different departments in the hospital. Eight senior urologists, two registered nurses and two nurse attendants participated in the experience. Prior to the start of the project, referral protocols had been agreed with the primary care physicians involved. Compliance with the protocols was periodically tested. Eventually 5537 first visits (January-December 2009) where evaluable. RESULTS: Overall, the 'one-stop' format proved feasible in 74.2 percent of the patients (4108/5537). Patients, who successfully used the 'one-stop' format, were significantly younger than those who required additional consultations (43 vs 50 years old, respectively, Student's t test < 0.001). For obvious reasons the 'one-stop' format was universally possible in male sterilization and penile phimosis patients. Similarly, the 'one-stop' policy was applied in most consultations due to male sexual dysfunction (75 percent) and urinary tract infection (73 percent). Other health problems, such as haematuria (62 percent) and renal colic (46 percent), required more than one visit so that care of the patient reverted to the traditional, outpatient care model. CONCLUSION: A 'one-stop' philosophy is feasible for a number of procedures in a urological outpatient clinic. The costs to implement such an approach would be limited to managerial expenditure.

Relationships between heavy metal concentrations in three different body fluids and male reproductive parameters: a pilot study.

BACKGROUND: Animal studies have shown the reproductive toxicity of a number of heavy metals. Very few human observational studies have analyzed the relationship between male reproductive function and heavy metal concentrations in diverse biological fluids. METHODS: The current study assessed the associations between seminal and hormonal parameters and the concentration of the 3 most frequent heavy metal toxicants (lead, cadmium and mercury) in three different body fluids. Sixty one men attending infertility clinics that participated in a case-control study to explore the role of environmental toxins and lifestyles on male infertility were analyzed. Concentration of lead, cadmium and mercury were measured in blood and seminal plasma and whole blood using anodic stripping voltammetry and atomic absorption spectrophotometry. Serum samples were analyzed for follicle-stimulating hormone, luteinizing hormone and testosterone. Semen analyses were performed according to World Health Organization criteria. Mann-Whitney test and Spearman's rank correlations were used for unadjusted analyses. Multiple linear regression models were performed controlling for age, body mass index and number of cigarettes per day. RESULTS: There were no significant differences between cases and controls in the concentrations of heavy metals in any of the three body fluids. In multivariate analyses using all subjects no significant associations were found between serum hormone levels and metal concentrations. However there was a significant positive association between the percentage of immotile sperms and seminal plasma levels of lead and cadmium. CONCLUSIONS: Our results suggest that the presence of lead and cadmium in the reproductive tract of men may be related to a moderate alteration of their seminal parameters.

Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects.

Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix alpha-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix alpha-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.

Dominant negative mutants of Bacillus thuringiensis Cry1Ab toxin function as anti-toxins: demonstration of the role of oligomerization in toxicity.

BACKGROUND: Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields. METHODOLOGY/PRINCIPAL FINDINGS: We isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix alpha-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins. CONCLUSIONS/SIGNIFICANCE: This is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms.

Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae.

Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a prepore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R(1) receptor. We show the helix alpha-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R(1) receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae.

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin.

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.

Alkali and halide-resistant catalysis by the multipotent oxidase from Marinomonas mediterranea.

The incorporation of fungal laccases into novel applications has been delayed mainly due to their intrinsic sensitivity towards halides and alkaline conditions. In order to explore new sources of enzymes we evaluated the multipotent polyphenol oxidase PPO1 from the marine bacterium Marinomonas mediterranea. Here we report that, in contrast to its fungal counterparts, PPO1 remained functional above neutral pH presenting high specificity for phenolic compounds, in particular for methoxyl-substituted mono-phenols and catechols. These properties, in addition to its tolerance towards chloride (up to 1 M) and its elevated redox potential at neutral pH (0.9 V), suggest this enzyme may be an interesting candidate for specific applications such as the Amperometric determination of phenolic compounds and bio-fuel cells.