Structure of the active form of human Origin Recognition Complex and its ATPase motor module

Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations

Harnessing insulin- and leptin-induced oxidation of PTP1B for therapeutic development

The protein tyrosine phosphatase PTP1B is a major regulator of glucose homeostasis and energy metabolism, and a validated target for therapeutic intervention in diabetes and obesity. Nevertheless, it is a challenging target for inhibitor development. Previously, we generated a recombinant antibody (scFv45) that recognizes selectively the oxidized, inactive conformation of PTP1B. Here, we provide a molecular basis for its interaction with reversibly oxidized PTP1B. Furthermore, we have identified a small molecule inhibitor that mimics the effects of scFv45. Our data provide proof-of-concept that stabilization of PTP1B in an inactive, oxidized conformation by small molecules can promote insulin and leptin signaling. This work illustrates a novel paradigm for inhibiting the signaling function of PTP1B that may be exploited for therapeutic intervention in diabetes and obesity

Dinamika funkcije pluća kod azbestne bolesti

As a rule, asbestosis is a disease of workers who are occupationally exposed to inhalation of asbestos dust, leaving permanent alterations on the lung parenchyma or pleura. In our ten-year study, we investigated 318 workers with pleural asbestosis from whom we took medical history which included occupational exposure to asbestos, radiological examinations and lung function, which is mandatory for the diagnosis and the follow up of the disease. We analysed functional parameters such as forced vital capacity (FVC) and forced expiratory volume in the first second (FEV1) and intermediate forced expiratory flow at 25 % to 75 % (FEF25 %-75 %). In addition, we investigated the predicted values of functional parameters according to smoking and non-smoking habits. We found a significant reduction in vital capacity, particularly in smokers after 25 years of exposure to asbestos. During the first 15 years, values of vital capacity on the group basis remained inside the 80 % of the normal values and were not significant for assessing the dynamics of the lung function. To better assess the effects of occupational asbestos exposure, it is necessary to interpret lung function data not only on the group basis, but also for each subject individually.Azbestoza je bolest izazvana udisanjem azbestnih čestica koje ostavljaju trajne promjene na parenhimu pluća i/ili pleuri. Dijagnoza se postavlja na osnovi anamnestičkih podataka, uvidom u profesionalnu izloženost azbestu i radiološkom obradom te patohistološkom potvrdom promjena na plućima i/ili pleuri. Funkcionalna obrada pluća obavezna je u postavljanju dijagnoze i praćenju bolesti. Tijekom desetogodišnjeg istraživanja funkcionalno smo obradili 318 osoba profesionalno izloženih azbestu s dokazanom azbestozom pleure. Analizirane su vrijednosti funkcionalnih parametara, i to forsiranoga vitalnog kapaciteta (FVC), forsiranoga ekspiracijskog volumena u prvoj sekundi (FEV1) i srednjega ekspiracijskog protoka (FEF25 %-75 %). Dokazan je statistički signifikantan pad vrijednosti FVC i FEV1. Dodatno smo istražili vrijednosti funkcionalnih parametara kod naših ispitanika s navikom pušenja i nepušača. U obje skupine prisutno je značajno sniženje vrijednosti vitalnog kapaciteta tijekom istraživanja, s tim da nakon 25 godina izloženosti azbestu kod pušača dolazi do naglog pada vrijednosti vitalnog kapaciteta u odnosu na nepušače. Bitno je uočiti da tijekom prvih 15 godina vrijednosti vitalnog kapaciteta ostaju unutar 80 % normalnih vrijednosti te nemaju značenja za praćenje dinamike funkcije pluća kod azbestne bolesti. Individualnim praćenjem profesionalno izloženih radnika ostvaruje se bolji uvid u dinamiku funkcije pluća kod azbestne bolesti

Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance

BACKGROUND:In recent years, an idiosyncratic new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts and, to date, only few substrates of BY-kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides. METHODOLOGY/PRINCIPAL FINDINGS:Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc and Etk. The regulatory role of Tyr71 phosphorylation on Ugd activity was then assessed and Tyr71 mutation was found to prevent Ugd activation by phosphorylation. Further, Ugd phosphorylation by Wzc or Etk was shown to serve distinct physiological purposes. Phosphorylation of Ugd by Wzc was found to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin. CONCLUSIONS/SIGNIFICANCE:Ugd phosphorylation seems to be at the junction between two distinct biosynthetic pathways, illustrating the regulatory potential of tyrosine phosphorylation in bacterial physiology

Interopservacijske razlike u dijagnosticiranju azbestoze prema klasifikaciji ILO

Inhalation of asbestos fibres leads to asbestosis of the pleura and the lung, with possible progression to lung cancer and malignant pleural or peritoneal mesothelioma. Asbestosis remains difficult to diagnose, especially in its early stages. The most important role in its diagnosis is that of chest radiographs. The aim of this cross-sectional study was to address interobserver variations in interpreting chest radiographs in asbestos workers, which remain to be an issue, despite improvements in the International Labour Office (ILO) classification system. In our ten-year study, we investigated 318 workers occupationally exposed to asbestos, and in 210 workers with diagnosed asbestos-related changes we compared interpretations of chest radiographs according to ILO by two independent radiologists. The apparent degree of interobserver variation in classifying lung fibrosis was 26.66 % for the diameter of changes and 42.2 % for the profusion of the changes. In cases with diffuse pleural thickening, the interobserver variation using ILO procedures was 34.93 %. This investigation raises the issue of standardisation and objectivity of interpretation of asbestosis according to the ILO classification system. This study has revealed a significant disagreement in the estimated degree of pleural and parenchymal asbestos pulmonary disease. This is why we believe highresolution computed tomography (HRCT) should also be used as a part of international classification.Azbestoza je bolest koja nastaje kao posljedica inhalacije azbestnih vlakana. Zahvaća pluća i pleuru, a ponekad može dovesti do karcinoma pluća, pleure i peritoneuma. Azbestozu je teško dijagnosticirati, osobito u početnom stadiju bolesti. U dijagnostici azbestoze presudna je radiološka obrada. Cilj ovog rada je utvrditi interopservacijske razlike u analizi radiograma prema kriterijima klasifikacije ILO kod profesjonalne azbestoze. U istraživanje je bilo uključeno 318 ispitanika profesionalno izloženih azbestnim česticama. U 210 ispitanika kojima je postavljena dijagnoza azbestoze usporedili smo radiološke nalaze analizirane od dvaju neovisnih radiologa eksperata u skladu s važećom klasifikacijom ILO. Uspoređujući rezultate analize malih zasjenjenja parenhima pluća prema njihovoj veličini, našli smo da se rezultati razlikuju u 26,66 % slučajeva, dok je prema prožetosti intersticija neslaganje bilo još učestalije, tj. zabilježeno je u 42,22 % slučajeva. U ispitanika s pleuralnim promjenama usporedili smo rezultate analize difuznih pleuralnih zadebljanja i našli neslaganje u debljini i/ili širini pleuralnih zadebljanja u 34,93 % slučajeva. Uočili smo značajno neslaganje u procjeni stupnja azbestne bolesti na pleuri i na parenhimu pluća. Zbog toga smatramo da je u kvantificiranju promjena izazvanih azbestom i u procjeni stupnja tjelesnog oštećenja nužno rabiti kompjutoriziranu tomografiju visoke rezolucije kao znatno osjetljiviju i pouzdaniju slikovnu tehniku

Nuclear DNA Replication in Trypanosomatids:There Are No Easy Methods for Solving Difficult Problems

In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens

The structure of human argonaute-2 in complex with miR-20a

Argonaute proteins lie at the heart of the RNA-induced silencing complex (RISC), wherein they use small RNA guides to recognize targets. Initial insight into the architecture of Argonautes came from studies of prokaryotic proteins, revealing a crescent-shaped base made up of the amino-terminal, PAZ, middle, and PIWI domains. The recently reported crystal structure of human Argonaute-2 (hAgo2), the "slicer" in RNA interference, in complex with a mixed population of RNAs derived from insect cells provides insight into the architecture of a eukaryotic Argonaute protein with defined biochemical and biological functions. Here, we report the structure of human Ago2 bound to a physiologically relevant microRNA, microRNA-20a, at 2.2 Å resolution. The miRNA is anchored at both ends by the Mid and PAZ domains and makes several kinks and turns along the binding groove. Interestingly, miRNA binding confers remarkable stability on hAgo2, locking this otherwise flexible enzyme into a stable conformation. © 2012 Elsevier Inc