Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit

Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coil, and purified the AHAS regulatory subunit of Ambidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.

Development of electrospun Ion jelly fibers® for drug delivery

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Engineering modular viral scaffolds for targeted bacterial population editing

Bacteria are central to human health and disease, but existing tools to edit microbial consortia are limited. For example, broad-spectrum antibiotics are unable to precisely manipulate bacterial communities. Bacteriophages can provide highly specific targeting of bacteria, but assembling well-defined phage cocktails solely with natural phages can be a time-, labor- and cost-intensive process. Here, we present a synthetic biology strategy to modulate phage host ranges by engineering phage genomes in Saccharomyces cerevisiae. We used this technology to redirect Escherichia coli phage scaffolds to target pathogenic Yersinia and Klebsiella bacteria, and conversely, Klebsiella phage scaffolds to target E. coli by modular swapping of phage tail components. The synthetic phages achieved efficient killing of their new target bacteria and were used to selectively remove bacteria from multi-species bacterial communities with cocktails based on common viral scaffolds. We envision this approach accelerating phage biology studies and enabling new technologies for bacterial population editing.

Nutrição mineral de hortaliças LXX: acumulação de matéria seca e recrutamento de macronutrientes pelo melão (Cucimis Melo L. cv. Valenciano Amarelo CAC) cultivado em latossolo vermelho amarelo em presidente Venceslau, SP

Com o objetivo de determinar: - a curva de crescimento do melão (Cucumis melo L.), através da acumulação de matéria seca; - os teores e consequentes acúmulos de nutrientes nos órgãos aéreos da planta, em diferentes estádios de crescimento; - a exportação de nutrientes na colheita de frutos, no ponto de consumo. Foi conduzido um ensaio em condições de campo o qual consistiu da amostragem em cinco estádios de crescimento - 15, 30, 45, 60 e 75 dias após a emergência - de plantas competitivas, as quais eram cortadas rente ao solo, divididas em caule e ramos, folhas, flores e frutos, para determinação de quantidade de matéria seca e análise química para macronutrientes minerais. Os tratamentos, então representados por épocas de amostragem, constaram de um delineamento em blocos casualizados com quatro repetições. Concluiu-se que: - o crescimento das plantas é inicialmente lento, intensificando-se posteriormente, com maiores incrementos entre 30 e 45 dias; - no final do ciclo, a planta acumulou 905,88 g de matéria seca, assim distribuída; caule e ramos 19,38%, folhas 30,32% e flores e frutos 50,30%; - a concentração dos nutrientes na parte aérea variou com a idade e o órgão considerado; - verifica-se nos frutos uma tendência de redução nos teores dos macronutrientes (com exceção do enxofre); - a parte aérea acumulou, aos 75 dias, em miligramas por planta: 23,08 de N; 3,46 de P; 28,90 de K; 12,74 de Ca; 5,55 de Mg; 1,59 de S. - são exportados numa colheita de uma tonelada de frutos, cerca de 1,78 kg de N; 0,33 kg de P; 2,65 kg de K; 0,14 kg de Ca; 0,21 kg de Mg; 0,09 kg de S.

Acúmulo de macronutrientes pela bananeira cv. prata em diferentes estádios de desenvolvimento

Visando estabelecer a marcha de absorção, quantidade exportada e reciclada de nitrogênio, fósforo, potássio e cálcio na bananeira cultivar Prata, instalou-se um ensaio no Estado do Espírito Santo, em janeiro de 1980, em terreno com declividade média de 40% e solo cambissólico distrófico. Sorteou-se três plantas matrizes do plantio até 465 dias após (colheita),sendo bimensal até 300 dias e mensal até o final, totalizando doze estádios de desenvolvimento, e três plantas rebento, mensalmente, da emissão até 300 dias após, atingindo nove estádios. Cada bananeira foi dividida em folha, pecíolo, pseudocaule, rizoma, engaço, botão floral e fruto para se analisar os macronutrientes. A partir dos dados coletados ajustou-se um programa de regressão e obteve-se as curvas de absorção de macronutrientes, cujos resultados permitiram concluir que, as plantas matriz e rebento: necessitam de expressivas quantidades de macronutrientes para desenvolver e produzir; apresentam uma absorção de macronutrientes que acompanha a matéria seca acumulada pelos órgãos, exceto para o potássio no período; absorvem mais de setenta e cinco por cento de nitrogênio, fósforo e potássio após o estádio de 180 dias e acima de setenta por cento do cálcio e magnésio, a partir de 240 e 120 dias; podem repor ao solo grande parte dos macronutrientes absorvidos; apresentam uma razão de absorção de macronutrientes de 20 K:12 N:10 Mg: 9 Ca: 1 P e de 26,5:8 N:4,5 Mg: 4,5 Ca: 1 P. Conclui-se ainda que: deve-se considerar a quantidade de macronutrientes exportados com a colheita e consumo dos frutos, principalmente nitrogênio, fósforo e potássio, em um programa de adubação; a planta rebento acumula mais macronutrientes do que a planta matriz.

Coleformes fecais em águas de esgoto: II- transferência de marcadores e presença de plasmidial

Investigou-se a transferência de marcadores genéticos e a presença de DNA plasmidial em 240 culturas de Escherichia coli originárias de água de esgoto (afluente e fluentes) da Estação de Tratamento da Ilha do Governador, na cidade do Rio de Janeiro, RJ. Experimentos de conjugação com E. coli K 12 permitiram o isolamento de transconjugantes com resistência a antibióticos (Su, Sm, Tc, Cm e Ap); a metais pesados (Cu, Hg e Zn) e fatores colicinogênicos (Col Ia, Ib e V) principalmente para os coliformes isolados nos setores terminais da estação de tratamento. A distribuição de plasmídeos foi prevalente nas culturas de E. coli advindas dos efluentes, com percentuais superiores a 65.

Rising to Greatness An Imperative for Improving Iowa’s Schools, 2011

In the early 1990s, the Cold War ended, Back to the Future III was in theaters, and Iowa led the nation in reading and mathematics. Times have changed. A decade into the 21st century, Iowa has conceded its place at the top. During the past 20 years, achievement trends illustrate Iowa’s slide from a national leader in PK-12 education to a national average―sometimes below average―performer as other states (and nations) have accelerated past the state. Iowa students’ futures are at risk. Collectively, Iowa students are not hitting the mark in mathematics and reading competency. Sure, Iowa has its share of super-achievers. But the mass of Iowa students—not just underprivileged or minority students, but many of the majority white, relatively affluent students as well—are falling short of what is needed to attain quality jobs, growing incomes, and secure livelihoods in today’s globally competitive world. The world has moved beyond the industrial age and information age and is now in the innovation age. Students must be armed not only with knowledge, but also with skills and insights needed to critically analyze and innovate. The pressing problems and grand opportunities the world faces require that many more people contribute as innovators and problem solvers, not order takers and implementers. Innovators will prosper. Order takers will stagnate. The days of an abundance of low-skill jobs have come to an end.

Effect of fertilization at sowing on nutrition and yield of crambe in second season

The interest in crambe (Crambe abyssinica ) cultivation in Brazil is on the rise, whereas information on the nutrient requirements for this crop is scarce. The objective of this work was to evaluate the effect of nitrogen-phosphorus-potassium (N-P2O5-K2O formula 8:28:16) fertilization (0, 150, and 300 kg ha-1) on crambe shoot biomass production, grain and oil yields, and nutrient extraction and exportation in the second growing season after soybean. The experiment with a Haplorthox (Dystroferric Red Latosol) was carried out for two years in Botucatu, São Paulo State, Brazil. A randomized complete block design with eight replications was used. Fertilization with NPK at sowing increased the shoot biomass production, grain yield, grain oil content, as well as nutrient extraction and exportation at harvest. In the fertilized treatments, the average amounts of nutrients extracted per hectare were 91 kg K, 71 kg N, 52 kg Ca, 9.4 kg P, 9.4 kg Mg, 7.9 kg S, 2,348 g Fe, 289 g Zn, 135 g Mn, and 18.2 g Cu; while the average values of nutrient exportation per hectare were 54 kg N, 20 kg K, 12.3 kg Ca, 10 kg P, 6.6 kg S, 3.2 kg Mg, 365 g Zn, 60 g Fe, 50 g Mn, and 7.3 g Cu, with NPK fertilizer application.

Early intervention for conversion disorder: neurologists and psychiatrists working together

It has been shown that for the reaction catalyzed by "biodegradative" L-threonine dehydratase from E. coli strains K-12 and 980 in 0.5 M phosphate-carbonate buffer, pH 8.4 and pH 9.5, the plots of initial reaction rate (v) versus the initial substrate concentration ([S]0 are characterized by several inflection points, i. e. an intermediate plateau. The plot of v versus the allosteric activator (AMP) concentration have very complicated shapes: there are several inflection points, and also the maximum at L-threonine concentration equal to 3-10(2) and 5-10(-2) M. High AMP concentrations inhibit the enzyme at high substrate concentrations. The reduced glutathion dose not influence the enzyme and does not alter the activating effect of AMP. On the basis of the data obtained it is proposed that the substrate and AMP shift the equilibrium between multiple oligomeric enzyme forms differing in catalytic activity and kinetic manifestations of allosteric interactions between the active and allosteric AMP-binding sites towards polymerization. Thus, the functioning the enzyme under study is discussed in the frames of the model of dissociating regulatory enzymes with multiple intermediate oligomeric forms.

Efeito da omissão de macronutrientes no crescimento, nos sintomas de deficiências nutricionais e na composição mineral em gravioleiras (Annona muricata)

Com o objetivo de avaliar o efeito da omissão de macronutrientes no crescimento, nos sintomas de deficiências nutricionais e na composição mineral em gravioleiras, conduziu-se experimento em casa de vegetação, mediante a técnica do elemento faltante. O delineamento foi o inteiramente casualizado, com quatro repetições e sete tratamentos, sendo completo (N, P, K, Ca, Mg, S e micronutrientes) e com omissão individual de N, P, K, Ca, Mg e S. As omissões dos macronutrientes afetaram o crescimento em altura, diâmetro das plantas e produção de matéria seca, quando comparados ao tratamento completo. Com base nos teores em g/kg dos macronutrientes nas folhas, encontraram-se os seguintes valores adequados nos tratamento completo e com omissão: N = 14,70 e 8,82; P = 0,92 e 0,47; K = 12,35 e 2,62; Ca = 14,11 e 3,44; Mg = 3,59 e 1,09; S = 5,32 e 2,30.

Canth, Minna

Minna Canth, eg. Ulrika Wilhelmina Canth, f. Johnsson f. 19.3.1844 i Tammerfors d. 12.5.1897 i Kuopio Minna Canth har uppfattats som en av de viktigaste författarna inom 1880-talets finländska realism. I sin produktion behandlade hon många av sin tids samhälleliga missförhållanden, så som arbetarklassens fattigdom och kvinnornas svaga ställning i samhället. Canths drama Työmiehen vaimo (1885, sv. Arbetarens hustru, översatt till svenska 1886) markerar inledningen på en samhällskritisk period i författarskapet. Skådespelet Kovan onnen lapsia (1888, sv. Olyckans barn, tidigare kallad De vanlottade) väckte upprörda känslor och ströks ur repertoaren vid Finska teatern efter den första föreställningen. Sedermera har man lyft fram kombinationen av samhälleliga och psykologiska tendenser i Canths produktion. Canth var även verksam som tidningsskribent och skrev många polemiska artiklar om kvinnans ställning. Hon deltog också aktivt i den litterära diskussionen och organiserade litterära salonger för tidens framväxande bildade klass, den s.k. ”Minnan salonki” (sv. Minnas salong) i sin hemgård Kanttila. Biografiskt lexikon för Finland: http://www.blf.fi/artikel.php?id=2816

Topelius, Zachris

Zachris Topelius s. 14.1.1818 Kuddnäsissä, Uusikaarlepyyn lähellä k. 12.3.1898 Sipoossa Zachris Topelius on yksi aikansa suurimpia suomalaisia kirjailijoita, kulttuuripersoonia ja yhteiskunnallisia vaikuttajia. Topeliuksen pääosin 1800-luvun jälkipuoliskolla syntyneessä tuotannossa vallitsee kansallisromantiikan henki, mutta teoksista löytyy myös realistisia piirteitä. Ominaista Topeliuksen kansankuvaukselle on naiivi tunteellisuus ja isänmaallisuus. Kirjailijan teoksiin vaikutti myös hänen kristillinen elämänkatsomuksensa. Topeliuksen mittavan tuotannon tärkeimpiin teoksiin kuuluvat lapsille suunnatut teokset, esimerkiksi Lukemisia lapsille (Läsning för barn I-VIII, 1865‒1896) sekä oppikirja Maamme kirja (Boken om vårt land, 1875), jotka vaikuttivat ajan ruotsinkielisen lastenkirjallisuuden uudistumiseen. Suomessa Topelius oli edelläkävijä myös historiallisten romaanien ja novellien kirjoittajana. Hänen teoksiaan ovat esimerkiksi Suomen herttuatar (Hertiginnan af Finland, 1850) ja Välskärin kertomuksia (Fältskärns berättelser I-V, kirjana 1853‒1867). Topelius tunnettiin lisäksi tuotteliaana lyyrikkona, jonka useita runoja on sävelletty. Virsi- ja laulutuotantoon kuuluvat sellaiset edelleen tunnetut virret ja laulut kuten ”En etsi valtaa loistoa” (”Julvisa”/”Gif mig ej glans”, 1887), ”Kesäpäivä Kangasalla” (”En sommardag i Kangasala”, 1853), ”Sylvian joululaulu” (”Sylvias hälsning från Sicilien”/”Sylvias julvisa”, 1853) ja ”Pajulaulu” (”Videvisan”/”Sof, du lilla vide ung”, 1869). - - - - - Zachris Topelius f. 14.1.1818 i Kuddnäs, nära Nykarleby d. 12.3.1898 i Sibbo Zachris Topelius är en av de största finländska författarna, kulturpersonligheterna och samhällspåverkarna under sin tid. Hans litterära produktion, främst tillkommen under den senare halvan av 1800-talet, präglas av den nationella romantikens anda men även av realistiska drag. Topelius fosterländskhet och bild av det finländska karakteriseras av en naiv känslosamhet och är starkt präglad av hans kristna livssyn. Till det mest betydelsefulla i hans omfattande författarskap hör hans produktion för barn (t.ex. Läsning för barn I-VIII, 1865-1896 samt läroboken Boken om vårt land, 1875) som innebar en förnyelse av tidens svenskspråkiga barnlitteratur. Han var också en föregångare som finländsk författare till historiska romaner och noveller (t.ex. Hertiginnan af Finland, 1850 och Fältskärns berättelser I-V, i bokform 1853-1867), samt mycket produktiv som lyriker. Topelius lyrik har i flera fall tonsatts och han är upphovsman till en rad klassiska och fortfarande sjungna psalmer och andra sånger (t.ex. Julvisa/Gif mig ej glans…, 1887, En sommardag i Kangasala, 1853, Sylvias hälsning från Sicilien/Sylvias julvisa, 1853 och Videvisan/Sof, du lilla vide ung, 1869). Kansallisbiografia: http://www.kansallisbiografia.fi/kb/artikkeli/2854/ http://www.blf.fi/artikel.php?id=2854

Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.

Une approche conceptuelle pour l’interprétation des graphiques en cinématique au secondaire

Cette recherche tente de déterminer si, dans le cadre d’un cours de physique de cinquième secondaire, l’utilisation d’un laboratoire par enquête guidée (comme complément d’enseignement basé sur une approche conceptuelle) permet aux élèves de mieux comprendre des notions de cinématique, que le laboratoire traditionnel. Elle s’inscrit dans une série d’études, réalisées au collégial ou à l’université, qui portent sur des approches d’enseignement exploitant le laboratoire comme moyen de transmission des concepts mécaniques en physique (McDermott, 1996; Beichner, 1994). Le laboratoire par enquête est associé à une approche conceptuelle axée sur le raisonnement qualitatif alors que celui qui est traditionnel est associé à une approche traditionnelle de l’enseignement de la physique. Le test TUG-K, «Test of Understanding Graphs in Kinematics » (Beichner, 1994), ainsi que des entrevues individuelles ont été utilisés afin d’évaluer la compréhension des concepts de cinématique. Il semble d’abord que le laboratoire par enquête guidé soit efficace pour enseigner la plupart des notions de cinématique. De plus, en comparant deux groupes d’une trentaine d’élèves de 5e secondaire ayant suivi deux types de laboratoires différents, l’étude a permis d’entrevoir une piste concernant la compréhension du concept d’accélération. Les résultats suggèrent qu’un laboratoire associé à une approche conceptuelle permettrait aux étudiants, à long terme, de mieux s’approprier les notions d’accélération qu’un laboratoire traditionnel.

Caractérisation et surexpression des fimbriae de type chaperon-placier de Salmonella enterica sérovar Typhi

Salmonella enterica sérovar Typhi (S. Typhi) est l’agent responsable de la fièvre typhoïde et cause environ 200 000 morts et 27 millions de cas annuellement. C’est un pathogène entérique dont le réservoir est restreint à l’Homme. Les raisons de cette restriction d’hôte sont méconnues et pourraient dépendre de l’expression de facteurs d’adhésion à des étapes importantes au cours de la pathogenèse. L’annotation bioinformatique du génome de S. Typhi identifie 12 fimbriae de type chaperon-placier (FCP), un curli ainsi qu’un pilus de type IV. L’objectif de ce projet de recherche est d’étudier ces systèmes d’adhésion peu caractérisés. D’abord, le niveau d’expression de ces gènes a été évalué dans différentes conditions de culture in vitro en utilisant une approche de gènes rapporteurs. L’expression des 14 systèmes d’adhésion a été détectée. Nos résultats indiquent qu’une carence en fer favorise l’expression des opérons bcf et csg. Indépendamment du fer, l’expression de bcf, csg, pil, sef, sta, stc, stg et sth est influencée par la richesse nutritive du milieu. L’incubation en milieu LB liquide favorise l’expression de la plupart des systèmes d’adhésion par rapport à un milieu LB liquide sans agitation ou un milieu LB solide. En somme, l’expression des systèmes d’adhésion de S. Typhi a été observée et est influencée par des conditions environnementales. Dans un second volet, nous avons tent de surexprimer les différents systèmes d’adhésion chez une souche d’E. coli ou de S. Typhi afimbriaire. Avec cette approche, nous avons été en mesure de démontrer que l’opéron tcf encode pour un fimbria fonctionnel que l’on a pu observer en microscopie électronique. L’expression de tcf chez une souche afimbriaire d’E. coli et S. Typhi a également diminué leur capacité d’adhésion à des cellules épithéliales intestinales humaines lors d’essais in vitro. Nos observations démontrent que l’expression des systèmes d’adhésion retrouvés chez S. Typhi est influencée par les conditions enviroi9onnementales. Au moins un de ces systèmes est fonctionnel. Ceci suggère une contribution des systèmes d’adhésion retrouvés chez S. Typhi lors de l’interaction de ce pathogène avec l’humain.