Betidamino acids: versatile and constrained scaffolds for drug discovery.

Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. Betidamino acids are most conveniently generated on solid supports used for the synthesis of peptides by selective acylation of one of the two amino functions of orthogonally protected aminoglycine(s) to generate the side chain either prior to or after the elongation of the main chain. We have used unresolved Nalpha-tert-butyloxycarbonyl-N'alpha-fluorenylmethoxycarbonyl++ + aminoglycine, and Nalpha-(Nalpha-methyl)-tert-butyloxycarbonyl-N'alpha-fluo renylmethoxycarbonyl aminoglycine as the templates for the introduction of betidamino acids in Acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2, where 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Aph is 4-aminophenylalanine, and Ilys is Nepsilon-isopropyllysine], a potent gonadotropin-releasing hormone antagonist, in order to test biocompatibility of these derivatives. Diasteremneric peptides could be separated in most cases by reverse-phase HPLC. Biological results indicated small differences in relative potencies (<5-fold) between the D and L nonalkylated betidamino acid-containing Acyline derivatives. Importantly, most betide diastereomers were equipotent with Acyline. In an attempt to correlate structure and observed potency, Ramachandran-type plots were calculated for a series of betidamino acids and their methylated homologs. According to these calculations, betidamino acids have access to a more limited and distinct number of conformational states (including those associated with alpha-helices, beta-sheets, or turn structures), with deeper minima than those observed for natural amino acids.

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.

Taxtomina A e Piriformospora indica no controle de Phytophthora nicotianae em citros e Phytophthora plurivora em faia (Fagus sylvatica)

Espécies de Phytophthora tem se destacado ao longo da história devido ao seu potencial destrutivo, se iniciando com a devastadora P. infestans na Irlanda e se estende até os dias de hoje com P. nicotianae em citros e P. plurivora em faia. Uma característica importante deste grupo de patógenos é que as medidas de controle da doença se baseiam na prevenção da entrada do patógeno na área visto que, uma vez instalado, o produtor precisa conviver com o mesmo, pois não se dispõem de métodos efetivos de controle. Neste sentido, a busca por métodos de controle torna-se primordial. O endofítico radicular Piriformospora indica, tem-se destacado em vários patossistemas devido a sua habilidade de induzir resistência contra patógenos, aumentar a tolerância à estresses abióticos e promover o crescimento de plantas. Taxtomina A, produzida por Streptomyces scabies, é capaz de ativar mecanismos de defesa de plantas, os quais são efetivos contra agentes patogênicos. Objetivou-se com este trabalho avaliar o efeito de P. indica e da taxtomina A sobre P. nicotianae em citros e P. plurivora em faia. Ambos foram avaliados quanto ao seu efeito direto sobre os patógenos em questão. O indutor de defesa vegetal Bion® foi utilizado em alguns ensaios para fins de comparação. Plântulas de citros e faia foram tratadas com concentrações crescentes de taxtomina e parâmetros fisiológicos, bioquímicos e de controle da doença foram avaliados. Taxtomina A não apresenta efeito direto sobre os patógenos avaliados. Os dados de incidência da doença em plântulas de faia tratadas com taxtomina A nas concentrações de 10, 25, 50 e 100 μg se mostraram consistentes com a quantidade de DNA do patógeno no sistema radicular, demonstrando que, aparentemente, a toxina induziu suscetibilidade nas plântulas de faia. Em citros, para os parâmetros fisiológicos e bioquímicos avaliados, em linhas gerais, a taxtomina A nas concentrações de 50 e 100 μg demonstrou potencial de aplicação no patossistema citros - P. nicotianae. Quando avaliada a mortalidade de plantas inoculadas com o patógeno e tratadas com taxtomina, bem como, quando quantificado o DNA do oomiceto no sistema radicular, as referidas concentrações também apresentaram os melhores desempenhos. Plântulas das mesmas espécies foram submetidas a inoculação com P. indica, sendo avaliados os efeitos na promoção de crescimento, na atividade de enzimas e de genes relacionados ao processo de defesa, bem como, no controle da doença. Não foi observado efeito direto do endofítico radicular sobre os patógenos avaliados. Quando plântulas de citros foram inoculadas com P. indica e depois com P. nicotianae, não foi observada promoção de crescimento e controle da doença. As análises histológicas e moleculares demonstraram a presença do endofítico no sistema radicular de plântulas de citros e faia. Análises bioquímicas revelaram apenas aumentos pontuais no teor de proteínas e na atividade da β-1,3-glucanase e da peroxidase no tratamento com P. indica + P. nicotianae. Os genes PR-1.4, PR-1.8, PR-β-glucosidase e Hsp70 foram induzidos em plântulas inoculadas com P. indica e com o patógeno, bem como no tratamento com Bion® e patógeno, porém em menor magnitude. O endofítico P. indica ativa o sistema de defesa de plântulas de citros, no entanto, os mecanismos ativados não são efetivos para o controle da doença na interação citros - P. nicotianae.

Estudo de metodologias de controle de qualidade do Mo-99 utilizado no preparo de geradores de Mo-99/Tc-99m

O 99mTc é o radionuclídeo mais utilizado em medicina nuclear. No Brasil os geradores de 99Mo/99mTc são produzidos exclusivamente pelo Centro de Radiofarmácia do IPEN-CNEN/SP, com 99Mo importado de diferentes fornecedores. O 99Mo (t1/2 = 66 h), por ser um produto de fissão do 235U, pode conter impurezas radionuclídicas prejudiciais à saúde humana. Dessa forma, para que o gerador seja utilizado de forma segura, é necessário que o 99Mo seja avaliado por ensaios de controle de qualidade e atenda à alguma especificação descrita em farmacopeia. A Farmacopeia Europeia (FE) apresenta monografia, com parâmetros (identificação, pureza radioquímica e pureza radionuclídica), métodos de análise, e limites, para avaliação da qualidade da solução de [99Mo] na forma de molibdato de sódio, que é utilizada como matéria-prima no preparo dos geradores de 99Mo/99mTc. No entanto, observa-se uma dificuldade na implementação e execução dos métodos por parte dos produtores de geradores, com pouca literatura sobre o assunto, provavelmente devido à falta de praticidade dos métodos propostos e à extensa lista de reagentes utilizados. Nesse trabalho foram avaliados vários parâmetros de qualidade do 99Mo descritos na monografia da FE. Foram estudados métodos de separação do 99Mo de suas impurezas radionuclídicas por extração em fase sólida (SPE) e por TLC. Após separação por SPE, foi proposta a quantificação de metais por ICP-OES para avaliar a porcentagem de retenção de Mo e a porcentagem de recuperação de Ru e Te e Sr em diversos tipos de cartuchos, em substituição ao uso de radiotraçadores. Observou-se que a marca de cartucho de SPE para separação do 99Mo recomendada pela FE apresentou baixa recuperação para Ru, quando comparado aos outros cartuchos de troca aniônica disponíveis no mercado. Amostras de 99Mo de diferentes fornecedores mundiais foram analisadas. Observou-se que é possível realizar a quantificação de 103Ru em amostras de 99Mo mesmo com tempos de decaimento acima de 4 semanas. Um método alternativo de separação do 99Mo do 131I por TLC apresentou resultados promissores. Não foi feita a quantificação das impurezas radionuclídicas emissoras beta e alfa. Todas as amostras analisadas apresentaram resultados dentro das especificações da FE para pureza radioquímica (>95%) e pureza radionuclídica.

Structural Polymorphism in Tau Filaments: An Implication for Neurodegenerative Diseases

Tau filaments are the pathological hallmark of >20 neurodegenerative diseases including Alzheimer's disease, Pick's disease, and progressive supranuclear palsy. In the adult human brain, six isoforms of tau are expressed that differ by presence or absence of the second of the four semiconserved repeats. As a consequence, half of the tau isoforms have three repeats (3R tau), whereas the other half has four repeats (4R tau). Site-directed spin labeling of recombinant tau in conjunction with electron paramagnetic resonance spectroscopy was used to obtain structural insights into tau filaments. The studies showed that the filaments of 4R tau and 3R tau share a highly ordered core structure in the third repeat with parallel, in-register arrangement of beta-strands. This structure in 3R and 4R is conserved regardless of whether full-length isoforms (htau40 and htau23) or truncated constructs (K18 and K19) are used. When mixed, 3R tau and 4R tau coassembled into heterogeneous filaments. Hence, these findings indicate that there are at least three compositionally distinct types of filaments: homogeneous 3R tau, homogeneous 4R tau, and heterogeneous 3R/4R tau. In vitro experiments show that the seeded filament growth, a prerequisite for tau spreading in tissue culture and brain, is crucially dependent on the isoform composition of individual seeds. Seeds of 3R tau and 3R/4R tau recruit both types of isoforms whereas seeds of 4R tau can recruit 4R tau, but not 3R tau, establishing an asymmetric barrier. Conformational templating of 4R tau onto 3R tau seeds eliminates this barrier, giving rise to a new type of tau filament. Conformational studies at the molecular level of tau filaments were done using Double electron-electron resonance spectroscopy, which allows the determination of distances between pairs of spin labels. These studies revealed structural differences between filaments of 3R tau and 4R tau. Furthermore, they indicated that 4R tau assumed the conformation of 3R tau when templated on 3R tau seeds. Our measurements have also provided insights into the heterogeneity of tau filament structure. Conformational differences due to variation in filament composition and seeding properties of tau filaments have shown that they are structurally polymorphic in nature. This structural polymorphism of tau filaments has widespread implications in understanding and treatment of neurodegenerative diseases.

Exposição a substâncias organocloradas em São Paulo: níveis séricos em doadores de sangue e fatores associados

A utilização de pesticidas organoclorados é motivo de preocupação das entidades ligadas à área de saúde em todo o mundo. Apesar de as formas de contaminação serem bem conhecidas, não há um controle eficaz na fiscalização do seu uso no Brasil. Sabe-se que altos níveis séricos destes compostos nos organismos de seres humanos e animais acarretam sérios problemas de saúde. Tendo em vista essa realidade, foi realizado, em 2009, o Projeto Piloto do I Inquérito Nacional de Populações Expostas a Substâncias Químicas, cujo subprojeto \"doadores de sangue\" teve como objetivo mensurar as concentrações de substâncias químicas no sangue de 547 residentes da região metropolitana de São Paulo, dentre elas os pesticidas organoclorados. Este trabalho teve como objetivos avaliar as concentrações dos pesticidas hexaclorobenzeno (HCB), alfa-HCH, ?-HCH, beta-HCH, beta-HCH, heptacloro, heptacloro epóxido, dieldrin, mirex, o,p\'-DDT, p,p\'-DDT, o,p\'-DDE, p,p\'-DDE, o,p\'-DDD e p,p\'-DDD nesta população e compará-las com as encontradas em outros países e determinar fatores associados aos níveis mais elevados destas substâncias. O método analítico utilizado foi de cromatografia a gás. Os resultados deste estudo indicam que a população adulta de São Paulo não está exposta a níveis preocupantes de pesticidas organoclorados, pois dentre os compostos analisados, apenas o beta-HCH e o p,p\'-DDE tiveram um número significante de amostras acima do limite de quantificação, 10,7% e 31,2% das amostras respectivamente. Quando utilizada a metade do limite de quantificação para substituir os valores abaixo do limite de quantificação do método, o valor médio encontrado para o beta-HCH foi de 0,028 ug/dL e para o p,p\'-DDE foi de 0,045 ug/dL. Este estudo propôs dois modelos multivariados para explicar os fatores associados aos compostos beta-HCH e p,p\'-DDE no sangue dos doadores. Segundo o modelo de Regressão Logística Ordinal Multivariado, os fatores associados a níveis mais altos de beta-HCH foram ter idade entre 26 e 45 anos e ser do sexo feminino. Para o p,p\'-DDE os fatores associados a níveis mais altos foram ter idade entre 26 e 45 anos, ser do sexo feminino e ter trabalhado com pesticidas, enquanto receber renda mensal de 3 a 5 salários mínimos e consumir derivados de origem animal uma ou mais vezes por semana foram associados a níveis mais baixos de p,p\'-DDE. Segundo o modelo de Regressão Linear Múltipla, os fatores associados a níveis mais altos de beta-HCH foram o sexo feminino, ter contato prévio com pesticidas na região agrícola, ter trabalhado com pesticidas em campanhas de saúde pública, ter trabalhado em empresas de capacitores ou transformadores, ter trabalhado em indústrias de solventes clorados, ter renda mensal de 3 a 5 salários mínimos, consumo de carnes uma ou duas vezes por semana e consumo de frutos do mar uma ou duas vezes por semana, enquanto consumo frequente de cerveja e ter renda mensal de 1 a 3 salários mínimos foram associado a níveis menores de beta-HCH. Já para o p,p\'-DDE, os fatores associados a níveis mais elevados foram ser do sexo feminino, ser não branco, ter trabalhado com pesticidas e consumir água de fontes que não sejam minerais ou de rede, enquanto o consumo frequente de bebidas alcoólicas foi associado a níveis mais baixos de p,p\'-DDE

Infecção concomitante experimental de Rattus norvegicus por Toxocara canis e Toxoplasma gondii: estudo comportamental e histopatológico

A hipótese de \"manipulação comportamental\" supõe que um parasito pode alterar o comportamento de seu hospedeiro visando aumentar a probabilidade de completar seu ciclo evolutivo. Tais alterações aumentariam a taxa de transmissão hospedeirohospedeiro, assegurando ao parasito ou a seus propágulos o encontro de novo hospedeiro. A possibilidade de infecções parasitárias provocarem mudanças comportamentais em seus hospedeiros e a elevada frequência com que o acometimento de seres humanos por larvas de Toxocara e cistos de Toxoplasma ocorre, têm chamado à atenção de pesquisadores interessados no estudo das relações hospedeiro-parasita. Na infecção por Toxoplasma gondii e Toxocara canis, cistos e larvas estão presentes em diversos locais anatômicos incluindo musculatura, coração, pulmões, olhos e cérebro. A presença de parasitos no cérebro dá oportunidade de manipulação do comportamento do hospedeiro. Entretanto, não se sabe qual ou quais mecanismos estão envolvidos no processo de manipulação do comportamento. Os objetivos do presente estudo foram verificar alterações na ansiedade, medo, memória e aprendizagem de Rattus norvegicus experimentalmente infectados por Toxocara canis e/ou Toxoplasma gondii em dois períodos após infecção, bem como a localização das larvas e cistos e presença de placas beta amiloide ( A) na região do hipocampo no tecido cerebral desses roedores corado pela técnica de Hematoxilina e Eosina (HE), e Vermelho de Congo, respectivamente. Foram utilizadas 40 exemplares fêmeas da espécie Rattus norvegicus, com seis a oito semanas. Os animais foram divididos em quatro grupos: Toxocara - 10 ratos infectados com 300 ovos de Toxocara canis, Toxoplasma -10 ratos infectados com 10 cistos de Toxoplasma gondii, Infecção dupla - 10 ratos infectados com 300 ovos de Toxocara canis e 10 cistos de Toxoplasma gondii, e controle - 10 ratos sem infecção. Nos dias 40, 41, 70 e 71 após a infecção, os animais dos grupos infectados e controle foram submetidos à avaliação no Labirinto em Cruz Elevado e Campo aberto. Aos 120 após infecção foi feita avaliação da memória, aprendizado e aversão a urina de gato dos animais no Labirinto de Barnes. No final das análises comportamentais os animais foram levados a eutanásia para retirada do cérebro e confecção dos cortes histológicos preparados em HE e Vermelho de Congo. Os resultados mostraram efeito ansiolítico para ambas as infecções, principalmente para Toxoplasma gondii. Não houve comprometimento da memória e aprendizado no LB, porém os animais infectados por Toxocara canis ou Toxoplasma gondii apresentaram menor tempo para encontrar a toca com urina e entrar nela. A leitura dos cortes histológicos corados com HE mostraram larvas de Toxocara canis e cistos de Toxoplasma gondii em regiões do sistema nervoso central dos animais relacionadas com memória e aprendizado. As lâminas coradas com Vermelho de Congo apresentaram placas beta amiloides ( A) em metade dos animais infectados por Toxoplasma gondii. Conclui-se que a infecção por ambos os parasitos apresenta efeito ansiolítico quando ocorre infecção única. Quando a Infecção ocorre concomitantemente há modulação no comportamento. Além disso, ratas infectadas com infecção única apresentam-se menos aversivas à urina de gatos.

A case in variant in cow's milk is atherogenic

Casein is a major protein in cow's milk that occurs in several variant forms, two of which are beta-casein A(1) and beta-casein A(2). The levels of these two proteins vary considerably in milk dependent on the breed of cow, and epidemiology studies suggest that there is a relationship between their consumption and the degree of atherosclerosis. In the present study, the direct effect of consumption of beta-casein A(1) vs beta-casein A(2) on atherosclerosis development was examined in a rabbit model. Sixty rabbits had their right carotid artery balloon de-endothelialised at t = 0, divided randomly into 10 groups (n = 6 per group), then for 6 weeks fed a diet containing 0, 5, 10 or 20% casein isolate, either beta-casein variant A(1) or A(2) made up to 20% milk protein with whey. Some groups had their diets supplemented with 0.5% cholesterol. Blood samples were collected at t = 0, 3 and 6 weeks and rabbits were sacrificed at t = 6 weeks. In the absence of dietary cholesterol, beta-casein A(1) produced significantly higher (P < 0.05) serum cholesterol, LDL, HDL and triglyceride levels than whey diet alone, which in turn produced higher levels than beta-casein A(2). Rabbits fed beta-casein A(1) had a higher percent surface area of aorta covered by fatty streaks than those fed beta-casein A(2) (5.2+/-0.81 vs 1.1+/-0.39, P < 0.05) and the thickness of the fatty streak lesions in the aortic arch was significantly higher (0.04+/-0.010 vs 0.00, P < 0.05). Similarly, the intima to media ratio (I:M) of the balloon injured carotid arteries in A(1) fed animals (0.77+/-0.07) was higher than in those that consumed A(2) (0.57+/-0.04) or whey (0.58+/-0.04), but this did not reach significance. In the presence of 0.5% dietary cholesterol, the thickness of the aortic arch lesions was higher (P < 0.05) in 5, 10 and 20% casein A(1) fed animals compared with their A(2) counterparts, while other parameters were not significantly different. It is concluded that beta-casein A(1)is atherogenic compared with beta-casein A(2). (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

The 2dF-SDSS LRG and QSO (2SLAQ) survey: the z < 2.1 quasar luminosity function from 5645 quasars to g=21.85

We have used the Two-Degree Field (2dF) instrument on the Anglo-Australian Telescope (AAT) to obtain redshifts of a sample of z < 3 and 18.0 < g < 21.85 quasars selected from Sloan Digital Sky Survey (SDSS) imaging. These data are part of a larger joint programme between the SDSS and 2dF communities to obtain spectra of faint quasars and luminous red galaxies, namely the 2dF-SDSS LRG and QSO (2SLAQ) Survey. We describe the quasar selection algorithm and present the resulting number counts and luminosity function of 5645 quasars in 105.7 deg(2). The bright-end number counts and luminosity functions agree well with determinations from the 2dF QSO Redshift Survey (2QZ) data to g similar to 20.2. However, at the faint end, the 2SLAQ number counts and luminosity functions are steeper (i.e. require more faint quasars) than the final 2QZ results from Croom et al., but are consistent with the preliminary 2QZ results from Boyle et al. Using the functional form adopted for the 2QZ analysis ( a double power law with pure luminosity evolution characterized by a second-order polynomial in redshift), we find a faint-end slope of beta =-1.78 +/- 0.03 if we allow all of the parameters to vary, and beta =-1.45 +/- 0.03 if we allow only the faint-end slope and normalization to vary (holding all other parameters equal to the final 2QZ values). Over the magnitude range covered by the 2SLAQ survey, our maximum-likelihood fit to the data yields 32 per cent more quasars than the final 2QZ parametrization, but is not inconsistent with other g > 21 deep surveys for quasars. The 2SLAQ data exhibit no well-defined 'break' in the number counts or luminosity function, but do clearly flatten with increasing magnitude. Finally, we find that the shape of the quasar luminosity function derived from 2SLAQ is in good agreement with that derived from Type I quasars found in hard X-ray surveys.

Iron-rich intermetallic phases and their role in casting defect formation in hypoeutectic Al-Si alloys

Iron is the most common and detrimental impurity in aluminum casting alloys and has long been associated with an increase in casting defects. While the negative effects of iron are clear, the mechanism involved is not fully understood. It is generally believed to be associated with the formation of Fe-rich intermetallic phases. Many factors, including alloy composition, melt superheating, Sr modification, cooling, rate, and oxide bifilms, could play a role. In the present investigation, the interactions between iron and each individual element commonly present in aluminum casting alloys, were investigated using a combination of thermal analysis and interrupted quenching tests. The Fe-rich intermetallic phases were characterized using optical microscope, scanning electron microscope, and electron probe microanalysis (EPMA), and the results were compared with the predictions by Thermocalc. It was found that increasing the iron content changes the precipitation sequence of the beta phase, leading to the precipitation of coarse binary beta platelets at a higher temperature. In contrast, manganese, silicon, and strontium appear to suppress the coarse binary beta platelets, and Mn further promotes the formation of a more compact and less harmful a phase. They are therefore expected to reduce the negative effects of the phase. While reported in the literature, no effect of P on the amount of beta platelets was observed. Finally, attempts are made to correlate the Fe-rich intermetallic phases to the formation of casting defects. The role of the beta phase as a nucleation site for eutectic Si and the role of the oxide bifilms and AIP as a heterogeneous substrate of Fe intermetallics are also discussed.

Ebola virus glycoprotein GP is not cytotoxic when expressed constitutively at a moderate level

Transient expression of Ebola virus (EBOV) glycoprotein GP causes downregulation of surface proteins, cell rounding and detachment, a phenomenon believed to play a central role in the pathogenicity of the virus. In this study, evidence that moderate expression of GP does not result in such morphological changes was provided. It was shown that GP continuously produced in 293T cells from the Kunjin virus replicon was correctly processed and transported to the plasma membrane without affecting the surface expression of beta 1 and alpha 5 integrins and major histocompatibility complex I molecules. The level of GIP expression in Kunjin replicon GP-expressing cells was similar to that observed in cells infected with EBOV early in infection and lower than that produced in cells transfected with plasmid DNA, phCMV-GP(1) expressing GP from a strong promoter. Importantly, transient transfection of Kunjin replicon GIP-expressing cells with GIP-coding plasmid DNA resulted in overexpression of GP, which lead to the downregulation of surface molecules and massive rounding and detachment of transfected cells. Here, it was also demonstrated that cell rounding and downregulation of the surface markers are the late events in EBOV infection, whereas synthesis and massive release of virus particles occur at early steps and do not cause significant cytotoxic effects. These findings indicate that the synthesis of EBOV GP in virus-infected cells is controlled well by several mechanisms that do not allow GP overexpression and hence the early appearance of its cytotoxic properties.

Patched1 functions as a gatekeeper by promoting cell cycle progression

Mutations in the Hedgehog receptor, Patched 1 (Ptch1), have been linked to both familial and sporadic forms of basal cell carcinoma (BCC), leading to the hypothesis that loss of Ptch1 function is sufficient for tumor progression. By combining conditional knockout technology with the inducible activity of the Keratin6 promoter, we provide in vivo evidence that loss of Ptch1 function from the basal cell population of mouse skin is sufficient to induce rapid skin tumor formation, reminiscent of human BCC. Elimination of Ptch1 does not promote the nuclear translocation of beta-catenin and does not induce ectopic activation or expression of Notch pathway constituents. In the absence of Ptch1, however, a large proportion of basal cells exhibit nuclear accumulation of the cell cycle regulators cyclin D1 and B1. Collectively, our data suggest that Ptch1 likely functions as a tumor suppressor by inhibiting G(1)-S phase and G(2)-M phase cell cycle progression, and the rapid onset of tumor progression clearly indicates Ptch1 functions as a gatekeeper. In addition, we note the high frequency and rapid onset of tumors in this mouse model makes it an ideal system for testing therapeutic strategies, such as Patched pathway inhibitors.

Elevated urinary neopterin suggests immune activation in Alzheimer’s disease and Down’s syndrome

Neopterin, an unconjugated pteridine, is secreted in large quantities by activated macrophages and can be used as a clinical marker of activated cellular immunity in a patient. Hence, neopterin levels were measured in urine samples taken from patients with Down’s syndrome (DS), non-hospitalized and hospitalized Alzheimer’s disease (AD) and age and sex matched controls. All subjects and patients were free from infectious and malignant disease. A significant effect of age on urinary neopterin levels was found in control subjects, levels being greater in younger and older subjects. No significant trends with age were found in AD and DS patients. The mean level of neopterin was significantly increased in DS and AD compared with age matched controls suggesting immune activation in these patients. In DS, elevated neopterin levels were present in individuals at least 17yrs old suggesting that immune activation could be associated with the initial deposition of beta/A4 in the brain.

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

Aims/hypothesis - Loss of the trophic support provided by surrounding non-endocrine pancreatic cell populations underlies the decline in beta cell mass and insulin secretory function observed in human islets following isolation and culture. This study sought to determine whether restoration of regulatory influences mediated by ductal epithelial cells promotes sustained beta cell function in vitro. Methods - Human islets were isolated according to existing protocols. Ductal epithelial cells were harvested from the exocrine tissue remaining after islet isolation, expanded in monolayer culture and characterised using fluorescence immunocytochemistry. The two cell types were co-cultured under conventional static culture conditions or within a rotational cell culture system. The effect of co-culture on islet structural integrity, beta cell mass and insulin secretory capacity was observed for 10 days following isolation. Results - Human islets maintained under conventional culture conditions exhibited a characteristic loss in structural integrity and functional viability as indicated by a diminution of glucose responsiveness. By contrast, co-culture of islets with ductal epithelial cells led to preserved islet morphology and sustained beta cell function, most evident in co-cultures held within the rotational cell culture system, which showed a significantly (p<0.05) greater insulin secretory response to elevated glucose compared with control islets. Similarly, insulin/protein ratio data suggested that the presence of ductal epithelial cells is beneficial for the maintenance of beta cell mass. Conclusions/interpretation - The data indicate a supportive role for ductal epithelial cells in islet viability. Further characterisation of the regulatory influences may lead to novel strategies to improve long-term beta cell function both in vitro and following islet transplantation.