Inactive endosialidase-based detection of bacterial and oncofetal polysialic acid

Polysialic acid is a carbohydrate polymer which consist of N-acetylneuraminic acid units joined by alpha2,8-linkages. It is developmentally regulated and has an important role during normal neuronal development. In adults, it participates in complex neurological processes, such as memory, neural plasticity, tumor cell growth and metastasis. Polysialic acid also constitutes the capsule of some meningitis and sepsis-causing bacteria, such as Escherichia coli K1, group B meningococci, Mannheimia haemolytica A2 and Moraxella nonliquefaciens. Polysialic acid is poorly immunogenic; therefore high affinity antibodies against it are difficult to prepare, thus specific and fast detection methods are needed. Endosialidase is an enzyme derived from the E. coli K1 bacteriophage, which specifically recognizes and degrades polysialic acid. In this study, a novel detection method for polysialic acid was developed based on a fusion protein of inactive endosialidase and the green fluorescent protein. It utilizes the ability of the mutant, inactive endosialidase to bind but not cleave polysialic acid. Sequencing of the endosialidase gene revealed that amino acid substitutions near the active site of the enzyme differentiate the active and inactive forms of the enzyme. The fusion protein was applied for the detection of polysialic acid in bacteria and neuroblastoma. The results indicate that the fusion protein is a fast, sensitive and specific reagent for the detection of polysialic acid. The use of an inactive enzyme as a specific molecular tool for the detection of its substrate represents an approach which could potentially find wide applicability in the specific detection of diverse macromolecules.Siirretty Doriast

Mixed-use -rakentamisen kaupunkitaloudelliset vaikutukset: Tarkastelussa joukkoliikennereittien varteen sijoittuvat hankkeet

Kaupunkirakennetta muokanneet trendit niin Suomessa kuin maailmalla ovat muuttuneet vuosikymmenten saatossa. 1800-luvulla rautatiet mahdollistivat kaupunkien levittäytymisen laajemmille alueille aiempaan jalankulkuun perustuvaan tiiviiseen kaupunkirakenteeseen verrattuna. Autojen yleistyttyä teollistumisen jälkeisinä vuosikymmeninä kaupunkirakenteen levittäytyminen saattoi jatkua edelleen ja kaupunkien toiminnot hajautuivat omille alueilleen. Keskustojen ulkopuolelle nousi erillisiä asuin- ja työpaikka-alueita sekä kauppakeskuksia. Nykyään kaupunkien rakenteesta keskustellaan tiiviin raken-tamisen näkökulmasta, jossa eri toimintojen saavutettavuus ei enää perustu pelkästään autoiluun. Tiiviissä kaupunkirakenteessa välimatkat toiminnosta ja alueelta toiseen on mahdollista toteuttaa jalan tai pyörällä. Toimintojen helppo ja nopea saavutettavuus eri liikennemuotoja hyödyntäen on olennaista tiiviissä kaupunkirakenteessa. Tällaiseen rakentamiseen tähtää mixed-use -rakentaminen, jossa ajatuksena on yhdistää ja integroida useampia erilaisia toimintoja samalle alueelle lähelle toisiaan. Erilaisia toimintoja voivat olla muun muassa asuminen, vähittäiskauppa, toimistotilat ja hotelli- tai kulttuuripalvelut. Kyseisten toimintojen täytyy olla fyysisesti ja käytännöllisesti yhdistyneet toisiinsa niin, että siirtyminen kohteesta toiseen on kävelyetäisyydellä. Tässä tutkimuksessa on tarkoituksena selvittää mixed-use -rakentamisen vaikutuksia kaupunkitalouden toimintaan ja tutkimuskysymyksenä on: Millaisia ovat joukkoliikennereitin varteen sijoittuvan mixed-use -hankkeen kaupunkitaloudelliset vaikutukset? Tutkimus on rajattu koskemaan vain joukkoliikennereitin varteen sijoittuvia hankkeita, jotta tarkastelusta voidaan poissulkea valmiin kaupunkirakenteen ulkopuolelle sijoittuvat mixed-use -alueet. Kaupunkitaloudellisia vaikutuksia käsitellään kaikkien eri kaupunkitalouden toimijoiden (maanomistajat, kotitaloudet, yritykset ja julkistalous) osalta erikseen. Tutkimuksen metodina käytetään kirjallisuuskatsausta, jonka avulla tutkittavasta aiheesta rakenne-taan mahdollisimman kattava kokonaiskuva. Kirjallisuuskatsauksessa esiin nousevia mixed-use -rakentamisen ja kaupunkitalouden toiminnan välisiä tekijöitä konkretisoidaan tutkimuksen lopussa case-tapauksen avulla. Aineistona toimivat sekä suomalaiset että kansainväliset kaupunkisuunnitteluun ja -talouteen kohdistuvia tutkimukset ja selvitykset sekä aihetta tukevat Tilastokeskuksen tilastot. Keskeisimpänä havaintona kirjallisuuskatsauksen ja case-esimerkin pohjalta nousi mixed-use -rakentamisen kaupunkitaloudellisten vaikutusten laaja-alaisuus ja useat toisiinsa kytkeytyvät vaikutusketjut. Maanomistajien osalta merkittävin ja useimmin esiin tullut vaikutus on maan arvon nousu. Kotitalouksille mixed-use -hankkeen toteuttamisesta ja sen käytön ajalta koituvia vaikutuksia ovat muun muassa työmarkkinoiden laajentuminen, asuntotarjonnan kasvu ja palvelutason monipuolistuminen. Yrityksiin kohdistuvien vaikutusten osalta merkittäviä tekijöitä ovat ainakin arvonlisäys, kokonaiskysynnän ja vetovoiman kasvu sekä tuottavuuden parantuminen. Julkistalouteen mixed-use -rakentaminen puolestaan vaikuttaa esimerkiksi verotulojen ja muiden veronluonteisten maksujen kautta. Kaiken kaikkiaan mixed-use -rakentaminen vastaa tämän hetken kaupunkikehityksen suurimpiin kehityskohtiin ja tutkimuksen tulokset sopivat käytettäviksi kaupunkikehityksessä esimerkiksi päätöksenteon tukena.fi=Opinnäytetyö kokotekstinä PDF-muodossa.|en=Thesis fulltext in PDF format.|sv=Lärdomsprov tillgängligt som fulltext i PDF-format

Time-resolved fluoroimmunoassay for bactericidal/permeability-increasing protein

Bactericidal/permeability-increasing protein (BPI) is a cationic antimicrobial protein produced by polymorphonuclear leukocytes, that specifically interacts with and kills Gram-negative bacteria. BPl competes with lipopolysaccharide-binding protein (LBP) secreted by liver cells into blood plasma for binding to lipopolysaccharide (LPS) and thus reduces the proinflammatory effects of LPS. We have developed a time-resolved fluoroimmunoassay for BPI and measured the concentration of BPI in human serum and plasma samples. The assay is based on a rabbit antibody against recombinant BPI. This antibody specifically adheres to polymorphonuclear leukocytes in immunostained human tissues. The difference in the serum concentration of BPI between unselected hospitalized patients with and without an infection was statistically significant. The mean concentration of BPI in serum samples was 28.3 μg/l (range 1.64–132, S.D. 26.8, n = 83). In contrast, there was no difference between the two groups in the BPI levels in plasma samples. For all individuals tested, BPI levels were consistently higher in plasma samples compared to the matched serum samples. The mean concentration of BPI in plasma samples was 52.3 μg/l (range 0.9–403, S.D. 60.6, n = 90). There was a positive correlation between the concentration of BPI and the white blood cell count as well as between the BPI concentration and C-reactive protein (CRP) in serum samples. In conclusion, the present study demonstrates that BPI can be quantified reliably by time-resolved fluoroimmunoassay in human serum samples

Design of a cytotoxic neuroblastoma-targeting agent using an enzyme acting on polysialic acid fused to a toxin

Polysialic acid, an abundant cell surface component of the developing nervous system, which declines rapidly postnatally to virtual absence in the majority of adult tissues, is highly expressed in some malignant tumors including neuroblastoma. We found that the binding of a noncatalytic endosialidase to polysialic acid causes internalization of the complex from the surface of neuroblastoma kSK-N-SH cells, a subline of SK-N-SH, and leads to a complete relocalization of polysialic acid to the intracellular compartment. The binding and uptake of the endosialidase is polysialic acid-dependent as it is inhibited by free excess ligand or removal of polysialic acid by active endosialidase, and does not happen if catalytic endosialidase is used in place of inactive endosialidase. Afusion protein composed of the noncatalytic endosialidase and the cytotoxic portion of diphtheria toxin was prepared to investigate whether the cellular uptake observed could be used for the specific elimination of polysialic acid-containing cells. The conjugate toxin was found to be toxic to polysialic acid-positive kSKN-SH with an IC50 of 1.0 nmol/L. Replacing the noncatalytic endosialidase with active endosialidase decreased the activity to the level of nonconjugated toxin. Normal nonmalignant cells were selectively resistant to the toxin conjugate. The results demonstrate that noncatalytic endosialidase induces a quantitative removal and cellular uptake of polysialic acid from the cell surface which, by conjugation with diphtheria toxin fragment, can be exploited for the selective elimination of polysialic acid-containing tumor cells.Peer reviewe

Receptor tyrosine kinase profiling of ischemic heart identifies ROR1 as a potential therapeutic target

BackgroundReceptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.MethodsRTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass,was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.ResultsIn addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.ConclusionsSeveral novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury.Peer reviewe

Combined genetic and chemical screens indicate protective potential for EGFR inhibition to cardiomyocytes under hypoxia

The return of blood flow to ischemic heart after myocardial infarction causes ischemia-reperfusion injury. There is a clinical need for novel therapeutic targets to treat myocardial ischemia-reperfusion injury. Here we screened for targets for the treatment of ischemia-reperfusion injury using a combination of shRNA and drug library analyses in HL-1 mouse cardiomyocytes subjected to hypoxia and reoxygenation. The shRNA library included lentiviral constructs targeting 4625 genes and the drug library 689 chemical compounds approved by the Food and Drug Administration (FDA). Data were analyzed using protein-protein interaction and pathway analyses. EGFR inhibition was identified as a cardioprotective mechanism in both approaches. Inhibition of EGFR kinase activity with gefitinib improved cardiomyocyte viability in vitro. In addition, gefitinib preserved cardiac contractility in zebrafish embryos exposed to hypoxia-reoxygenation in vivo. These findings indicate that the EGFR inhibitor gefitinib is a potential candidate for further studies of repurposing the drug for the treatment of myocardial infarction

An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation

Specificity in signaling activated by receptor tyrosine kinases is typically attributed to characteristics of their intracellular domains. Here, the authors demonstrate that an extracellular receptor sequence motif controls intracellular signaling as a result of extracellular glycan interactions.The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as beta 1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.Peer reviewe

Receptor tyrosine kinase profiling of ischemic heart identifies ROR1 as a potential therapeutic target

BackgroundReceptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.MethodsRTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass,was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.ResultsIn addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.ConclusionsSeveral novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury

Human Metaplastic Breast Carcinoma and Decorin

Metaplastic breast carcinoma (MBC) is a rare subtype of invasive breast cancer and has poor prognosis. In general, cancers are heterogeneous cellular masses comprised of different cell types and their extracellular matrix (ECM). However, little is known about the composition of the ECM and its constituents in MBC. Decorin is a ubiquitous ECM macromolecule known of its oncosuppressive activity. As such, it provides an intriguing molecule in the development of novel therapeutics for different malignancies such as MBC. In this study, decorin immunoreactivity and the effect of adenoviral decorin cDNA (Ad-DCN) transduction were examined in MBC. Multiple immunohistochemical stainings were used to characterize a massive breast tumour derived from an old woman. Furthermore, three-dimensional (3D) explant cultures derived from the tumour were transduced with Ad-DCN to study the effect of the transduction on the explants. The MBC tumour was shown to be completely negative for decorin immunoreactivity demonstrating that the malignant cells were not able to synthesize decorin. Ad-DCN transduction resulted in a markedly altered cytological phenotype of MBC explants by decreasing the amount of atypical cells and by inhibiting cell proliferation. The results of this study support approaches to develop new, decorin-based adjuvant therapies for MBC.</p