Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes

Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

Maternal feeding self-efficacy and fruit and vegetable intakes in infants. Results from the SAIDI study

Adequate consumption of fruits and vegetables (FV) is a characteristic of a healthy diet but remains a challenge in nutrition interventions. This cross-sectional study explored the multi-directional relationships between maternal feeding self-efficacy, parenting confidence, child feeding behaviour, exposure to new food and FV intake in a cohort of 277 infants. Mothers with healthy infants weighing ≥2500 g and ≥37 weeks gestation were recruited post-natally from 11 South Australian hospitals. Socio-demographic datawere collected at recruitment. At 6 months postnatal, infantswereweighed and measured, andmothers completed a questionnaire exploring their perceptions of child feeding behaviour and child exposure to newfoods. The questionnaire also included the Short Temperament Scale for Infants, Kessler 10 to measure maternal psychological distress and 5 items measuring maternal feeding self-efficacy. The number of occasions and variety of FV (number of subgroups within food groups) consumed by infants were estimated from a 24-hour dietary recall and 2 days food record. Structural equation modellingwas performed using Mplus version 6.11. Median (IQR) variety scores were 2 (1–3) for fruit and 3 (2–5) for vegetable intake. The most popular FV consumed were apple (n = 108, 45.0%) and pumpkin (n = 143, 56.3%). None of the variables studied predicted the variety of child fruit intake. Parenting confidence, exposure to new foods and child feeding behaviourwere indirectly related to child vegetable intake through maternal feeding self-efficacy while total number of children negatively predicted child vegetable variety (p < 0.05). This highlights the need for addressing antecedents of maternal feeding self-efficacy and the family eating environment as key strategies towards development of healthy eating in children.

A QUM partnership approach to regulating biosimilars in Australia

Despite the realisation of the potential implications from biosimilars is relatively recent, much has already been written about raising the awareness of differences between biosimilars and originating/ reference listed (innovator) pharmaceuticals. The European Medicines Agency has led the global charge in regulating biosimilars. Regardless of sufficient similarities across international regulations, differences do exist across jurisdictions. The consideration of regulating biosimilars demands a congruent approach across all stages: pre-registration (Australian copyright protection, patent, international obligations), registration (confidential information, international regulators, safety and efficacy), post-registration (Pharmaceutical Benefit Scheme, prescriber and dispenser awareness). Our National Medicines Policy could provide the necessary congruent framework and function for national and international regulation of biosimilars. The Policy concedes that pharmaceuticals will be affected by financial policies and trade considerations, international treaty obligations, industrial policies, education policies and the need for public-private partnerships.

Better half-dead than read? The Mezzomorto cases and their implications for literary culture in the 1930s

This article focusses on the two libel cases arising from Brian Penton's review of Vivian Crockett's novel Mezzomorto for the Bulletin in 1934, viewing them as points of entry into Australian literary politics in the 1930s, and as windows on to one of the most enduring and interesting feuds in Australian literary culture, that between P.R. 'Inky' Stephensen, self-styled 'Bunyip Critic,' and Brian Penton, arch exponent of 'destructive criticism' and scourge of parochial pretension. The cases are particularly interesting for what they reveal about the evolving positions of two influential figures in Australian writing of the 1930s and 1940s. They also play in to contemporary debates about the state and status of 'literature' in Australia. And while Penton's biographer Patrick Buckridge avers that the cases did not impact on any of the larger contemporary literary issues (meaning censorship and free speech), a case may be made for the significance of the libel actions in the context of attempts to establish an industrial and cultural presence for a diverse range of Australian writing.

Donald Ezekiel AM : passionate and innovative contact lens practitioner and manufacturer

Donald Ezekiel (known to all as ‘Don’) was born in Singapore on September 12, 1936, to a German mother and Iraqi father. His parents were Jewish refugees, who met in Batavia,1 married and alternately lived in Batavia and Singapore. The family established their primary residence in Singapore after Don’s older brother Eric (later to become a haematologist) was born in 1934. The Ezekiel family was forced to flee in 1941 when the Japanese bombed Singapore and were fortunate to obtain passage on a hospital ship to Perth. They returned to Singapore after the war but left again on their own accord in 1951 due to race riots. The Ezekiels sold up everything in Singapore and decided to settle in Perth...

The Down Under book and film remind us our copyright law’s still unfair for artists

Australian copyright law is broken, and the Australian Government isn’t moving quickly to fix it. Borrowing, quoting, and homage are fundamental to the creative process. This is how people are inspired to create. Under Australian law, though, most borrowing is copyright infringement, unless it is licensed or falls within particular, narrow categories. This year marks five years since the very real consequences of Australia’s restrictive copyright law for Australian artists were made clear in the controversial litigation over Men at Work’s 1981 hit Down Under. The band lost a court case in 2010 that found that the song’s iconic flute riff copied some of the 1934 children’s song Kookaburra Sits in the Old Gumtree. A new book and documentary tell us more about the story behind the anthem – and the court case. The book, Down Under by Trevor Conomy, and the documentary, You Better Take Cover by Harry Hayes, bring renewed interest and new perspectives on the tragic story.

Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A

The major histocompatibility complex (MHC) on chromosome 6 is associated with susceptibility to more common diseases than any other region of the human genome, including almost all disorders classified as autoimmune. In type 1 diabetes the major genetic susceptibility determinants have been mapped to the MHC class II genes HLA-DQB1 and HLA-DRB1 (refs 1–3), but these genes cannot completely explain the association between type 1 diabetes and the MHC region4, 5, 6, 7, 8, 9, 10, 11. Owing to the region's extreme gene density, the multiplicity of disease-associated alleles, strong associations between alleles, limited genotyping capability, and inadequate statistical approaches and sample sizes, which, and how many, loci within the MHC determine susceptibility remains unclear. Here, in several large type 1 diabetes data sets, we analyse a combined total of 1,729 polymorphisms, and apply statistical methods—recursive partitioning and regression...

Studies of Phosphazenes. Part 13.' Thermal Rearrangement Reactions of Some Met hoxycyclo phosp hazenes

The methoxycyclophosphazenes [NP(OMe),], (n = 3-6) rearrange on heating to give oxocyclophosphazanes, [N(Me)PO(OMe)],. Isomeric products are formed when n = 4-6. The lH, ,lP, and 13C n.m.r. data for the starting materials and the products are presented. The ethoxy- and n-propoxy-derivatives N,P,( OR)* do not undergo the above rearrangement. The geminal derivatives N,P,R,(OMe), (R = Ph or NHBut) on heating yield both fully and partially rearranged products, namely dioxophosphaz-1 -enes and oxophosphazadienes, as shown by 270- MHz lH n.m.r. spectroscopy. The non-geminal derivative N,P,( NMe,),(OMe), gives only the fully rearranged product N,Me,P,(NMe,),O,(OMe), whose structure has been established from its lH and 31P n.m.r. spectra.

Wipert von Blücher und Finnland : Alternativpolitik und Diplomatie im 'Dritten Reich'

This dissertation explores the role of the German minister to Helsinki, Wipert von Blücher (1883-1963), within the German-Finnish relations of the late 1930s and the Second World War. Blücher was a key figure – and certainly one of the constants – within German Finland policy and the complex international diplomacy surrounding Finland. Despite representing Hitler’s Germany, he was not a National Socialist in the narrower sense of the term, but a conservative civil servant in the Wilhelmine tradition of the German foreign service. Along with a significant number of career diplomats, Blücher attempted to restrict National Socialist influence on the exercise of German foreign policy, whilst successfully negotiating a modus vivendi with the new regime. The study of his political biography in the Third Reich hence provides a highly representative example of how the traditional élites of Germany were caught in an cycle of conformity and, albeit tacit, opposition. Above all, however, the biographical study of Blücher and his behaviour offers an hitherto unexplored approach to the history of the German-Finnish relations. His unusually long tenure in Helsinki covered the period leading up to the so-called Winter War, which left Blücher severely distraught by Berlin’s effectively pro-Soviet neutrality and brought him close to resigning his post. It further extended to the German-Finnish rapprochement of 1940/41 and the military cooperation of both countries from mid-1941 to 1944. Throughout, Blücher developed a diverse and ambitious set of policy schemes, largely rooted in the tradition of Wilhelmine foreign policy. In their moderation and commonsensical realism, his designs – indeed his entire conception of foreign policy – clashed with the foreign political and ideological premises of the National Socialist regime. In its theoretical grounding, the analysis of Blücher’s political schemes is built on the concept of alternative policy and indebted to A.J.P. Taylor’s definition of dissent in foreign policy. It furthermore rests upon the assumption, introduced by Wolfgang Michalka, that National Socialist foreign policy was dominated by a plurality of rival conceptions, players, and institutions competing for Hitler’s favour (‘Konzeptionen-Pluralismus’). Although primarily a study in the history of international relations, my research has substantially benefited from more recent developments within cultural history, particularly research on nobility and élites, and the renewed focus on autobiography and conceptions of the self. On an abstract level, the thesis touches upon some of the basic components of German politics, political culture, and foreign policy in the first half of the 20th century: national belonging and conflicting loyalties, self-perception and representation, élites and their management of power, the modern history of German conservatism, the nature and practice of diplomacy, and, finally, the intricate relationship between the ethics of the professional civil service and absolute moral principles. Against this backdrop, the examination of Blücher’s role both within Finnish politics and the foreign policy of the Third Reich highlights the biographical dimension of the German-Finnish relationships, while fathoming the determinants of individual human agency in the process.

Fernando Pessoan ortonyymi ja heteronyymit modernistis-romanttisena myyttinä

Tutkimuksessani selvitän portugalilaisrunoilija Fernando Pessoan (1888-1935) heteronyymejä ja ortonyymiä modernistis-romanttisena myyttinä. Myytin piirteet paljastuivat Pessoan omia kirjoituksia ja runoja tutkimalla ja käyttämällä apuna Pessoa-tutkimusta. Tutkimusaineistonani ovat Pessoa-ortonyymin Mensagem-runoelma ja ”Autopsykografia”-runo, Álvaro de Campoksen vuonna 1917 Portugal Futurista –lehdessä julkaistu ”Ultimatum”-runomanifesti, Alberto Caeiron ”Guardador de Rebanhos”-runosikermä sekä Ricardo Reisin ”Oodit”-kokonaisuuteen kuuluvat runot: n:ro 59 ”Acima da verdade estão os deuses” (”Totuuden yläpuolella ovat jumalat”, 1914), n:ro 66 ”Olho os campos, Neera” (”Katson peltoja, Neera”, 1917), n:ro 181 ”Amo o que vejo” (”Rakastan sitä mitä näen”, 1934), n:ro 175 ”Estas só. Ninguem o sabe.” (”Olet yksin. Kukaan ei tiedä sitä”, 1933). Tutkimuksessani romanttisen runouden keskeisinä piirteinä pidetään uskoa taiteilijaneroon (geniukseen), mimeettiseen illuusioon (taide tavoittelee ulkoisen todellisuuden toisintamista mahdollisimman autenttisena) ja tekijän ja kokijan eli subjektin ja objektin erottamiseen toisistaan. Käsitänkin modernin runon tutkimuksessani hylänneen monia entisiä käsityksiä ja vastakkainasetteluja muun muassa tekijän ja ulkopuolisen maailman, mielen ja kielen välillä. Näitä moderneja ”rikkomuksia” identifioin ja määrittelen Pessoan heteronyymien runoudessa. Tutkimukseni apukäsitteitä ovat vilpittömyys ja autenttisuus, jotka kuvaavat runoilijan suhdetta itseensä ja muihin ja joita on pohtinut mm. Lionel Trilling teoksessaan Sincerity and authencity. Romanttisessa runoudessa suhde yhteisöön muodostui ensisijaisesti suhteessa omaan itseen, yhtenäiseen minuuteen nojaavan vilpittömyyden kautta, modernissa runoudessa hajanaisuutta ja yksilön suhdetta myös yhteisöön korostavan autenttisuuden avulla. Tutkimuksessani havaitsin, että Pessoa on jo tuotantonsa alusta lähtien kehittänyt tietoisesti modernistis-romanttista myyttiä – päätelmää tukivat niin Pessoan omat kirjoitukset kuin runoanalyysini. Pessoa ennusti ensimmäisen kerran romanttiseen nerotaiteilijaan vertautuvaa ”supra-Camõesia” ”A nova poesia Portuguesa Sociologicamente considerada” –esseessään vuonna 1912. Campoksen ”Ultimatum”-manifestissa puhuja uskoi muutaman useita kymmeniä persoonallisuuksia hallitsevan ”Synteesi-Ihmisen” tai ”supra-Camõesin” ilmestymiseen. ”Synteesi-Ihmisen” oli määrä palauttaa kukoistukseensa lamaantuneessa tilassa ollut Portugalin kirjallisuus uuteen kukoistukseensa. Eräänlaisen perustan koko myytille loi vuotta ennen Pessoan kuolemaa julkaistu Mensagem (suom. Viesti), jossa runoilija esittää saudosistisen ja sebastianistisen perustan. Teixeira de Pascoaesin kehittämä saudosismi näkyy Mensagemissa uskona Portugalin valtion perustajien ja laajentajien – löytöretkeilijöiden – edustamaan voittoisaan lusofoniseen mentaliteettiin, jonka Pessoa asettaa niin kansakunnan, runoilijan kuin ihmisen ihanteeksi. Toinen Mensagemissa esiintyvä myytin rakennusaine on sebastianismi eli usko kuningas Sebastianin (1554-1578) paluuseen. ”Synteesi-Ihminen”/”supra-Camões” oli reinkarnoitunut Sebastian, jonka piti palauttaa kansa ja kulttuuri kukoistukseensa lusofonisen valloittajamentaliteetin avulla. ”Autopsykografia”-runossaan Pessoa esittää puolestaan runoilijaa koskevan modernin poetiikan ongelmineen: esittävyyteen perustuva taide etäännyttää kokijan – taiteilijan – aina väistämättä ensimmäisestä, spontaanista ja luonnollisesta kokemuksesta ja tekee hänestä teeskentelijän ja simulakrumien (Baudrillard), toisen tason merkityksen tuottajan. ”Autopsykografian” moderni poetiikka koskee myös lukijaa, joka ei tavoita runoilijan välittämää merkitystä vaan kolmannen tason merkityksen. ”Autopsykografian” runous- ja ihmiskäsitys on moderni: runoilija on ammattimainen teeskentelijä, lähtökohtaisesti vilpillinen ja epäautenttinen. Pessoa ja Campos edustavat Pessoan objektiivisia heteronyymejä ja myytin modernia puolta. Pessoan subjektiiviset heteronyymit Reis ja Caeiro täydentävät objektiviisia, modernin ihmisen ahdistusta ja levottomuutta ilmaisseita Pessoaa ja Camposta ollen luonteeltaan romanttisia vilpittömyyden kannattajia. Caeiro arvosti näköaistia yli muiden aistien. Hänen tavoitteenaan oli katsoa aina kuin ensimmäistä kertaa, spontaanisti ja välittömästi kuin lapsi. Reis puolestaan korosti harmoniaa jumalten, ihmisten ja luonnon välillä ja uskoi kohtuullisiin aistinautintoihin. Molemmat muodostivat Pessoan myytin romanttisen puolen yhteisessä halussaan hylätä sosiaaliset roolit ja opetukset ja palata kohti yksilöllistä minää. Vilpittömyys ja autenttisuus ovat Caeiron ja Reisin ohjeita runoilijalle ja ihmiselle. Objektiiviset ja subjektiiviset heteronyymit yhdessä muodostavat modernistis-romanttisen myytin, joka kokonaisuudessaan on looginen: objektiiviset heteronyymit esittivät teeskentelyn, vilpillisyyden ja moderniuden ongelmat; subjektiiviset tarjosivat ratkaisua niihin, poisopettelua sosiaalisista rooleista ja rohkeutta mennä kohti omaa yksilöllistä minuutta.

Kampen om Eros : Om kön och kärlek i Pahlensviten

The Struggle for Eros: On Love and Gender in the Pahlen Series The present dissertation examines how gender, sexuality and motherhood are constructed in the novel series Fröknarna von Pahlen (The Misses von Pahlen, I VII, 1930 1935) by the Swedish author Agnes von Krusenstjerna. The aim of the study is to analyze how the Pahlen series relates to the discourses on gender and sexuality circulating in the 1930s, and how the series opens a dialogue with the feminist thinking of the time especially with the book Lifslinjer I (Love and Marriage, 1903) by the Swedish author Ellen Key. Fröknarna von Pahlen holds a central position in the research on Agnes von Krusenstjerna partly due to the literary debate that the novel series triggered. The debate was connected to the development taking place in the Swedish society in the beginning of the 1930s, in the so-called second phase of the Modern Breakthrough. Sweden was at that time characterized by struggle over the definitions of gender, sexuality and parenthood, and this struggle is also visible in the Pahlen series. The literary debate took place in 1934 1935 and it began after an article by the modernist writer Karin Boye was published in Social-Demokraten on 28 January 1934. In her polemic article, Boye saw the Pahlen series as a sign that the family institution is on the verge of a breakdown and with it the whole moral system that has come to existence through it . Boye went on to state that Krusenstjerna only sees and describes and that she explores neither new literary forms nor new values. Boye wrote the article before the last two parts of the novel series were published, so obviously she could not discuss the utopian vision characterizing those parts. This study, however, strives to demonstrate that Krusenstjerna not only sees and describes, but that she like many of her contemporary female colleagues appears to take the request of Friedrich Nietzsche to revaluate all values seriously. Like the works of her contemporaries, Krusenstjerna s Pahlen series is marked by a double vision on the one hand a critique of the prevailing social order, and on the other hand a dream of a new world and a new human being. In this research the vision of the Pahlen series is characterized as queer in order to emphasize that the series not only criticizes the prevailing gender order and its morals, but is also open for new ways of doing gender, parenthood, and family.