Tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids as a new family of anti-Alzheimer agents targeting beta-amyloid, tau, and cholinesterase pathologies

Optimization of an essentially inactive 3,4-dihydro-2H-pyrano[3,2-c]quinoline carboxylic ester derivative as acetylcholinesterase (AChE) peripheral anionic site (PAS)-binding motif by double O → NH bioisosteric replacement, combined with molecular hybridization with the AChE catalytic anionic site (CAS) inhibitor 6-chlorotacrine and molecular dynamics-driven optimization of the length of the linker has resulted in the development of the trimethylene-linked 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine6-chlorotacrine hybrid 5a as a picomolar inhibitor of human AChE (hAChE). The tetra-, penta-, and octamethylene-linked homologues 5bd have been also synthesized for comparison purposes, and found to retain the nanomolar hAChE inhibitory potency of the parent 6-chlorotacrine. Further biological profiling of hybrids 5ad has shown that they are also potent inhibitors of human butyrylcholinesterase and moderately potent Aβ42 and tau anti-aggregating agents, with IC50 values in the submicromolar and low micromolar range, respectively. Also, in vitro studies using an artificial membrane model have predicted a good brain permeability for hybrids 5ad, and hence, their ability to reach their targets in the central nervous system. The multitarget profile of the novel hybrids makes them promising leads for developing anti-Alzheimer drug candidates with more balanced biological activities.

Tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids as a new family of anti-Alzheimer agents targeting beta-amyloid, tau, and cholinesterase pathologies

Optimization of an essentially inactive 3,4-dihydro-2H-pyrano[3,2-c]quinoline carboxylic ester derivative as acetylcholinesterase (AChE) peripheral anionic site (PAS)-binding motif by double O → NH bioisosteric replacement, combined with molecular hybridization with the AChE catalytic anionic site (CAS) inhibitor 6-chlorotacrine and molecular dynamics-driven optimization of the length of the linker has resulted in the development of the trimethylene-linked 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine6-chlorotacrine hybrid 5a as a picomolar inhibitor of human AChE (hAChE). The tetra-, penta-, and octamethylene-linked homologues 5bd have been also synthesized for comparison purposes, and found to retain the nanomolar hAChE inhibitory potency of the parent 6-chlorotacrine. Further biological profiling of hybrids 5ad has shown that they are also potent inhibitors of human butyrylcholinesterase and moderately potent Aβ42 and tau anti-aggregating agents, with IC50 values in the submicromolar and low micromolar range, respectively. Also, in vitro studies using an artificial membrane model have predicted a good brain permeability for hybrids 5ad, and hence, their ability to reach their targets in the central nervous system. The multitarget profile of the novel hybrids makes them promising leads for developing anti-Alzheimer drug candidates with more balanced biological activities.

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

”Käsitteitä tarpeen mukaan". Kirjallisuustieteelliset käsitteet lukion kirjallisuudenopetuksessa

Tavoitteeni tässä tutkimuksessa oli selvittää, miten kirjallisuustieteellisiä proosa-anayysin käsitteitä opetetaan opetussuunnitelman mukaisissa lukion äidinkielen ja kirjallisuuden oppikirjoissa ja miten hyvin kokelaat hallitsivat kertoja-käsitteen tekstitaidon ylioppilaskokeessa keväällä 2007. Samalla pohdin, minkälainen kirjallisuustieteellinen käsitteistö palvelisi tekstianalyysin opetusta koulussa, koska Lukion opetussuunnitelman perusteet 2003 ja äidinkielen ylioppilaskoe edellyttävät äidinkielen ja kirjallisuuden opetukselta ja oppilailta käsitteiden käyttöä. Tutkimusaineistonani olivat kaikki kuusi käytössä olevaa lukion äidinkielen ja kirjallisuuden oppikirjaa ja 440 kpl kevään 2007 äidinkielen tekstitaidon ylioppilaskokeen vastaustekstiä. Oppikirjoja tarkastelin soveltamalla niiden arviointiin Lev S. Vygotskin ajatuksia arkikäsitteiden ja tieteellisten käsitteiden opettamisesta ja Hans Aeblin esittämiä teoreettisia malleja käsitteiden opettamisesta ja oppimisesta. Tutkimukseni osoittaa, että opetussuunnitelmassa mainittujen proosa-analyysin käsitteiden kertoja, näkökulma, motiivi, aihe ja teema opetus on epätäsmällistä. Oppikirjoissa ei ole otettu huomioon sitä, että käsitteenoppiminen on monivaiheinen prosessi. Myöskään problematiikkaa, joka aiheutuu kyseisten käsitteiden määrittelyn kirjavuudesta ja käytöstä sekä arkikielen käsitteinä että tieteellisinä käsitteinä, ei oppikirjoissa käsitellä. Sama näkyy ylioppilaskoeaineistossa: oppilaat eivät hallitse käsitettä kertoja tieteellisenä käsitteenä. Tietoisuus kirjallisuustieteellisten käsitteiden määrittelyn problematiikasta ja arkikäsitteiden ja tieteellisten käsitteiden ontologisista kategorioista on onnistuneen käsitteenoppimisen edellytys. Kirjallisuustieteelliset käsitteet ovat metakäsitteitä, jotka edellyttävät oppilaiden metakäsitteellisen tietoisuuden ja motivaation hyödyntämistä opetuksessa, jossa olisi sovellettava monipuolisesti eri oppimiskäsitysten parhaita puolia hyödyntäviä lähestymistapoja, erilaisia pedagogisia diskursseja. Koulujen kirjallisuudenopetusta suunniteltaessa ja kirjallisuustieteellisiä käsitteitä opetettaessa on otettava huomioon niin kirjallisuustieteen kuin kasvatustieteen näkökulma. Opetussuunnitelman ja ylioppilaskokeen asettama vaatimus käsitteiden käytöstä on kohtuuton, mikäli ei sovita, miten käsitteet määritellään ja mitä käsitteitä kokelaiden oletetaan ylioppilaskokeessa hallitsevan. Kirjallisuustieteellisten käsitteiden puutteellisen opetuksen oppikirjoissa ja niiden epämääräisen käytön ylioppilaskokeen tehtävänannoissa ja arvioinnissa voi kärjistyneimmillään nähdä oppilaan oikeusturvakysymyksenä

Hjärtats vanor, tankens välvilja och handens gärning : dygd som vårdetiskt grundbegrepp

The main aim of the study is to elucidate the meaning and dimensions of the concept of „virtue‟, and to find the place of virtue in a caritative caring ethics, i.e. a caring ethics based on human love and mercy. The intention is to create a theory model which utilizes the possibilities of virtue in developing the caritative caring ethics as a whole. The caritative caring ethics has a universal potential – it is primarily not a professional ethics, but it may form a frame of reference and basis for formulating ethical codes, and for ethical discussions within different caring contexts. The hermeneutic approach of the study is inspired by Gadamer‟s philosophical hermeneutics combined with the view of hermeneutics as a hypothetical-deductive process. The study is guided by Eriksson‟s model of definition of concepts. The concept of „virtue‟ is studied focusing on its ethical dimensions. These ethical dimensions of virtue are seen as anchored to an inner ethos, whereas ethos stands for the ontological goodness, a basic notion of the Good that permeates the entity of the human being, and forms the base of the culture where he lives and acts. The overarching research questions are: 1. What is virtue? 2. What is „virtue‟ as a basic concept in caring science? 3. What place does virtue have in caritative caring ethics? The answer of the first question is mainly searched for by an ontological determination comprising partly an etymologic and semantic analysis of „virtue‟, and partly a determination of the essence of virtue. The answer to the second and third questions are mainly searched for using a contextual determination, where the purposive context and pragmatic features of virtue are studied in relation to caring ethics. The ontological and contextual determinations are brought together through hermeneutical interpretation, forming a new whole, which constitutes the results of the study. The results of the study are depicted in a theory model, in which the movement of virtue from ethos to deed is moulded as caritative caring ethics. The material of the study consists of dictionaries, texts written by Aristotle and St. Thomas Aquinas, articles, dissertations, and books, as well as parts of a pilot survey answered by 33 nurses. The results of the study show that the essence of virtue is primarily functional, not ethical. The ethical emerges when virtue is contextualized in a human communion. Virtue makes something fulfil its function well; makes the human being good, and gives him morals and morality. The human being needs prudence, love, and humility to acquire and develop the moral virtues. Virtue is a power, related to a value, which considering a caritative caring ethics consists of the caritas motif. Human love is shown through deeds, making the human being do what he is expected to do. Virtue, as an active power of becoming, affirms and clarifies the human being‟s ability to develop in the direction of the Good. Virtue becomes essential and unifying when morality appears in the human mind as auctoritas, an inner, prompting power based on divinity or a transcendental ethos. Together ethos and virtue create opportunities for an inner ethics based on voluntariness and joy in being and doing the true, the good, and the beautiful.

Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Jäsenen psykologinen side Etelä-Karjalan Eekoon Osuuskaupassa

Pro gradu -tutkielman tavoitteena on selvittää asiakasomistajien psykologisen omistajuuden tila Etelä-Karjalan Eekoon Osuuskaupassa. Tavoitteena on selvittää miten psykologista sidettä voi vahvistaa ja mitä keinoja on syytä välttää sitoutettaessa asiakasomistajia. Tutkielma on toteutettu laadullisin menetelmin. Tutkielmassa käsitellään psykologista omistajuutta ja identifioitumista sen lähikäsitteiden sitoutumisen ja sisäistämisen avulla. Tutkielman tuloksista ilmenee, että jäsenten psykologinen side Osuuskauppaa kohtaan on melko heikko ja jäsenet eivät tunne olevansa omistajia Osuuskaupassa. Jäsenten psykologinen side vahvistuu Eekoossa parhaiten niin, että jäsenet kokevat voivansa konkreettisesti vaikuttaa Osuuskaupan toimintaan.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Aspectos clínicos, histopatológicos e moleculares da dermatosparaxia em ovinos White Dorper

Dermatosparaxia em animais é uma doença autossômica recessiva do tecido conjuntivo caracterizada por fragilidade e hiperextensibilidade cutânea. A doença em ovinos White Dorper é provocada pela mutação c.421G>T no gene ADAMmetalopeptidase com trombospondina tipo 1 motif, 2 (ADAMTS2). O objetivo deste estudo foi descrever os achados clínicos, moleculares e histopatológicos da dermatosparaxia em ovinos White Dorper de um rebanho localizado no Centro-Oeste Paulista. O rebanho era composto por nove animais, sendo um reprodutor, quatro matrizes e seus respectivos borregos. Dos nove animais examinados, dois apresentavam sinais clínicos compatíveis com dermatosparaxia. O exame histopatológico de amostras cutâneas das lesões destes dois animais revelou também achados compatíveis com dermatosparaxia, sendo caracterizados por epiderme e anexos cutâneos preservados e sem características atípicas; colágeno displásico arranjado em feixes pequenos, fragmentados e com focos de degeneração, anexos cutâneos proeminentes e na região da derme foco hemorrágico intenso associado a moderado infiltrado neutrofílico na derme profunda. Com o objetivo de realizar o diagnóstico molecular da enfermidade, uma PCR foi padronizada utilizando primers específicos desenhados para amplificar a região do gene ADAMTS2 que continha a mutação c.421G>T e o DNA obtido de amostras de sangue de todos os animais do rebanho. O sequenciamento direto dos produtos da PCR, comprovou que os dois animais clinicamente afetados possuíam a mutação responsável pela dermatosparaxia. A metodologia descrita neste estudo possibilitou o diagnóstico definitivo da doença. Segundo a literatura consultada, esta é a primeira vez que a dermatosparaxia é descrita em ovinos White Dorper no Brasil. A metodologia aqui descrita poderá ser empregada em estudos futuros que avaliem a prevalência desta mutação no Brasil, possibilitando a adoção de medidas que previnam a disseminação dessa mutação no rebanho brasileiro de ovinos White Dorper.

Prevalence study of SNP c.421G>T in the ADAMTS2 gene responsible for dermatosparaxis in White Dorper sheep in Brazil

Abstract: Dermatosparaxis is an autosomal recessive disorder of connective tissue; the disorder is clinically characterized by skin fragility and hyperextensibility. Dermatosparaxis in White Dorper sheep is caused by a single nucleotide polymorphism (SNP) (c.421G>T) in the ADAM metalloproteinase with thrombospondin type 1 motif, 2 (ADAMTS2) gene. The aim of this study was to investigate the prevalence of this SNP in a White Dorper herd in São Paulo state, Brazil. In this study, we collected blood DNA samples from 303 White Dorper sheep and performed polymerase chain reaction to amplify the SNP region. The samples were sequenced to determine the presence of the SNP in the ADAMTS2 gene. The SNP prevalence in the studied population was 15.5%; this finding indicates that more effective control measures should be used to prevent the inheritance of SNP c.421G>T in the ADAMTS2 gene in Brazilian White Dorper herds.

Gamification as a means for employee motivation, personal engagement and behavioral outcomes: a gamification system developers’ perspective

A major challenge faced by companies today is the engagement gap at the workplace and how to motivate employees to engage in less intrinsically motivating work activities that are valuable for the organization. The objective of this study is to investigate gamification as a means for employee motivation and personal engagement that result in behavioral outcomes from the gamification developers’ perspective. Theories of work motivation and engagement are viewed in relation to gamification. The empirical part conducts a qualitative multiple-case study. The data is analyzed with the CAQDAS NVivo. The empirical findings suggest that gamification can enhance employee motivation, but careful consideration of extrinsic motivators is necessary to avoid their detrimental effect on intrinsic motivation. Employee self-determination is built through internalization of gamified system’s goals reaching autonomous motivation to engage in the target behavior. Employee engagement is built by fulfilling the psychological conditions of meaningfulness, safety and availability. The results suggest that gamification can build employee motivation and engagement leading to behavior change if designed with the business objectives in mind. Moreover, the gamified system needs to be renewed to address the changes in the business environment and reflect them in the employee behavior.

Influence of surface functionalization on the behavior of silica nanoparticles in biological systems

Personalized nanomedicine has been shown to provide advantages over traditional clinical imaging, diagnosis, and conventional medical treatment. Using nanoparticles can enhance and clarify the clinical targeting and imaging, and lead them exactly to the place in the body that is the goal of treatment. At the same time, one can reduce the side effects that usually occur in the parts of the body that are not targets for treatment. Nanoparticles are of a size that can penetrate into cells. Their surface functionalization offers a way to increase their sensitivity when detecting target molecules. In addition, it increases the potential for flexibility in particle design, their therapeutic function, and variation possibilities in diagnostics. Mesoporous nanoparticles of amorphous silica have attractive physical and chemical characteristics such as particle morphology, controllable pore size, and high surface area and pore volume. Additionally, the surface functionalization of silica nanoparticles is relatively straightforward, which enables optimization of the interaction between the particles and the biological system. The main goal of this study was to prepare traceable and targetable silica nanoparticles for medical applications with a special focus on particle dispersion stability, biocompatibility, and targeting capabilities. Nanoparticle properties are highly particle-size dependent and a good dispersion stability is a prerequisite for active therapeutic and diagnostic agents. In the study it was shown that traceable streptavidin-conjugated silica nanoparticles which exhibit a good dispersibility could be obtained by the suitable choice of a proper surface functionalization route. Theranostic nanoparticles should exhibit sufficient hydrolytic stability to effectively carry the medicine to the target cells after which they should disintegrate and dissolve. Furthermore, the surface groups should stay at the particle surface until the particle has been internalized by the cell in order to optimize cell specificity. Model particles with fluorescently-labeled regions were tested in vitro using light microscopy and image processing technology, which allowed a detailed study of the disintegration and dissolution process. The study showed that nanoparticles degrade more slowly outside, as compared to inside the cell. The main advantage of theranostic agents is their successful targeting in vitro and in vivo. Non-porous nanoparticles using monoclonal antibodies as guiding ligands were tested in vitro in order to follow their targeting ability and internalization. In addition to the targeting that was found successful, a specific internalization route for the particles could be detected. In the last part of the study, the objective was to clarify the feasibility of traceable mesoporous silica nanoparticles, loaded with a hydrophobic cancer drug, being applied for targeted drug delivery in vitro and in vivo. Particles were provided with a small molecular targeting ligand. In the study a significantly higher therapeutic effect could be achieved with nanoparticles compared to free drug. The nanoparticles were biocompatible and stayed in the tumor for a longer time than a free medicine did, before being eliminated by renal excretion. Overall, the results showed that mesoporous silica nanoparticles are biocompatible, biodegradable drug carriers and that cell specificity can be achieved both in vitro and in vivo.

Complementarity of radioautographic and immunohistochemical techniques for localizing neuroreceptors at the light and electron microscopy level

To assess relationships between neuropeptide-binding sites and receptor proteins in rat brain, the distribution of radioautographically labeled somatostatin and neurotensin-binding sites was compared to that of immunolabeled sst2A and NTRH receptor subtypes, respectively. By light microscopy, immunoreactive sst2A receptors were either confined to neuronal perikarya and dendrites or diffusely distributed in tissue. By electron microscopy, areas expressing somatodendritic sst2A receptors displayed only low proportions of membrane-associated, as compared to intracellular, receptors. Conversely, regions displaying diffuse sst2A labeling exhibited higher proportions of membrane-associated than intracellular receptors. Furthermore, the former showed only low levels of radioautographically labeled somatostatin-binding sites whereas the latter contained high densities of somatostatin-binding suggesting that membrane-associated receptors are preferentially recognized by the radioligand. In the case of NTRH receptors, there was a close correspondence between the light microscopic distribution of NTRH immunoreactivity and that of labeled neurotensin-binding sites. Within the substantia nigra, the bulk of immuno- and autoradiographically labeled receptors were associated with the cell bodies and dendrites of presumptive DA neurons. By electron microscopy, both markers were detected inside as well as on the surface of labeled neurons. At the level of the plasma membrane, their distribution was highly correlated and characterized by a lack of enrichment at the level of synaptic junctions and by a homogeneous distribution along the remaining neuronal surface, in conformity with the hypothesis of an extra-synaptic action of this neuropeptide. Inside labeled dendrites, there was a proportionally higher content of immunoreactive than radiolabeled receptors. Some of the immunolabeled receptors not recognized by the radioligand were found in endosome-like organelles suggesting that, as in the case of sst2A receptors, they may have undergone endocytosis subsequent to binding to the endogenous peptide

Snake venom metalloproteinases and disintegrins: interactions with cells

Metalloproteinases and disintegrins are important components of most viperid and crotalid venoms. Large metalloproteinases referred to as MDC enzymes are composed of an N-terminal Metalloproteinase domain, a Disintegrin-like domain and a Cys-rich C-terminus. In contrast, disintegrins are small non-enzymatic RGD-containing cysteine-rich polypeptides. However, the disintegrin region of MDC enzymes bears a high degree of structural homology to that of the disintegrins, although it lacks the RGD motif. Despite these differences, both components share the property of being able to recognize integrin cell surface receptors and thereby to inhibit integrin-dependent cell reactions. Recently, several membrane-bound MDC enzymes, closely related to soluble venom MDC enzymes, have been described in mammalian cells. This group of membrane-anchored mammalian enzymes is also called the ADAM family of proteins due to the structure revealing A Disintegrin And Metalloproteinase domains. ADAMs are involved in the shedding of molecules from the cell surface, a property which is also shared by some venom MDC enzymes.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.