Time course proteomic profiling of human myocardial infarction plasma samples: An approach to new biomarker discovery

Background: The aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology. Methods: Ten individuals with recent acute ischemic-type chest pain (< 12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T(0)) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T(2)), and the next four stages occurred at 12 h intervals after T(0). Individuals (n = 7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent). Results: Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p < 0.05). The 2STEMI group, had similar to 85% fewer differently expressed protein peaks than those without previous history of AMI (76, p < 0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T(0)), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM. Conclusions: Proteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery. (C) 2011 Elsevier B.V. All rights reserved.

Lower respiratory tract infections among human immunodeficiency virus-exposed, uninfected infants

Objectives: To evaluate whether maternal HIV disease severity during pregnancy is associated with an increased likelihood of lower respiratory tract infections (LRTIs) in HIV-exposed, uninfected infants. Methods: HIV-exposed, uninfected, singleton, term infants enrolled in the NISDI Perinatal Study, with birth weight >2500 g were followed from birth until 6 months of age. LRTI diagnoses, hospitalizations, and associated factors were assessed. Results: Of 547 infants, 103 (18.8%) experienced 116 episodes of LRTI (incidence = 0.84 LRTIs/100 child-weeks). Most (81%) episodes were bronchiolitis. Forty-nine (9.0%) infants were hospitalized at least once with an LRTI. There were 53 hospitalizations (45.7%) for 116 LRTI episodes. None of these infants were breastfed. The odds of LRTI in infants whose mothers had CD4% <14 at enrollment were 4.4 times those of infants whose mothers had CD4% >= 29 (p = 0.003). The odds of LRTI in infants with a CD4+ count (cells/ mm(3)) <750 at hospital discharge were 16.0 times those of infants with CD4+ >= 750 (p = 0.002). Maternal CD4+ decline and infant hemoglobin at the 6-12 week visit were associated with infant LRTIs after 6-12 weeks and before 6 months of age. Conclusions: Acute bronchiolitis is common and frequently severe among HIV-exposed, uninfected infants aged 6 months or less. Lower maternal and infant CD4+ values were associated with a higher risk of infant LRTIs. Further understanding of the immunological mechanisms of severe LRTIs is needed. (C) 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Preventing human trafficking for the purpose of sexual exploitation: the need for a bottom-up approach : the case-study of Brazilians trafficked to Portugal

Masters Thesis – Academic Year 2007/2008 - European Master’s Degree in Human Rights and Democratization (E.MA) - European Inter-university Centre for Human Rights and Democratization (EIUC) -Faculdade de Direito, Universidade Nova de Lisboa (UNL)

Enhancing human security through crisis management : opportunities and challenges for learning

Inhimilliseen turvallisuuteen kriisinhallinnan kautta – oppimisen mahdollisuuksia ja haasteita Kylmän sodan jälkeen aseelliset konfliktit ovat yleensä alkaneet niin sanotuissa hauraissa valtioissa ja köyhissä maissa, ne ovat olleet valtioiden sisäisiä ja niihin on osallistunut ei-valtiollisia aseellisia ryhmittymiä. Usein ne johtavat konfliktikierteeseen, jossa sota ja vakaammat olot vaihtelevat. Koska kuolleisuus konflikteissa voi jäädä alle kansainvälisen määritelmän (1000 kuollutta vuodessa), kutsun tällaisia konflikteja ”uusiksi konflikteiksi”. Kansainvälinen yhteisö on pyrkinyt kehittämään kriisinhallinnan ja rauhanrakentamisen malleja, jotta pysyvä rauhantila saataisiin aikaiseksi. Inhimillinen turvallisuus perustuu näkemykseen, jossa kunnioitetaan jokaisen yksilön ihmisoikeuksia ja jolla on vaikutusta myös kriisinhallinnan ja rauhanrakentamisen toteuttamiseen. Tutkimukseen kuuluu kaksi empiiristä osaa: Delfoi tulevaisuuspaneeliprosessin sekä kriisinhallintahenkilöstön haastattelut. Viisitoista eri alojen kriisinhallinta-asiantuntijaa osallistui paneeliin, joka toteutettiin vuonna 2008. Paneelin tulosten mukaan tulevat konfliktit usein ovat uusien konfliktien kaltaisia. Lisäksi kriisinhallintahenkilöstöltä edellytetään vuorovaikutus- ja kommunikaatiokykyä ja luonnollisesti myös varsinaisia ammatillisia valmiuksia. Tulevaisuuspaneeli korosti vuorovaikutus- ja kommunikaatiotaitoja erityisesti siviilikriisinhallintahenkilöstön kompetensseissa, mutta samat taidot painottuivat sotilaallisen kriisinhallinnan henkilöstön kompetensseissakin. Kriisinhallinnassa tarvitaan myös selvää työnjakoa eri toimijoiden kesken. Kosovossa työskennelleen henkilöstön haastatteluaineisto koostui yhteensä 27 teemahaastattelusta. Haastateltavista 9 oli ammattiupseeria, 10 reservistä rekrytoitua rauhanturvaajaa ja 8 siviilikriisinhallinnassa työskennellyttä henkilöä. Haastattelut toteutettiin helmi- ja kesäkuun välisenä aikana vuonna 2008. Haastattelutuloksissa korostui vuorovaikutus- ja kommunikaatiotaitojen merkitys, sillä monissa käytännön tilanteissa haastateltavat olivat ratkoneet ongelmia yhteistyössä muun kriisinhallintahenkilöstön tai paikallisten asukkaiden kanssa. Kriisinhallinnassa toteutui oppimisprosesseja, jotka usein olivat luonteeltaan myönteisiä ja informaalisia. Tällaisten onnistumisten vaikutus yksilön minäkuvaan oli myönteinen. Tällaisia prosesseja voidaan kuvata ”itseä koskeviksi oivalluksiksi”. Kriisinhallintatehtävissä oppimisella on erityinen merkitys, jos halutaan kehittää toimintoja inhimillisen turvallisuuden edistämiseksi. Siksi on tärkeää, että kriisinhallintakoulutusta ja kriisinhallintatyössä oppimista kehitetään ottamaan huomioon oppimisen eri tasot ja ulottuvuudet sekä niiden merkitys. Informaaliset oppimisen muodot olisi otettava paremmin huomioon kriisinhallintakoulutusta ja kriisinhallintatehtävissä oppimista kehitettäessä. Palautejärjestelmää olisi kehitettävä eri tavoin. Koko kriisinhallintaoperaation on saatava tarvittaessa myös kriittistä palautetta onnistumisista ja epäonnistumisista. Monet kriisinhallinnassa työskennelleet kaipaavat kunnollista palautetta työrupeamastaan. Liian rutiininomaiseksi koettu palaute ei edistä yksilön oppimista. Spontaanisti monet haastatellut pitivät tärkeänä, että kriisinhallinnassa työskennelleillä olisi mahdollisuus debriefing- tyyppiseen kotiinpaluukeskusteluun. Pelkkä tällainen mahdollisuus ilmeisesti voisi olla monelle myönteinen uutinen, vaikka tilaisuutta ei hyödynnettäisikään. Paluu kriisinhallintatehtävistä Suomeen on monelle haasteellisempaa kuin näissä tehtävissä työskentelyn aloittaminen ulkomailla. Tutkimuksen tulokset kannustavat tutkimaan kriisinhallintaa oppimisen näkökulmasta. On myös olennaista, että kriisinhallinnan palautejärjestelmiä kehitetään mahdollisimman hyvin edistämään sekä yksilöllistä että organisatorista oppimista kriisinhallinnassa. Kriisinhallintaoperaatio on oppimisympäristö. Kriisinhallintahenkilöstön kommunikaatio- ja vuorovaikutustaitojen kehittäminen on olennaista tavoiteltaessa kestävää rauhanprosessia, jossa konfliktialueen asukkaatkin ovat mukana.

Gender and human security : the challenge facing Canada's human security policy /

During the 1980's and for much of the 1990's, many countries in the Asia Pacific were renowned for their economic development and prosperity. The Asian tigers were a source of great interest for many economists and international investors. The 1997 Asian financial crisis, however, dramatically altered the growth and the performance of these economies. The crisis sent several ofAsia's best performing economies on a downward spiral from which many have yet to fully recover. The crisis exposed the financial and the political weaknesses ofmany countries in the region. Moreover, the crisis severely affected the wellbeing and the security ofmany ofthe region's citizens. This text will examine the economic crisis in greater detail and explore current debates in the study of international relations theory. More specifically, this paper will examine recent challenges posed to traditional international relations theory and address alternative approaches to this field of study. This paper will examine Critical theory and its role in shifting the referent object of security from the state to the individual. In this context, this paper will also assess Critical theory's role in enabling such issues as gender and human security to find a place on the agendas of international relations scholars and foreign policy makers. The central focus ofthis study will be the financial crisis and its impact on human security in the Southeast Asia. Furthermore, this paper will assess the recovery efforts ofthe domestic governments, international organizations and various Canadian sponsored initiatives in the context ofhuman security.

Towards sustainable peace : peace-building and human security

Failed and fragile states that result from intrastate war pose severe threats to the security of both the international system and individual states alike. In the post-Cold War era, the international community has come to recognize the reality of these threats and the difficulty involved in ending violence and building sustainable peace in failed and fragile states. This work focuses upon the development of a comprehensive strategy for sustainable peace-building by incorporating the tenets of the human security doctrine into the peace-building process. Through the use of case studies of The Former Yugoslav Republic of Macedonia and East Timor, the development and refinement of the doctrine of human security will occur, as well as, an understanding of how and where human security fits into the sustainable peace-building equation. The end result of the analysis is the development of a hierarchical pyramid formation that brings together human security and peace-building into one framework that ultimately creates the foundation and structure of sustainable peace-building. With the development of a sustainable peace-building structure based upon the human security doctrine, the role of Canada in the support of sustainable peace-building is analyzed in relation to the form and level of involvement that Canada undertakes and contributes to in the implementation and support of sustainable peace-building initiatives. Following from this, recommendations are provided regarding what role(s) Canada should undertake in the sustainable peace-building process that take into consideration the present and likely future capabilities of Canada to be involved in various aspects of the peace-building process. ii This paper outlines the need for a peace-building strategy that is designed to be sustainable in order that failed and fragile states resulting from intrastate conflict do not regress or collapse back into a condition of civil war, and subsequently designs such a strategy. The linking of peace-building and human security creates the required framework from which sustainable peace-building is derived. Creating sustainable peace is necessary in order to increase the likelihood that both present and future generations existing in failed and fragile states will be spared from the scourge of intrastate war.

Triangulation of rights, balancing of interests : exploring the tensions between freedom of conscience and freedom of religion in comparative constitutional law

La liberté de religion, souvent reconnue comme étant la « première liberté » dans de nombreuses traditions juridiques, reflète également les différentes conceptions de la place de l’individu et de la communauté dans la société. Notre étude analysera les modèles constitutionnels canadien, américain et européen de liberté de religion et conscience. Dans un premier chapitre, nous examinerons les conceptions théoriques de la religion dans les sciences sociales ainsi les approches juridiques afin de mieux cerner comment la religion est conçue et de plus, comprendre les diverses influences sur sa conceptualisation. Dans un second et troisième chapitre, nous tenterons d’une part, de qualifier la relation entre la liberté de conscience et la liberté de religion au Canada en nous livrant à une analyse approfondie des deux libertés et d’autre part, d’identifier les questions qui demeurent irrésolues. Dans le chapitre final, nous observerons comment la liberté de conscience a été interprétée dans les contextes américain et dans l’Union Européenne, par le biais de la Cour Européenne des droits de l’Homme. Notre hypothèse est que l’on peut arriver à une meilleure compréhension de la relation entre les libertés de conscience et religion en clarifiant les conceptions théoriques de la religion et de la conscience en droit constitutionnel comparé.

The Assault on Human Rigths and the Resilience of the Andean Dream: Health Inequities in Ecuador. IV Annual Distinguished Lecture sponsored by the Center for Research on Ethnicity, Culture and Health; School of Public Health; University of Michigan

The point of departure for these reflections is life, since its protection is the central purpose encouraging the defense of human rights and of public health. Life in the Andes has an exceptional diversity. Particularly in Ecuador, my country, this diversity constitutes a characteristic sign that is expressed in two main forms: natural megadiversity and multiculturalism. Indeed, Ecuador’s small territory synthesizes practically all types of lifezones that exist on Earth, having received the gift of high average rates of solar energy and abundant nutritional sources, which have facilitated the natural reproduction of countless species that show their beautiful vitality in the variety of ecosystems that compose the Andean mountain range, the tropical plains, the Amazon humid forests, and the Galapagos Islands. But besides being a highly biodiverse country, it is also a plurinational and multi-cultural society, in which the activity of human beings, organized into social conglomerates of different historical and cultural backgrounds, have formed more than a dozen nations and peoples. Regrettably this natural and human wealth has not been able to bear its best fruits due to the violent operation of a deep social inequity – unfortunately also one of the highest in the Americas—which conspires against life and is reproduced in national and international inequitable relations. This structural inequity has changed its form throughout the centuries and currently has reached its highest and most perverse level of development.

A framework for revitalisation of rural education and training systems in sub-Saharan Africa: Strengthening the human resource base for food security and sustainable livelihoods

Recent studies of the current state of rural education and training (RET) systems in sub-Saharan Africa have assessed their ability to provide for the learning needs essential for more knowledgeable and productive small-scale rural households. These are most necessary if the endemic causes of rural poverty (poor nutrition, lack of sustainable livelihoods, etc.) are to be overcome. A brief historical background and analysis of the major current constraints to improvement in the sector are discussed. Paramount among those factors leading to its present 'malaise' is the lack of a whole-systems perspective and the absence of any coherent policy framework in most countries. There is evidence of some recent innovations, both in the public sector and through the work of non-governmental organisations (NGOs), civil society organisations (CSOs) and other private bodies. These provide hope of a new sense of direction that could lead towards meaningful 'revitalisation' of the sector. A suggested framework offers 10 key steps which, it is argued, could largely be achieved with modest internal resources and very little external support, provided that the necessary leadership and managerial capacities are in place. (C) 2006 Elsevier Ltd. All rights reserved.

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion. which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and RI stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS RI also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10-24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS RI and with the increases in Bacteroides in cultures with both microbial EPS (RI and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids. (C) 2009 Elsevier B.V. All rights reserved.

Prospects for biodiversity conservation in the Atlantic Forest: Lessons from aging human-modified landscapes

Recent global assessments have shown the limited coverage of protected areas across tropical biotas, fuelling a growing interest in the potential conservation services provided by anthropogenic landscapes. Here we examine the geographic distribution of biological diversity in the Atlantic Forest of South America, synthesize the most conspicuous forest biodiversity responses to human disturbances, propose further conservation initiatives for this biota, and offer a range of general insights into the prospects of forest species persistence in human-modified tropical forest landscapes worldwide. At the biome scale, the most extensive pre-Columbian habitats across the Atlantic Forest ranged across elevations below 800 masl, which still concentrate most areas within the major centers of species endemism. Unfortunately, up to 88% of the original forest habitat has been lost, mainly across these low to intermediate elevations, whereas protected areas are clearly skewed towards high elevations above 1200 masl. At the landscape scale, most remaining Atlantic Forest cover is embedded within dynamic agro-mosaics including elements such as small forest fragments, early-to-late secondary forest patches and exotic tree mono-cultures. In this sort of aging or long-term modified landscapes, habitat fragmentation appears to effectively drive edge-dominated portions of forest fragments towards an early-successional system, greatly limiting the long-term persistence of forest-obligate and forest-dependent species. However, the extent to which forest habitats approach early-successional systems, thereby threatening the bulk of the Atlantic Forest biodiversity, depends on both past and present landscape configuration. Many elements of human-modified landscapes (e.g. patches of early-secondary forests and tree mono-cultures) may offer excellent conservation opportunities, but they cannot replace the conservation value of protected areas and hitherto unprotected large patches of old-growth forests. Finally, the biodiversity conservation services provided by anthropogenic landscapes across Atlantic Forest and other tropical forest regions can be significantly augmented by coupling biodiversity corridor initiatives with biota-scale attempts to plug existing gaps in the representativeness of protected areas. (C) 2010 Elsevier Ltd. All rights reserved.

Clinical correlations of human cytomegalovirus strains and viral load in kidney transplant recipients

Little is known about clinical differences associated with cytomegalovirus (CMV) infection by distinct strains in renal transplant patients. Different clinical pictures may be associated with specific viral genotypes. viral load, as well as host factors. The objective of this study was to identify CMV strains to determine viral load (antigenemia), and their correlation with clinical data in renal transplant recipients. Seventy-one patients were enrolled, comprising 91 samples. After selection, polymorphonuclear cells were used to amplify and sequence the gB region of CMV DNA. The sequences were analyzed to ascertain the frequency of different genotypes. Additionally, the results of this Study showed that the gB coding gene presents a great variability, revealing a variety of patterns: classical gB (1.4%), gB1V (46.4%), classical gB2 (35.2%), gB2V (2.8%), gB3 (1.4%), classical gB4 (4.9%) and gB4V (4.9%). The mean viral load in kidney transplant patient was 75.1 positive cells (1-1000). A higher viral load was observed in patients with genotype 4 infection. Statistically significant differences were detected between gB1 and gB4 (p=0.010), and between gB2 and gB4 (p=0.021). The average numbers of positive cells in relation to clinical presentation were: 34.5 in asymptomatic, 49.5 in CMV associated syndrome and 120.7 in patients with invasive disease (p=0.048). As a group, gB1 was the most frequent strain and revealed a potential risk for developing invasive disease. Viral load also seemed to be important as a marker associated with clinical presentation of the disease. (C) 2008 Elsevier B.V. All rights reserved.

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta 1 integrin

We studied the induction of protease activity by the laminin alpha 1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha 1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M I cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta 1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta 1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta 1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta 1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells. (c) 2008 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Factors associated with increased prevalence of human papillomavirus infection in a cohort of HIV-Infected Brazilian women

Objectives: Human papillomavirus (HPV) infection is a major risk factor for cervical disease. Using baseline data from the HIV-infected cohort of Evandro Chagas Clinical Research Institute at Fiocruz, Rio de Janeiro, Brazil, factors associated with an increased prevalence of HPV were assessed. Methods: Samples from 634 HIV-infected women were tested for the presence of HPV infection using hybrid capture 11 and polymerase chain reaction. Prevalence ratios (PR) were estimated using Poisson regression analysis with robust variance. Results: The overall prevalence of HPV infection was 48%, of which 94% were infected with a high-risk HPV. In multivariate analysis, factors independently associated with infection with high-risk HPV type were: younger age (<30 years of age; PR 1.5, 95% confidence interval (CI) 1.1-2.1), current or prior drug use (PR 1.3, 95% CI 1.0-1.6), self-reported history of HPV infection (PR 1.2, 95% CI 0.96-1.6), condom use in the last sexual intercourse (PR 1.3, 95% CI 1.1-1.7), and nadir CD4+ T-cell count <100 cells/mm(3) (PR 1.6, 95% CI 1.2-2.1). Conclusions: The estimated prevalence of high-risk HPV-infection among HIV-infected women from Rio de Janeiro, Brazil, was high. Close monitoring of HPV-related effects is warranted in all HIV-infected women, in particular those of younger age and advanced immunosuppression. (C) 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of similar to 120 kDa that could be solubilized by phospholipase C or Polidocanol. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.