The use of fluorescent probes to characterize conformational changes in the interaction between vitronectin and plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central β- sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational effects of vitronectin on the reactive center loop of PAI-1 have not been documented. Mutant forms of PAI-1 were designed with a cysteine substitution at either position P1\u27 or P9 of the reactive center loop. Labeling of the unique cysteine with a sulfhydryl-reactive fluorophore provides a probe that is sensitive to vitronectin binding. Results indicate that the scissile P1-P1\u27 bond of PAI-1 is more solvent exposed upon interaction with vitronectin, whereas the N- terminal portion of the reactive loop does not experience a significant change in its environment. These results were complemented by labeling vitronectin with an arginine-specific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation constants of approximately 100 nM are calculated for the vitronectin/PAI-1 interaction from titrations using both fluorescent probes. Furthermore, experiments in which PAI-1 failed to compete with heparin for binding to vitronectin argue for separate binding sites for the two ligands on vitronectin

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

TYYTYVÄISYYS SUKANVETOLAITTEESEEN : QUEST 2.0-kyselyn toteuttaminen Peruspalvelukeskus Oiva-liikelaitoksessa ja Parkanon kaupungissa

Opinnäytetyön tavoitteena oli selvittää sukanvetolaitteen käyttäjien tyytyväisyyttä saa¬maansa apuvälineeseen ja apuvälineprosessiin. Tutkimus toteutettiin hyödyntämällä QUEST 2.0-mittaria. QUEST (Quebec User Evaluation of Satisfaction with Assistive Technology) on 12 -kohtainen arviointimenetelmä, jonka avulla arvioidaan käyttäjän tyytyväisyyttä käyttämäänsä apuvälineeseen ja siihen liittyviin palveluihin. Toimintaterapian perusajatuksena on, että aktiivinen osallistuminen merkityksellisiin toimintoihin tukee ihmisen terveyttä sekä hyvinvointia. Apuvälineen avulla voidaan edistää toimintaan osallistumista ja sen suorittamista. Apuvälineen käytön ja tyytyväisyyden tutkiminen on tärkeää, koska siten ammattilaiset saavat tietoonsa käyttöä lisääviä tekijöitä. Tutkimus toteutettiin Peruspalvelukeskus Oiva-liikelaitoksessa ja Parkanon kaupungissa. Kysely lähetettiin 78 henkilölle, jotka olivat saaneet sukanvetolaitteen käyttöönsä vuoden 2004 jälkeen. 46 henkilöä vastasi ja palautti kyselyn, joten vastausprosentti oli 59 %. Niistä hyväksyttyjä vastauksia oli 36 vastaajalla. Keskiarvolla mitaten vastaajat olivat tyytyväisiä sekä sukanvetolaitteeseen että siihen liittyneeseen apuvälineprosessiin. Tyytyväisyydessä oli kuitenkin paljon hajontaa vastaajien välillä. Vastaajien mukaan tärkeimmät tekijät liittyen apuvälineeseen ja sen prosessiin olivat käytön helppous, tarkoituksenmukaisuus, paino, turvallisuus ja luotettavuus, kestävyys sekä apuvä¬lineen käyttöön saamisen prosessi. Tutkimuksen avulla saatujen tulosten perusteella yhteis¬työtahot pystyivät muokkaamaan sukanvetolaitteen apuvälineprosessia tarpeelliseen suun¬taan. He myös saivat käyttökokemuksen QUEST 2.0-kyselystä.The purpose of this study was to survey the satisfaction of stocking aid users with the device and with the related service process. The survey was conducted by using the QUEST 2.0 measurement tool. QUEST (Quebec User Evaluation of Satisfaction with Assistive Technology) is a 12-item assessment method and it is used to assess users’ satisfaction with their assistive devices and the related services. The basic principle in occupational therapy is that active participation in meaningful occupations supports people’s health as well as their well-being. By using assistive devices it is possible to support participation in and the performance of occupations. It is important to study the use of a device and the related satisfaction because this is how professionals will learn about the factors that will increase their use. The survey was conducted in the Basic Service Centre Oiva Ltd and the town of Parkano. A questionnaire was sent to 78 persons who had received a stocking aid for their use after the year 2004. 46 persons returned the questionnaire, which gave a response rate of 59 per cent. Out of them 36 persons had acceptable answers. Based on the mean value, the respondents were satisfied with the stocking aid and the related services. There was, however, a great deal of dispersion between the respondents. According to the respondents, the most important factors concerning the device and its services were simplicity of use, effectiveness, weight, safety, durability and delivery. With the results of this study the cooperation partners were able to modify the service process of the stocking aid to a necessary direction. They were also able to have a user experience of the QUEST 2.0 surve

The mechanism underlying activation of factor IX by factor XIa

Factor XI (fXI) is the zymogen of a plasma protease, factor XIa (fXIa), that contributes to thrombin generation during blood coagulation by proteolytic conversion of factor IX (fIX) to factor IXaβ (fIXaβ). There is considerable interest in fXIa as a therapeutic target because it contributes to thrombosis, while serving a relatively minor role in hemostasis. FXI/XIa has a distinctly different structure than other plasma coagulation proteases. Specifically, the protein lacks a phospholipid-binding Gla-domain, and is a homodimer. Each subunit of a fXIa dimer contains four apple domains (A1 to A4) and one trypsin-like catalytic domain. The A3 domain contains a binding site (exosite) that largely determines affinity and specificity for the substrate fIX. After binding to fXIa, fIX undergoes a single cleavage to form the intermediate fIXα. FIXα then rebinds to the A3 domain to undergo a second cleavage, generating fIXaβ. The catalytic efficiency for the second cleavage is ~ 7-fold greater than that of the first cleavage, limiting fIXα accumulation. Residues at the N-terminus and C-terminus of the fXIa A3 domain likely form the fIX binding site. The dimeric conformation of fXIa is not required for normal fIX activation in solution. However, monomeric forms of fXI do not reconstitute fXI-deficient mice in arterial thrombosis models, indicating the dimer is required for normal function in vivo. FXI must be a dimer to be activated normal by the protease fXIIa. It is also possible that the dimeric structure is an adaptation that allows fXI/XIa to bind to a surface through one subunit, while binding to its substrate fIX through the other

Metals affect the structure and activity of human plasminogen activator inhibitor-1. I. Modulation of stability and protease inhibition

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1–2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ∼50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ∼30 min with copper alone. Nickel had the largest effect, reducing the half-life to ∼5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability

Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation

Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems1, 2. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis3, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting4, 5. Here we present the 2.2 Å crystal structures of human alpha-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase6. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors7, 8, 9. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as 'molecular sexuality'1

Nerve ultrasound for diagnosing chronic inflammatory neuropathy A multicenter validation study

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