Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.

Resurssien ja kyvykkyyksien vastaavuus alan muuttuviin vaatimuksiin

Tämän diplomityön tavoitteena on tutkia kohdeyrityksen strategista muutosta perinteisestä tuotetoimittajasta ratkaisujen toimittajaksi. Toimialan muutosta arvioidaan tarkemmin, jotta voidaan selvittää kuinka yrityksen tulisi mukauttaa resurssejaan ja kyvykkyyksiään sopeutuakseen muutoksen tuomiin uusiin haasteisiin. Tarkastelun alla on erityisesti yrityksen strateginen muutos ja kuinka löytää strategian kannalta ne dynaamiset kyvykkyydet joiden avulla yritys saa pysyvää kilpailuetua. Yritykset pyrkivät strategisessa suunnittelussaan pysyvään kilpailuetuun. Strategista johtamisen teorioita on esitetty useita ja niiden kautta on vaikea selittää nykyisin nopeasti muuttuvassa toimintaympäristössä toimivien yritysten menestystä. Dynaamisten kyvykkyyksistä on haettu selitystä menestyksen taakse. Yritysten tavoitellessa kokonaan uusia liiketoiminta-alueita niihin kohdistuu suuria muutoksia niin ulkoisesti kuin sisäisestikin. Tällöin on kriittistä tunnistaa ne ydinkyvykkyydet joiden avulla voidaan menestyä uusilla liiketoiminta-alueilla. Työn ensimmäisessä osassa esitetään strategiaan ja kyvykkyyksiin liittyviä teorioita. Teoriaosiossa tarkastellaan myös palveluyritykseksi muuntautumisen haasteita, jonka jälkeen esitellään kohdeyrityksen strategiset tavoitteet ja kuinka puuttuvia resursseja voidaan tunnistaa. Resurssipohjaisen lähestymistavan avulla päästiin hyvin käsiksi toimialan muutoksen aiheuttamiin puuttuviin resursseihin. Analyysin aikana tunnistettiin puutteita tavoitteisiin nähden ja tässä työssä nousi yhtenä keskeisimpänä esille ICT-osaaminen kohdeyrityksen siirtyessä laitevalmistajan roolista kohti ratkaisun toimittajaa. Tutkimuksen perusteella voidaan todeta, että resurssipohjaisen lähestymistavan avulla voidaan tunnistaa tämän tyyppisessä yrityksessä resurssipuutteita joita kehittämällä yritys voi saavuttaa pysyvää kilpailuetua.

Exposure to Tobacco Smoke and Markers of Subclinical Atherosclerosis in Children and Adolescents. The STRIP Study.

Background: Measurement of serum cotinine, a major metabolite of nicotine, provides a valid marker for quantifying exposure to tobacco smoke. Exposure to tobacco smoke causes vascular damage by multiple mechanisms, and it has been acknowledged as a risk factor for atherosclerosis. Multifactorial atherosclerosis begins in childhood, but the relationship between exposure to tobacco smoke and arterial changes related to early atherosclerosis have not been studied in children. Aims: The aim of the present study was to evaluate exposure to tobacco smoke with a biomarker, serum cotinine concentration, and its associations with markers of subclinical atherosclerosis and lipid profile in school-aged children and adolescents. Subjects and Methods: Serum cotinine concentration was measured using a gas chromatographic method annually between the ages 8 and 13 years in 538-625 children participating since infancy in a randomized, prospective atherosclerosis prevention trial STRIP (Special Turku coronary Risk factor Intervention Project). Conventional atherosclerosis risk factors were measured repeatedly. Vascular ultrasound studies were performed among 402 healthy 11-year-old children and among 494 adolescents aged 13 years. Results: According to serum cotinine measurements, a notable number of the school aged children and adolescents were exposed to tobacco smoke, but the exposure levels were only moderate. Exposure to tobacco smoke was associated with decreased endothelial function as measured with flow-mediated dilation of the brachial artery, decreased elasticity of the aorta, and increased carotid and aortic intima-media thickness. Longitudinal exposure to tobacco smoke was also related with increased apolipoprotein B and triglyceride levels in 13-year-old adolescents, whose body mass index and nutrient intakes did not differ. Conclusions: These findings suggest that exposure to tobacco smoke in childhood may play a significant role in the development of early atherosclerosis. Key Words: arterial elasticity, atherosclerosis, children, cotinine, endothelial function, environmental tobacco smoke, intima-media thickness, risk factors, ultrasound

Ajankohtainen kokoelma visuaalisen kulttuurin tutkimuksesta

Kirjallisuusarvostelu

Seksuaalivähemmistöjen tiedontarpeet ja tiedonhankinta

Artikkeli perustuu kirjoittajan samannimiseen pro gradu -tutkielmaan Tampereen yliopistossa 2006.