Protease inhibitor from Moringa oleifera leaves: Isolation, purification, and characterization

Protease inhibitors have great demand in medicine and biotechnology. We report here the purification and characterization of a protease inhibitor isolated from mature leaf extract of Moringa oleifera that showed maximum inhibitor activity. The protease inhibitor was purified to 41.4-fold by Sephadex G75 and its molecular mass was calculated as 23,600 Da. Inhibitory activity was confirmed by dot-blot and reverse zymogram analyses. Glycine, glutamic acid, alanine, proline and aspartic acid were found as the major amino acids of the inhibitor protein. Maximal activity was recorded at pH 7 and at 40 ◦C. The inhibitor was stable over pH 5–10; and at 50 ◦C for 2 h. Thermostability was promoted by CaCl2, BSA and sucrose. Addition of Zn2+ and Mg2+, SDS, dithiothreitol and -mercaptoethanol enhanced inhibitory activity, while DMSO and H2O2 affected inhibitory activity. Modification of amino acids at the catalytic site by PMSF and DEPC led to an enhancement in the inhibitory activity. Stoichiometry of trypsin–protease inhibitor interaction was 1:1.5 and 0.6 nM of inhibitor effected 50% inhibition. The low Ki value (1.5 nM) obtained indicated scope for utilization of M. oliefera protease inhibitor against serine proteases

Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine

Trypsin Inhibitor from Edible Mushroom Pleurotus floridanus Active against Proteases of Microbial Origin

Protease inhibitors can be versatile tools mainly in the fields of medicine, agriculture and food preservative applications. Fungi have been recognized as sources of protease inhibitors, although there are only few such reports on mushrooms. This work reports the purification and characterization of a trypsin inhibitor from the fruiting body of edible mushroom Pleurotus floridanus (PfTI) and its effect on the activity of microbial proteases. The protease inhibitor was purified up to 35-fold by DEAE-Sepharose ion exchange column, trypsin-Sepharose column and Sephadex G100 column. The isoelectric point of the inhibitor was 4.4, and its molecular mass was calculated as 37 kDa by SDS-PAGE and 38.3 kDa by MALDI-TOF. Inhibitory activity confirmation was by dot-blot analysis and zymographic activity staining. The specificity of the inhibitor toward trypsin was with Ki of 1.043×10−10 M. The inhibitor was thermostable up to 90 °C with maximal stability at 30 °C, active over a pH range of 4–10 against proteases from Aspergillus oryzae, Bacillus licheniformis, Bacillus sp. and Bacillus amyloliquefaciens. Results indicate the possibility of utilization of protease inhibitor from P. floridanus against serine proteases

Lipid Droplets in Dictyostelium discoideum

Lipid Droplets dienen zur Speicherung von Neutrallipiden wie z. B. Triglyceriden und Sterolestern. Im ersten Teil der vorliegenden Arbeit wurde die Bildung dieser zellulären Fettspeicher in D. discoideum untersucht. Es konnte herausgefunden werden, dass Lipid Droplets entstehen, wenn die Zellen entweder in einer Suspension von Bakterien oder in Gegenwart von Palmitinsäure kultiviert werden. Die Bildung der Lipidtröpfchen wird dabei von einem schnelleren Zellwachstum, einem Anstieg des Triglyceridgehalts, einer Reduktion der Phagozytoserate und einer Abnahme des Zellvolumens begleitet. Wurde die Lipid Droplet-Bildung durch Kultivierung der Zellen mit Palmitinsäure angeregt, entsteht neben Triglyceriden noch eine weitere Verbindung, bei der es sich entweder um Fettsäureethylester oder Wachsester handelt. Eine weitere Eigenschaft von Zellen, die in Gegenwart der Palmitinsäure inkubiert wurden, ist die Fähigkeit exogene Fettsäuren schneller aufzunehmen, als normal kultivierte Zellen. Aus der vorliegenden Arbeit wurde gefolgert, dass dies durch eine zusätzliche Aufnahme der Fettsäuren über die Plasmamembran hervorgerufen wird. In Zellen, die ohne Fettsäuren inkubiert wurden, findet hingegen der Fettsäureimport über die Endosomen statt. Ein Protein, das nicht direkt am Prozess der Fettsäureaufnahme beteiligt ist, aber importierte Fettsäuren mit CoA aktiviert, ist die LC-FACS1. Aus Versuchen mit der Knockout-Mutante ging hervor, dass die aktivierten Fettsäuren, in Zellen, die zuvor mit Palmitinsäure oder Bakterien inkubiert wurden, in Triglyceride eingebaut werden. Der reduzierte Triglyceridgehalt im Knockout rief eine Erhöhung der Phagozytoserate hervor. Im zweiten Teil dieser Arbeit wurden die Lipidtröpfchen mit einem Saccharosegradienten aufgereinigt. Mit Hilfe der Massenspektrometrie konnten 281 Proteine in der Lipid Droplet-Fraktion identifiziert werden. Ein Teil dieser Proteine könnte durch die Interaktion der Lipidtröpfchen mit anderen Organellen in die Lipid Droplet-Fraktion gelangt sein und ist ebenso wenig Teil des Lipid Droplet-Proteoms wie die zytoplasmatischen Proteine, die eine Verunreinigung darstellen. Vier der zehn Proteine aus der Lipid Droplet-Fraktion, die in der vorliegenden Arbeit untersucht wurden, konnten nach Kultivierung in palmitinsäurehaltigem Medium tatsächlich auf der Oberfläche der Lipidtröpfchen beobachtet werden. Eines dieser Proteine ist LSD1. Es stellt das einzige PAT-Protein in D. discoideum dar und gehört der Kategorie der CPATs an. Analog zu Perilipin/PLIN1 und Adipophilin/PLIN2 könnte LSD1 eine Schutzfunktion der Lipid Droplets vor zytoplasmatischen Lipasen haben. Neben DdLSD1 konnten auch die Proteine ADH und ALI auf den Lipidtröpfchen lokalisiert werden. Bei beiden handelt es sich um 17beta-Hydroxysteroid-Dehydrogenasen - Proteine, die eine Funktion im Lipid- oder Fettsäuremetabolismus besitzen können. Das Protein SMT katalysiert die C24-Methylierung des Sterolgerüsts in D. discoideum und war nach Inkubation der Zellen mit exogenen Fettsäuren ebenfalls auf den Lipid Droplets zu beobachten.

Private Higher Education in the Sultanate of Oman: Rationales, Development and Challenges

Diese Studie stellt die Entwicklung des privaten Hochschulbereichs im Oman dar und analysiert sie auf die damit verbundenen Erwartungen. Sie untersucht die wesentlichen Herausforderungen, denen sich dieser Sektor zu stellen hat, und formuliert einige Empfehlungen, um die Rolle der privaten Hochschulbildung im Oman zu fördern. Um die Situation in Oman einordnen zu konnen, wurde die Literatur zu Systemen der privaten Hochschulbildung in verschiedenen Ländern vergleichend aufgearbeitet. Der Autor dieser Dissertation hat zudem zahlreiche offizielle Dokumente, Statistiken der Regierung, Berichte, Korrespondenzen und auch unveröffentlichtes Material zum Thema Bildung, Wirtschaft und zur Personalentwicklung geprüft und analysiert. Halb-strukturierte Interviews wurden mit Präsidenten und Dekanen privater Hochschulen sowie mit einigen externen Akteuren durchgeführt, um die Stärken und Schwächen, Herausforderungen und Ziele des privaten Hochschulsektors in Oman zu analysieren.

Characterization of Lipid Droplets and Functional Analysis of Lipid Droplet-Associated Proteins in Dictyostelium discoideum

Lipid droplets (LDs) are the universal storage form of fat as a reservoir of metabolic energy in animals, plants, bacteria and single celled eukaryotes. Dictyostelium LD formation was investigated in response to the addition of different nutrients to the growth medium. LDs were induced by adding exogenous cholesterol, palmitic acid (PA) as well as growth in bacterial suspension, while glucose addition fails to form LDs. Among these nutrients, PA addition is most effective to stimulate LD formation, and depletion of PA from the medium caused LD degradation. The neutral lipids incorporated into the LD-core are composed of triacylglycerol (TAG), steryl esters, and an unknown neutral lipid (UKL) species when the cells were loaded simultaneously with cholesterol and PA. In order to avoid the contamination with other cellular organelles, the LD-purification method was modified. The isolated LD fraction was analysed by mass spectrometry and 100 proteins were identified. Nineteen of these appear to be directly involved in lipid metabolism or function in regulating LD morphology. Together with a previous study, a total of 13 proteins from the LD-proteome were confirmed to localize to LDs after the induction with PA. Among the identified LD-proteins, the localization of Ldp (lipid droplet membrane protein), GPAT3 (glycerol-3-phosphate acyltransferase 3) and AGPAT3 (1-acylglycerol-3-phosphate-acyltransferase 3) were further verified by GFP-tagging at the N-termini or C-termini of the respective proteins. Fluorescence microscopy demonstrated that PA-treatment stimulated the translocation of the three proteins from the ER to LDs. In order to clarify DGAT (diacylglycerol acyltransferase) function in Dictyostelium, the localization of DGAT1, that is not present in LD-proteome, was also investigated. GFP-tagged DGAT1 localized to the ER both, in the presence and absence of PA, which is different from the previously observed localization of GFP-tagged DGAT2, which almost exclusively binds to LDs. The investigation of the cellular neutral lipid level helps to elucidate the mechanism responsible for LD-formation in Dictyostelium cells. Ldp and two short-chain dehydrogenases, ADH (alcohol dehydrogenase) and Ali (ADH-like protein), are not involved in neutral lipid biosynthesis. GPAT, AGPAT and DGAT are three transferases responsible for the three acylation steps of de novo TAG synthesis. Knock-out (KO) of AGPAT3 and DGAT2 did not affect storage-fat formation significantly, whereas cells lacking GPAT3 or DGAT1 decreased TAG and LD accumulation dramatically. Furthermore, DGAT1 is responsible for the accumulation of the unknown lipid UKL. Overexpression of DGAT2 can rescue the reduced TAG content of the DGAT1-KO mutant, but fails to restore UKL content in these cells, indicating that of DGAT1 and DGAT2 have overlapping functions in TAG synthesis, but the role in UKL formation is unique to DGAT1. Both GPAT3 and DGAT1 affect phagocytic activity. Mutation of GPAT3 increases it but a DGAT1-KO decreases phagocytosis. The double knockout of DGAT1 and 2 also impairs the ability to grow on a bacterial lawn, which again can be rescued by overexpression of DGAT2. These and other results are incorporated into a new model, which proposes that up-regulation of phagocytosis serves to replenish precursor molecules of membrane lipid synthesis, whereas phagocytosis is down-regulated when excess fatty acids are used for storage-fat formation.  

Wild food trees in Eastern Nuba Mountains, Sudan: Use diversity and threatening factors

This study was conducted in 2010 in Eastern Nuba Mountains, Sudan to investigate ethnobotanical food and non-food uses of 16 wild edible fruit producing trees. Quantitative and qualitative information was collected from 105 individuals distributed in 7 villages using a semi-structured questionnaire. Also gathering of data was done using a number of rapid rural appraisal techniques, including key informant interviews, group discussion, secondary data sources and direct observations. Data was analysed using fidelity level and informant consensus factor methods to reveal the cultural importance of species and use category. Utilizations for timber products were found of most community importance than food usages, especially during cultivated food abundance. Balanites aegyptiaca, Ziziphus spina-christi and Tamarindus indica fruits were asserted as most preferable over the others and of high marketability in most of the study sites. Harvesting for timber-based utilizations in addition to agricultural expansion and overgrazing were the principal threats to wild edible food producing trees in the area. The on and off prevailing armed conflict in the area make it crucial to conserve wild food trees which usually play a more significant role in securing food supply during emergency times, especially in times of famine and wars. Increasing the awareness of population on importance of wild food trees and securing alternative income sources, other than wood products, is necessary in any rural development programme aiming at securing food and sustaining its resources in the area.

Dairy farms typology and management of animal genetic resources in the peri-urban zone of Bamako (Mali)

Facing growth in demand, dairy production in peri-urban areas of developing countries is changing rapidly. To characterise this development around Bamako (Mali), this study establishes a typology of dairy production systems with a special focus on animal genetic resources. The survey included 52 dairy cattle farms from six peri-urban sites. It was conducted in 2011 through two visits, in the dry and harvest seasons. The median cattle number per farm was 17 (range 5-118) and 42% of farmers owned cropland (8.3 +/- 7.3 ha, minimum 1 ha, maximum 25 ha). Feeding strategy was a crucial variable in farm characterisation, accounting for about 85% of total expenses. The use of artificial insemination and a regular veterinary follow-up were other important parameters. According to breeders’ answers, thirty genetic profiles were identified, from local purebreds to different levels of crossbreds. Purebred animals raised were Fulani Zebu (45.8%), Maure Zebu (9.2%), Holstein (3.0%), Azawak Zebu (1.3%), Mere Zebu (0.5%) and Kuri taurine (0.1%). Holstein crossbred represented 30.5% of the total number of animals (19.0% Fulani-Holstein, 11.2% Maure-Holstein and 0.3% Kuri-Holstein). Montbéliarde, Normande and Limousin crossbreds were also found (6.6%, 0.7% and 0.3%, respectively). A multivariate analysis helped disaggregate the diversity of management practices. The high diversity of situations shows the need for consideration of typological characteristics for an appropriate intervention. Although strongly anchored on local breeds, the peri-urban dairy systems included a diversity of exotic cattle, showing an uncoordinated quest of breeders for innovation. Without a public intervention, this dynamic will result in an irremediable erosion of indigenous animal genetic resources.

Alimenta tu salud.

Materiales de trabajo elaborados por un grupo de profesores de secundaria que presentan modelos de actividades y fichas para tratar el tema transversal de educación para la salud en las siguientes áreas: ciencias de la naturaleza, francés, informática, inglés, lengua y literatura, tecnología y tutoría.

Robot Motion Vision by Fixation

In many motion-vision scenarios, a camera (mounted on a moving vehicle) takes images of an environment to find the "motion'' and shape. We introduce a direct-method called fixation for solving this motion-vision problem in its general case. Fixation uses neither feature-correspondence nor optical-flow. Instead, spatio-temporal brightness gradients are used directly. In contrast to previous direct methods, fixation does not restrict the motion or the environment. Moreover, fixation method neither requires tracked images as its input nor uses mechanical tracking for obtaining fixated images. The experimental results on real images are presented and the implementation issues and techniques are discussed.

Detecció, anàlisi de partícules provocades pel desgast en olis lubricant usats com a eina de manteniment de motors de combustió interna

A la física o a l’enginyeria es defineix com fregament o fricció a la resistència que s'oposa a la rotació o al lliscament d'un cos sobre un altre, o també a la força que apareix en la superfície de contacte de dos cossos quan s'intenta lliscar un sobre un altre. El fregament ha estat fins avui dia un gran problema físic, això és degut a que genera imperfeccions, especialment microscòpiques, entre les superfícies en contacte. Aleshores és quan apareix la tribologia. La tribologia és la ciència que estudia la fricció, el desgast i la lubricació de superfícies en contacte. El lliscament entre superfícies sòlides es caracteritza generalment per un alt coeficient de fricció i un gran desgast a causa de les propietats específiques de les superfícies. La lubricació consisteix en la introducció d'una capa intermèdia d'un material aliè entre les superfícies en moviment. En aquest projecte hem intentat aplicar tècniques i mètodes de control els quals puguin arribar a millorar i perfeccionar els sistemes de mesura i control a nivell industrial i/o particular. En el nostre cas ha estat l’estudi dels olis lubricants usats en motors de combustió interna de variats vehicles com motocicletes, automòbils, camions o vaixells. Hem introduït una millora d’automatització mitjançant un circuit pneumàtic al captador de partícules, hi hem introduït una tècnica per captar partícules a partir de filtres de membrana, hem estudiat les mostres i llurs ferrogrames i membranes amb la finalitat de detectar les anomalies dels motors. L’objectiu del treball és l’estudi del desgast originat en motors de combustió interna, majoritàriament, Dièsel de camions, automòbils i vaixells. Això, comprèn la captació de partícules en ferrografies, la seva observació i anàlisi en microscopi, la seva classificació, comparació i la detecció d’anomalies en els motors. Per altra banda, també s’aprofundirà en les tècniques d’anàlisi, la lubricació i manteniment dels motors i el nou disseny i validació d’un captador de partícules automatitzat

Conversa a Utopia

Joan Manuel del Pozo acaba de publicar la primera traducció catalana directa d’Utopia. Apareguda per primer cop en llatí el 1516, és l’obra més destacable de l’humanista anglès Tomas More, decapitat per protestar contra la pretensió d’Enric VIII d’envair un espai de poder que fins aleshores li havia estat aliè: l’espiritual. Utopia és una illa, i More la va imaginar com un paradís de tolerància religiosa, on no faltava la feina ni els recursos per a tothom. Amb el pretext de l’edició del llibre, Engega ha convidat el filòsof Joan Manuel del Pozo, el químic Miquel Costas i la doctora en Medicina Marta Aymerich, a mantenir una conversa gens utòpica, que us presentem a continuació

Incidencia de la política exterior de China y la Federación Rusa en el mantenimiento del desarrollo de armas nucleares y enriquecimiento de uranio iraníes, durante la primera administración del Presidente Mahmoud Ahmadinejad (2002-2005)

El giro que el sistema internacional dio tras los atentados perpetrados el 11 de septiembre de 2001 fue de enorme importancia para la política exterior de Irán y su posicionamiento en el sistema internacional. En efecto, la lucha guerra contra el terrorismo que Estados Unidos inició y que finalmente desembocaría en las incursiones armadas a Afganistán e Irak -ambos países limítrofes de Irán, en 2001 y 2003 respectivamente, así como la inclusión de Irán por parte de la administración Bush en 2002 como parte del denominado Eje del mal, generaron en el imaginario iraní una idea de amenaza internacional y por lo mismo, la necesidad de hacerse fuerte con el fin de mantener su integridad territorial y gubernamental. Así pues, una compleja situación económica y social sumada a una fuerte coalición del partido conservador iraní -uno de los más fuertes del país, soportado por el Ayatolá Alí Jamenei, autoridad suprema de la nueva República, un nuevo líder de corte conservador, llegaría a la presidencia: Mahmoud Ahmadinejad, un líder controversial y problemático que desde su primera campaña presidencial en 2002 defendería el derecho de la República a desarrollar armas nucleares para fines pacíficos. El verdadero inconveniente surge cuando occidente, en cabeza de los Estados Unidos –en compañía de algunas potencias europeas como lo son Alemania, Francia y Gran Bretaña, duda sobre esta última afirmación. Esta investigación pretende profundizar cada uno de los elementos anteriormente mencionados así como establecer la forma en la cual China y Rusia –países que ven de forma diferenciada el manejo de arsenal nuclear, logran configurarse y acreditar este desafío ante la comunidad internacional.

Incidencia de las identidades transnacionales en las comunidades de origen. Estudio de caso : Túnez a la luz de la primavera árabe

El presente trabajo de investigación busca responder cuestiones relativas a los procesos de formación de identidades bajo la lógica de un modelo transnacional de las migraciones. De manera más específica, es un intento por analizar el desarrollo de los sucesos que provocaron en enero de este año el revocamiento del mandato de Zine El Abidine Ben Alí, quien se mantuvo en el poder desde 1987, a la luz de conceptos como la identidad nacional, la migración transnacional y la formación de identidades. ¿Cuál es el proyecto nacional que se está planteando la sociedad tunecina actual y sobre cuáles han sido los factores sociales, políticos, económicos y culturales que han determinado las actuaciones de los tunecinos luego de su independencia de Francia. ¿Han sido estas protestas el reflejo de un cambio en la manera en la cual los tunecinos se identifican unos con otros?, o, en otras palabras, ¿el proyecto político emprendido por la sociedad tunecina actual responde a un cambio en la construcción de identidad de la nación, y cuáles han sido los factores que han determinado ese proceso?

Computer Misuse Act Presentation

This is one of two presentations we have produced for the resource set part of the INFO2009 Assignment 2 Group Poster.