Wounding of Arabidopsis halleri leaves enhances cadmium accumulation that acts as a defense against herbivory.

Approximately 0.2 % of all angiosperms are classified as metal hyperaccumulators based on their extraordinarily high leaf metal contents, for example >1 % zinc, >0.1 % nickel or >0.01 % cadmium (Cd) in dry biomass. So far, metal hyperaccumulation has been considered to be a taxon-wide, constitutively expressed trait, the extent of which depends solely on available metal concentrations in the soil. Here we show that in the facultative metallophyte Arabidopsis halleri, both insect herbivory and mechanical wounding of leaves trigger an increase specifically in leaf Cd accumulation. Moreover, the Cd concentrations accumulated in leaves can serve as an elemental defense against herbivory by larvae of the Brassicaceae specialist small white (Pieris rapae), thus allowing the plant to take advantage of this non-essential trace element and toxin. Metal homeostasis genes are overrepresented in the systemic transcriptional response of roots to the wounding of leaves in A. halleri, supporting that leaf Cd accumulation is preceded by systemic signaling events. A similar, but quantitatively less pronounced transcriptional response was observed in A. thaliana, suggesting that the systemically regulated modulation of metal homeostasis in response to leaf wounding also occurs in non-hyperaccumulator plants. This is the first report of an environmental stimulus influencing metal hyperaccumulation.

Ash2 acts as an Ecdysone Receptor coactivator by stabilizing the histone methyltransferase Trr.

The molting hormone ecdysone triggers chromatin changes via histone modifica- tions that are important for gene regulation. On hormone activation, the ecdysone receptor (EcR) binds to the SET domain-containing histone H3 methyltransferase trithorax-related protein (Trr). Methylation of histone H3 at lysine 4 (H3K4me), which is associated with tran- scriptional activation, requires several cofactors, including Ash2. We find that ash2 mutants have severe defects in pupariation and metamorphosis due to a lack of activation of ecdy- sone-responsive genes. This transcriptional defect is caused by the absence of the H3K4me3 marks set by Trr in these genes. We present evidence that Ash2 interacts with Trr and is re- quired for its stabilization. Thus we propose that Ash2 functions together with Trr as an ecdysone receptor coactivator.

Clinical determinants of early parasitological response to ACTs in African patients with uncomplicated falciparum malaria: a literature review and meta-analysis of individual patient data.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum has emerged in the Greater Mekong sub-region and poses a major global public health threat. Slow parasite clearance is a key clinical manifestation of reduced susceptibility to artemisinin. This study was designed to establish the baseline values for clearance in patients from Sub-Saharan African countries with uncomplicated malaria treated with artemisinin-based combination therapies (ACTs). METHODS: A literature review in PubMed was conducted in March 2013 to identify all prospective clinical trials (uncontrolled trials, controlled trials and randomized controlled trials), including ACTs conducted in Sub-Saharan Africa, between 1960 and 2012. Individual patient data from these studies were shared with the WorldWide Antimalarial Resistance Network (WWARN) and pooled using an a priori statistical analytical plan. Factors affecting early parasitological response were investigated using logistic regression with study sites fitted as a random effect. The risk of bias in included studies was evaluated based on study design, methodology and missing data. RESULTS: In total, 29,493 patients from 84 clinical trials were included in the analysis, treated with artemether-lumefantrine (n = 13,664), artesunate-amodiaquine (n = 11,337) and dihydroartemisinin-piperaquine (n = 4,492). The overall parasite clearance rate was rapid. The parasite positivity rate (PPR) decreased from 59.7 % (95 % CI: 54.5-64.9) on day 1 to 6.7 % (95 % CI: 4.8-8.7) on day 2 and 0.9 % (95 % CI: 0.5-1.2) on day 3. The 95th percentile of observed day 3 PPR was 5.3 %. Independent risk factors predictive of day 3 positivity were: high baseline parasitaemia (adjusted odds ratio (AOR) = 1.16 (95 % CI: 1.08-1.25); per 2-fold increase in parasite density, P <0.001); fever (>37.5 °C) (AOR = 1.50 (95 % CI: 1.06-2.13), P = 0.022); severe anaemia (AOR = 2.04 (95 % CI: 1.21-3.44), P = 0.008); areas of low/moderate transmission setting (AOR = 2.71 (95 % CI: 1.38-5.36), P = 0.004); and treatment with the loose formulation of artesunate-amodiaquine (AOR = 2.27 (95 % CI: 1.14-4.51), P = 0.020, compared to dihydroartemisinin-piperaquine). CONCLUSIONS: The three ACTs assessed in this analysis continue to achieve rapid early parasitological clearance across the sites assessed in Sub-Saharan Africa. A threshold of 5 % day 3 parasite positivity from a minimum sample size of 50 patients provides a more sensitive benchmark in Sub-Saharan Africa compared to the current recommended threshold of 10 % to trigger further investigation of artemisinin susceptibility.

Nuclear Factor I-C acts as a regulator of hepatocyte proliferation at the onset of liver regeneration.

BACKGROUND & AIMS: Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed abnormal skin wound healing and growth of its appendages, suggesting a role in controlling cell proliferation in adult regenerative processes. Liver regeneration following partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes lead to their rapid and phased proliferation. Although NFI-C is highly expressed in the liver, no hepatic function was yet established for this transcription factor. This study aimed to determine whether NFI-C may play a role in hepatocyte proliferation and liver regeneration. METHODS: Liver regeneration and cell proliferation pathways following two-thirds PH were investigated in NFI-C knockout (ko) and wild-type (wt) mice. RESULTS: We show that the absence of NFI-C impaired hepatocyte proliferation because of plasminogen activator I (PAI-1) overexpression and the subsequent suppression of urokinase plasminogen activator (uPA) activity and hepatocyte growth factor (HGF) signalling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wt mice. The subsequent transient down regulation of NFI-C, as can be explained by a self-regulatory feedback loop with transforming growth factor beta 1 (TGF-ß1), may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. CONCLUSION: NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration.

The Drosophila TNF Eiger Is an Adipokine that Acts on Insulin-Producing Cells to Mediate Nutrient Response.

Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. Here we show that Eiger, the Drosophila TNF-α, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-α converting enzyme (TACE) family is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, we have identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-α that mediates adaptive response to nutrient deprivation.

Improving the effectiveness of supply chain development work – an expert role perspective

This study focuses to the intersection of three sets of activities in a company: expert work, development work and supply chain management, SCM. Experts and expert work represent a set of individuals whose efficiency and impact this study is intended to improve, while development work defines the set of organizational activities to focus on. SCM as an expertise area acts as the platform on which this study is built. The study has two aims. Firstly, it aims to derive a model helping an SCM expert to increase the effectiveness of expert work in development tasks by understanding the encountered organizational situations and processes better, reflecting his/her past and future actions to organizational processes and selecting and adjusting the processes and contents of his/her work accordingly. Secondly, it aims to develop applicable approaches and methods to understand, evaluate and manage the organizational processes and situations in development work. The integrative model on approaches and methods to improve the effectiveness of development processes is split to two aggregate dimensions: technical performance of the developed solution and consumption of resources of the development process. Six potential approaches and methods aiming at helping in the management of organizational dimensions are presented in enclosed publications. The approaches focus on three subtasks of development work: decision making, implementation and change, and knowledge accumulation. The approaches and methods have been tested in case studies representing typical development processes in the area of supply chain management. As a result, four suggestions are presented. Firstly, SCM experts are advised to consider the SCM development work to be consisting of development processes. Secondly, inside these processes they should identify and evaluate the risk of difficult decision-making related to organizational factors. Thirdly, they are prompted for an active role in implementation and change, supporting the implementation through whole process. Finally, the development should be seen in a holistic view, taking into account the stage of knowledge and organizational issues related to it, and adopt a knowledge development strategy.

Manon Lescaut : a lyric drama in four acts

Oletettavasti sama levytys kuin CD-julkaisussa RCA Victor Gold Seal Opera Series, 60573-2-RG, jossa tosin Plinio Clabassin tilalle merkitty Enrico Campi.

Beyond the obvious : three acts in educational drama

Researching research is not a common theme in educational drama. Nor is the educational drama process from a participant perspective a typical focus of research, at least not if the participants are disabled. Yet this is the theme of this thesis, a drama in three acts. The aim of this thesis is to describe, analyse, and discuss both the ways in which research within educational drama can be carried out and represented, and the experiences of the participants of the educational drama process. The theoretical framework that steers the research process is built up of two pairs of frames, each of them, like Russian nesting dolls, containing further frames. The first frame, relating to the outcomes of conducting research in educational drama, comprises philosophical, representational, and personal theories. As the second question asks what educational drama is, the subject related frame is built up of pedagogical, drama educational, and aesthetic theories. The study in its entirety follows the structure of the researcher’s hermeneutical learning process and takes the form of a journey starting from what is familiar, stretching towards what is new and different, and finally returning back to the beginning with a new view on what was there at the start. The thesis consists of two separate but related studies. The first, a familiar study conducted earlier, Alpha in Act I, was carried out among upper secondary school pupils. In the second, the new and therefore unfamiliar study, Omega in Act III, the participants are adult individuals who are physically and communicatively disabled. In between these two Acts an element of “Verfremdung” where the Alpha study is systematically scrutinized as the purpose is to teach and to manage the reader to think. Meta-discussions on the philosophical issues of the study are conducted throughout the text, parallel to the empirical parts. The outcomes of the first research question show that philosophical, methodical, and representational consistency is crucial for research. While this may sound like stating the obvious, this has nevertheless not always been considered fact, especially not within qualitative research. The outcomes further stress that representational issues are also to be recognized when presenting non-rational aspects of educational drama. By wording the world, through the use of visualising language, the surplus of meanings of educational drama can be, as they are within this study, made visible, sensible, and almost tangible, not only cognitively understandable. The outcomes of the second question point to the different foci of the studies, with Alpha focusing on the rationally retold experiences and Omega focusing on nonrational experiences. The outcomes expose educational drama as a learning process comprising doing, reflecting, and being. The doing aspect communicates the concrete efforts in creating a piece of theatre, while the being aspect relates experiences of being as situated, embodied and sensuous, reciprocal, empowering, aesthetic and artistic, and existential. Reflection is the twine that runs throughout the process and connects both doing and being. In summary, the outcomes could be formulated as “learning from learning how to make theatre”.

Hallinnollinen sanktiointi

Tausta Suomessa lainsäätäjä reagoi helposti yhteiskunnalliseen häiriötekijään rikosoikeuden keinoin. Tästä on osoituksena se, että syksyllä 2010 varsinaisen rikoslain lisäksi rikoksesta oli säädetty 256 eri laissa. Suomalainen, lähtökohdiltaan maltillinen kontrolli- ja kriminaalipolitiikka on 1970-luvulla linjatun vastaisesti johtanut 2000-luvulle tultaessa rikosoikeudellisen kontrollin lisääntymiseen ja jopa kiristymiseen. Raja rikosten ja rikkomusten välillä on liudentunut ja paikoitellen kadonnut ja erityisesti turvallisuudesta on kehittynyt – käsitteeseen liittyvistä oikeudellisista jännitteistä huolimatta – keskeinen rikosoikeudellisen sääntelyn oikeutusperuste. Rikosoikeuden käyttäminen vähäisten rikosten kontrollointiin on aiheuttanut Suomeen viivästymisen ongelman. Yleisessä tiedossa on se, että Euroopan ihmisoikeustuomioistuin on antanut 2000-luvulla Suomelle kymmeniä tuomioita siksi, että rikosjuttuja on käsitelty viranomaisissa ja tuomioistuimissa liian kauan. Vähemmän on sen sijaan keskusteltu siitä, että myös vähäiset rikokset viivästyvät rikosoikeuden alan laajetessa ja viranomaisten voimavarojen ehtyessä. Suomessa ongelmaa on pyritty hoitamaan rikosoikeudellista toimivaltaa hallintoviranomaisille delegoimalla tilanteen ollessa nyt se, että uuden sakkomenettelylain voimaan tullessa noin 90 % rikosoikeudellisesta kompetenssista on poliisilla ja muilla hallintoviranomaisilla. Se taasen on ongelmallista perustuslain 3 §:ssä säädetyn vallanjakoperiaatteen kannalta. Välttämätöntä on korostaa myös sitä, että pääosin rikosoikeudelliseen kontrolliin perustuva systeemi aiheuttaa rikosoikeuden inflaatiota: rikosoikeuden arvostuksen vähenemisestä aiheutuvia ei-mittavissa olevia kuluja. Rikosoikeuden laajenemista vastaan esitetään kuitenkin harvoin kritiikkiä. Rikosoikeustieteen piirissäkään sille ei ole esitetty vakavasti otettavaa vaihtoehtoa. Esimerkiksi hallinnollisia sanktioita ei ole Suomessa – ei tosin muissa Pohjoismaissakaan – koettu rikosoikeuden vaihtoehtona, vaan usein rikosoikeutta täydentävänä järjestelmänä. Hallinnollisten sanktioiden käyttäminen rikosoikeuden ohessa ei ole ollut yksiselitteistä ja jäsenneltyä, yleisiin oppeihin perustuvaa. Kontrollin erityisille alueille, esimerkiksi sotilaskontrolliin, on lisäksi kehittynyt rikos- ja hallinto-oikeuden sekajärjestelmiä. Tutkimuskysymykset Käsillä olevassa artikkelimuotoisessa väitöskirjatutkimuksessa pyritään hahmottelemaan rikos- ja hallinto-oikeudellisten rangaistusjärjestelmien välisiä eroavaisuuksia Suomessa. Tutkimuksen keskeisin tehtävä on sen pohtiminen, millä tavalla 2010-luvulle tultaessa olisi tutkimuksen empiiriset ja teoreettiset lähtökohdat huomioon ottaen järkevää ja tarkoituksenmukaista rangaista. Tällä perusteella kehitetään yksikertaista hallintosanktiointiteoreettista mallia, että sitä hyväksikäyttäen lainvalmisteluprosessin sanktiointitilanteessa, sanktiosta päätettäessä, olisi mahdollista asettaa oikeusturvan vähimmäistakeet täyttävällä tavalla oikeus- ja muilta vaikutuksiltaan rationaalinen seuraamus sekä säätää sen määräämiseksi yhteiskunnallisilta vaikutuksiltaan tehokas ja riittävän oikeusturvallinen menettely. Tutkimustehtävä on tiivistetysti 1) hallinnollisten sanktioiden ja rikosoikeudellisten rangaistusten ja niiden alan määrittäminen, 2) hallinnollisten sanktioiden sääntelymallien valintaperusteluiden selvittäminen, 3) hallintosanktioteoreettisen mallin kehittäminen lainvalmisteluprosessia varten sekä 4) de lege ferenda -suositusten tekeminen rangaistavuuden hyväksyttävyyden ja joutuisuuden parantamiseksi. Menetelmä ja aineistot Tutkimus kytkeytyy teoreettisen rikosoikeustieteen, hallinto-oikeustieteen ja prosessioikeustieteen alalle ja se on luonteeltaan ongelmakeskeinen. Tieteenalojen väliset painotukset ja niiden vaikutus vaihtelevat tutkimuksessa. Vaikka tutkimus on monella tavalla luonteeltaan teoreettinen, siinä rakennetaan nimenomaan lainsäädäntötyötä ja lainvalmistelua tukevaa tutkimusta ja teoriaa. Tältä osin näkökulma liittyy kriminalisointiteoreettisessa tutkimuksessa hahmotettuun lainsäätäjän oikeuslähdeoppia koskevaan uuteen näkökulmaan, jolla on yhteyksiä lainsäädäntötutkimukseen ja oikeusteoriassa hahmotettuun legisprudence-tutkimusuuntaukseen, näkökulman suuntautuessa lainsäädäntötasolle. Tutkimuksen lähteistä suurin osa on oikeustieteellistä kirjallisuutta, jonka pääosa muotoutuu suomalaisesta oikeustieteellisestä kirjallisuudesta. Myös pohjoismaalaista, saksalaista ja hieman myös anglosaksista kirjallisuutta on käytetty. Kansainvälisten kirjallisuuslähteiden merkitys ei ole ollut merkittävä, koska kattava eurooppalainen näkemys aihepiiristä on pyritty saamaan Euroopan ihmisoikeustuomioistuimen ratkaisukäytäntöä hyödyntäen. Tutkimuksen lähteinä on käytetty laajasti myös kansallista lainvalmisteluaineistoa, erityisesti hallituksen esityksiä ja perustuslakivaliokunnan lausuntokäytäntöä. Johtopäätökset Kriminaali- ja/tai kontrollipolitiikassa on juuri nyt tärkeää pohtia, millä tavalla 2010-luvulle tultaessa on järkevää ja tarkoituksenmukaista rangaista. Tähän voisi olla mahdollista vastata tutkimuksessa kehitettävällä hallintosanktiointiteoreettisella mallilla. Mallin kehittäminen on edellyttänyt yhteiskunnallisten sanktiokäytäntöjen oikeusvaltioopillista tarkastelua, jolloin sanktioiden yhteiskunnalliseen merkitykseen pureutuminen kuvailee myös sitä, millainen modernin oikeusvaltion tila tällä hetkellä on. Perinteisten oikeusvaltioiden paradigmojen käyttäminen ei välttämättä edistä relevantin oikeusvaltiollisen sanktiointiteoreettisen mallin kehittämistä. Voi olla niinkin, että klassinen oikeusvaltio-oppi ja oletetunlaiset rikosoikeuden erityiset elementit eivät enää vastaa yhteiskuntatodellisuutta. Näin rankaisemisen yleiset opit voivat nousta kehittämisen kohteeksi oikeusvaltiollisuuden elementtien toteuttamiseksi valtavasta kasvaneessa hallintoyhteiskunnassa – hallinto-johtoisessa yhteiskunnallisessa todellisuudessa. Suomen rikoslaki sisältää laajassa merkityksessä sadoittain erilaatuisia kriminalisointeja, joiden merkitys on usein hallinnon toimintaa subventoiva, symbolinen tai jonkin toisen kriminalisoinnin vaikutusta tehostamaan pyrkivä. Tällainen rikoslaki on tehtävänsä, vaikuttavuutensa ja hyväksyttävyytensä kannalta ongelmallinen. Suomalainen hallinnollisten sanktioiden järjestelmä ei sekään ole järkevä ja tarkoituksenmukainen, yleisiin oppeihin perustuva systeemi. Myös yleiseurooppalainen kehitys näyttäisi viittaavan siihen, että Suomessa on tarve kehittää sekä rikosoikeutta että sille vaihtoehtoista hallintosanktioiden järjestelmää, sanktio-oikeutta. Tämä edellyttää lainsäädäntöuudistuksia.

I hälsans spelrum : lek på vårdandets scen

With caring science as its foundation and by means of the perioperative dialogue, the intended contribution and overall aim of this present thesis is to describe what play is and could be in the caring reality, an ideal model. The perioperative dialogue is the nurse anaesthetists’ pre-, intra- and post-operative dialogues with the children they care for in connection with anesthesia. The thesis is composed according to Schopenhauer’s notion that the road to science presupposes the world seen as performances, and has an all-pervading hermeneutic approach. The performances of the thesis are: the performance of all performances, the empirical performance, the transcendental performance and the universal performance. The performance of all performances originates in the theoretical perspective of the thesis and describes what play and its characteristics are. This performance is realized through the hermeneutic interpretation of the etymology and original meaning of the word play along with texts from caring science, philosophy, anthropology and the history of religion. The empirical performance originates in four empirical studies where caring is organized as a perioperative dialogue. In study I, the material was collected with the help of participating observations and semi-structured interviews, in study II, with the help of the critical incident method and in study III, with the help of conversation interviews. In study IV, play develops into a clinical caring science research method. The research participants consist of children with special needs, children with a pronounced fear of anaesthesia, parents of children with severe autism and nurse anaesthetists. The empirical performance relates in what way play manifests in a perioperative child context by interpreting the results from the empiric in the light of the characteristics of play. The transcendental performance is enacted in the playhouse of health and presents a picture of the essence of play, the playing. In the playhouse of health, the light, winged movement of play is actualized when what was previously too difficult, too heavy and pinioned instead is as easy as anything. The eye of love and compassion knows the art of deciphering the secret script where the Other’s holiness resides, even if mere glimpses of it appear. The universal performance depicts three caring acts where the entrance consists of entering play, the ideal of which is realized in the unmasked openness face to face, that which protects the playing human being against encroachment and an unwanted audience. In the second caring act, entering play plays on to the finely-tuned interplay between human beings in the winged play of beauty and dignity. In the third caring act, the world’s deepest plays are staged on the stage of caring, in the sense that the innermost being of each individual, the universal will joins in and allows individuals to live as playing human beings who are at home with themselves and the world. The captivating, graceful and friendly play works from within itself, as long as it illumined by the light of claritas can play undisturbed on the stage of caring where it – like an unclouded mirror of its own ideal watches over children’s health.

Intended Quality: Relationships between Development, Testing and Quality

An empirical study was conducted in the area of software engineering to study relationships between development, testing and intended software quality. International standards served as a starting point of the study. For analysis a round of interviews was kept and transcribed. It was found that interaction between humans is critical, especially in transferring knowledge and standards’ processes. The standards are communicated through interaction and learning processes are involved before compliance. One of the results was that testing is the key to sufficient quality. The outcome was that successful interaction, sufficient testing and compliance with the standards combined with good motivation may provide most repeatable intended quality.

New and Emerging Challenges of the ICT-Mediated Health and Well-Being Services

This monograph dissertation looks into the field of ICT-mediated health and well-being services. Through six chapters that extend the work done in the reviewed and published articles, the dissertation focuses on new and emerging technologies, and to impact of their use on the beneficiary; the individual who eventually derives advantage from the services. As the field is currently going through major changes particularly in the OECD countries, the focus is on shortterm developments in the field and the analysis on the long term developments is cursory by nature. The dissertation includes theoretical and empirical elements. Most of the empirical elements are linked to product development and conceptualization performed in the national MyWellbeing project that ended in 2010. In the project, the emphasis was on conceptualization of a personal aid for the beneficiary that could be used for managing information and services in the field of health and well-being services. This work continued the theme of developing individual-centric solutions for the field; a work that started in the InnoElli Senior program in 2006. The nature of this thesis is foremost a conceptual elaboration based on a literature review, illustrated in empirical work performed in different projects. As a theoretical contribution, this dissertation elaborates the role of a mediator, i.e. an intermediary, and it is used as an overarching theme. The role acts as a ‘lens’ through which a number of technology-related phenomena are looked at, pinned down and addressed to a degree. This includes introduction of solutions, ranging from anthropomorphic artefacts to decision support systems that may change the way individuals experience clinical encounters in the near-future. Due to the complex and multiform nature of the field, it is impractical and effectively impossible to cover all aspects that are related to mediation in a single work. Issues such as legislation, financing and privacy are all of equal importance. Consideration of all these issues is beyond the scope of this dissertation and their investigation is left to other work. It follows from this that the investigation on the role is not intended as inclusive one. The role of the mediator is also used to highlight some of the ethical issues related to personal health information management, and to mediating health and well-being related issues on behalf of another individual, such as an elderly relative or a fellow member of a small unit in the armed forces. The dissertation concludes in a summary about the use and functions of the mediator, describing some potential avenues for implementing such support mechanisms to the changing field of ICT-mediated health and well-being services. The conclusions also describe some of the limitations of this dissertation, including remarks on methodology and content.

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 µg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease.