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1.
World J Urol ; 35(7): 1125-1132, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27783146

RESUMO

PURPOSE: Urethral strictures are a common disease of the lower urinary tract in men. At present, the use of buccal mucosa is the method of choice for long or recurrent strictures. However, autologous tissue-engineered grafts are still under investigation for reconstructive urological surgery. The aim of this pilot study was to evaluate the use of human urothelial cells (HUC) seeded on bovine collagen type I-based cell carriers (CCC) in an animal model and to evaluate short-term outcome of the surgical procedure. METHODS: Four male Göttingen minipigs were used with immunosuppression (cyclosporine A) for this pilot xenograft study. HUC obtained from human benign ureteral tissue were stained by PKH26 and seeded on a collagen cell carrier (CCC). Seven weeks after urethral stricture induction and protective vesicostomy, cell-seeded CCC was implanted in the urethra with HUC luminal and antiluminal, respectively. After two weeks animals were euthanized, urethrography and histological assessment were performed. RESULTS: Surgery was technically feasible in all minipigs. Stricture was radiologically established 7 weeks after induction. CCC was visible after two weeks and showed good integration without signs of inflammation or rejection. In the final urethrography, no remaining stricture could be detected. Near porcine urothelium, PKH26-positive areas were found even if partially detached from CCC. Although diminished, immunofluorescence with pankeratin, CK20, E-cadherin and ZO-1 showed intact urothelium in several areas on and nearby CCC. CONCLUSION: Finally, this study demonstrates that the HUC-seeded CCC used as a xenograft in minipigs is technically feasible and shows promising results for further studies.


Assuntos
Transplante de Células/métodos , Procedimentos de Cirurgia Plástica/métodos , Engenharia Tecidual/métodos , Estreitamento Uretral/cirurgia , Procedimentos Cirúrgicos Urológicos Masculinos/métodos , Urotélio/citologia , Animais , Bovinos , Colágeno Tipo I/fisiologia , Modelos Animais de Doenças , Xenoenxertos , Humanos , Masculino , Modelos Anatômicos , Suínos , Porco Miniatura , Resultado do Tratamento
2.
Mol Biol Cell ; 22(1): 153-64, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21119001

RESUMO

Cand1 inhibits cullin RING ubiquitin ligases by binding unneddylated cullins. The Cand1 N-terminus blocks the cullin neddylation site, whereas the C-terminus inhibits cullin adaptor interaction. These Cand1 binding sites can be separated into two functional polypeptides which bind sequentially. C-terminal Cand1 can directly bind to unneddylated cullins in the nucleus without blocking the neddylation site. The smaller N-terminal Cand1 cannot bind to the cullin neddylation region without C-terminal Cand1. The separation of a single cand1 into two independent genes represents the in vivo situation of the fungus Aspergillus nidulans, where C-terminal Cand1 recruits smaller N-terminal Cand1 in the cytoplasm. Either deletion results in an identical developmental and secondary metabolism phenotype in fungi, which resembles csn mutants deficient in the COP9 signalosome (CSN) deneddylase. We propose a two-step Cand1 binding to unneddylated cullins which initiates at the adaptor binding site and subsequently blocks the neddylation site after CSN has left.


Assuntos
Aspergillus nidulans/metabolismo , Proteínas Culina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fusão Gênica Artificial , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , Citoplasma/metabolismo , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fatores de Transcrição/química , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação , Ubiquitinas/metabolismo
3.
Mol Microbiol ; 78(4): 964-79, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21062371

RESUMO

The COP9 signalosome complex (CSN) is a crucial regulator of ubiquitin ligases. Defects in CSN result in embryonic impairment and death in higher eukaryotes, whereas the filamentous fungus Aspergillus nidulans survives without CSN, but is unable to complete sexual development. We investigated overall impact of CSN activity on A. nidulans cells by combined transcriptome, proteome and metabolome analysis. Absence of csn5/csnE affects transcription of at least 15% of genes during development, including numerous oxidoreductases. csnE deletion leads to changes in the fungal proteome indicating impaired redox regulation and hypersensitivity to oxidative stress. CSN promotes the formation of asexual spores by regulating developmental hormones produced by PpoA and PpoC dioxygenases. We identify more than 100 metabolites, including orsellinic acid derivatives, accumulating preferentially in the csnE mutant. We also show that CSN is required to activate glucanases and other cell wall recycling enzymes during development. These findings suggest a dual role for CSN during development: it is required early for protection against oxidative stress and hormone regulation and is later essential for control of the secondary metabolism and cell wall rearrangement.


Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/metabolismo , Parede Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Hormônios/metabolismo , Complexos Multiproteicos/metabolismo , Estresse Oxidativo , Peptídeo Hidrolases/metabolismo , Transdução de Sinais , Aspergillus nidulans/genética , Complexo do Signalossomo COP9 , Proteínas Fúngicas/genética , Deleção de Genes , Perfilação da Expressão Gênica , Metaboloma , Complexos Multiproteicos/genética , Peptídeo Hidrolases/genética , Proteoma
4.
Eukaryot Cell ; 7(6): 1041-52, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18390647

RESUMO

Nuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC in Aspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (for binding to NUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.


Assuntos
Aspergillus nidulans/citologia , Aspergillus nidulans/metabolismo , Núcleo Celular/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Sequência de Aminoácidos , Dineínas/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Técnicas do Sistema de Duplo-Híbrido
5.
J Biol Chem ; 282(37): 26666-26674, 2007 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-17636265

RESUMO

Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Delta12 desaturase and a processive bifunctional oleoyl-Delta12/linoleoyl-omega3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Delta12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Delta12/omega3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model.


Assuntos
Aspergillus nidulans/enzimologia , Ácidos Graxos Dessaturases/química , Sequência de Bases , Sítios de Ligação , Membrana Celular/enzimologia , Ácidos Graxos Dessaturases/genética , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Especificidade por Substrato
6.
Proc Natl Acad Sci U S A ; 104(19): 8089-94, 2007 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-17470786

RESUMO

Fruit body formation in filamentous fungi is a complex and yet hardly understood process. We show here that protein turnover control is crucial for Aspergillus nidulans development. Deletion of genes encoding COP9 signalosome (CSN) subunits 1, 2, 4, or 5 resulted in identical blocks in fruit body formation. The CSN multiprotein complex controls ubiquitin-dependent protein degradation in eukaryotes. Six CSN subunits interacted in a yeast two-hybrid analysis, and the complete eight-subunit CSN was recruited by a functional tandem affinity purification tag fusion of subunit 5 (CsnE). The tagged CsnE was unable to recruit any CSN subunit in a strain deleted for subunit 1 or subunit 4. Mutations in the JAMM metalloprotease core of CsnE resulted in mutant phenotypes identical to those of csn deletion strains. We propose that a correctly assembled CSN including a functional JAMM links protein turnover to fungal sexual development.


Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Complexos Multiproteicos/química , Peptídeo Hidrolases/química , Motivos de Aminoácidos , Aspergillus nidulans/genética , Complexo do Signalossomo COP9 , Genoma Fúngico , Complexos Multiproteicos/fisiologia , Peptídeo Hidrolases/fisiologia , Fenótipo , Subunidades Proteicas
7.
Mol Microbiol ; 64(4): 873-6, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17501912

RESUMO

It is a challenge in biology to explore the molecular and cellular mechanisms necessary to form a complex three-dimensional structure composed of different cell types. Interesting models to study the underlying processes are fungi that can transform their wire-like hyphal filaments into complex and sometimes container-like fruit bodies. In the past, the role of developmental triggers and transcription factors was a major focus of research on fungal model organisms. In this issue of Molecular Microbiology, Nowrousian and collaborators report that fruit body development of the model organism Sordaria macrospora includes a novel player, a specific membrane protein of the endoplasmic reticulum that is not required for vegetative growth. This finding represents an important step towards connecting regulation of development with the co-ordinated changes in cellular compartments.


Assuntos
Carpóforos/crescimento & desenvolvimento , Morfogênese/fisiologia , Sordariales/crescimento & desenvolvimento , Retículo Endoplasmático/química , Proteínas Fúngicas/fisiologia , Proteínas de Membrana/fisiologia
8.
Mol Microbiol ; 61(1): 76-88, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16824096

RESUMO

Cellular differentiation relies on precise and controlled means of gene expression that act on several levels to ensure a flexible and defined spatio-temporal expression of a given gene product. In our aim to identify transcripts enriched during fruiting body formation of the homothallic ascomycete Aspergillus (Emericella) nidulans, the grrA gene could be identified in a negative subtraction hybridization screening procedure. It encodes a protein similar to fungal F-box proteins, which function as substrate receptors for ubiquitin ligases, and that is highly related to the Saccharomyces cerevisiae regulatory protein Grr1p. Expression studies confirmed induction of grrA transcription and expression of its gene product during cleistothecial development of A. nidulans. Functional complementation of a yeast grr1Delta mutant was achieved by overexpression of the grrA coding sequence. A grrADelta deletion mutant resembles the wild-type in hyphal growth, asexual sporulation, Hülle cell formation or development of asci-containing cleistothecia, but is unable to produce mature ascospores due to a block in meiosis as demonstrated by cytological staining of cleistothecial contents. Our results specify a particular involvement of the E3 ubiquitin ligase SCFGrrA in meiosis and sexual spore formation of an ascomyceteous fungus and shed light on the diverse functions of ubiquitin-proteasome-mediated protein degradation in eukaryotic development.


Assuntos
Aspergillus nidulans/genética , Proteínas F-Box/genética , Meiose/genética , Sequência de Aminoácidos , Aspergillus nidulans/metabolismo , Aspergillus nidulans/fisiologia , Sequência de Bases , Northern Blotting , Proteínas F-Box/metabolismo , Proteínas F-Box/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Meiose/fisiologia , Dados de Sequência Molecular , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
9.
Int J Cancer ; 119(3): 549-55, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16506207

RESUMO

The adhesion molecule L1 is expressed in primary melanomas and cutaneous metastases in contrast to melanocytic nevi and melanocytes, and is significantly associated with metastatic spread. Recent studies have demonstrated that in carcinomas L1 expression is associated with sustained activation of the extracellular signal-regulated kinase (ERK) pathway and upregulation of ERK-dependent, motility- and invasion-associated gene products including alphavbeta3 integrin. The objective of this study was to further investigate the role of the adhesion molecule L1 in melanoma progression, and to evaluate whether targeting the L1 adhesion molecule would have therapeutic effects against invasive melanoma growth. Using human melanoma cells from different stages of progression in monolayer and organotypic human skin culture mimicking the pathophysiological environment of cutaneous melanoma, we found that (1) L1 expression mostly correlates with melanoma progression and alphavbeta3 integrin expression, (2) overexpression of L1 in early radial growth phase melanoma cells promotes conversion from radial to vertical growth phase melanoma without upregulation of alphavbeta3 integrin expression, and (3) suppression of L1 function significantly reduces migration and invasion of melanoma cells, but does not completely block invasive melanoma growth. Altogether, L1 plays a critical role in melanoma invasion and progression and offers therapeutic potential in combination with conventional anticancer agents.


Assuntos
Melanoma/patologia , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Animais , Anticorpos/farmacologia , Western Blotting , Células CHO , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Progressão da Doença , Humanos , Imuno-Histoquímica , Integrina alfaVbeta3/análise , Masculino , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Molécula L1 de Adesão de Célula Nervosa/análise , Molécula L1 de Adesão de Célula Nervosa/imunologia , Pele/metabolismo , Pele/patologia , Técnicas de Cultura de Tecidos/métodos
10.
Nature ; 438(7071): 1105-15, 2005 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-16372000

RESUMO

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.


Assuntos
Aspergillus fumigatus/genética , Aspergillus nidulans/genética , Aspergillus oryzae/genética , Genoma Fúngico/genética , Genômica , Aspergillus fumigatus/fisiologia , Aspergillus nidulans/fisiologia , Aspergillus oryzae/fisiologia , Sequência de Bases , Sequência Consenso/genética , Sequência Conservada/genética , Evolução Molecular , Genes Fúngicos Tipo Acasalamento/genética , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Proteoma/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA , Sintenia/genética
11.
Front Biosci ; 10: 2986-3001, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15970553

RESUMO

Malignant melanoma is a highly aggressive tumor of the pigment-producing cells in the skin with a rapidly increasing incidence and a poor prognosis for patients with advanced disease that is resistant to current therapeutic concepts. Therefore, the development of novel strategies for treating melanoma are of utmost importance. In melanoma, both the Ras-Raf-MEK-ERK (MAPK) and the PI3K-AKT (AKT) signaling pathways are constitutively activated through multiple mechanisms, and thus exert several key functions in melanoma development and progression. Conversely, several molecules known to play key roles in melanoma development and progression such as the adhesion molecules E-/N-cadherin, MelCAM and alphavbeta3 integrin are regulated by these pathways and/or activate the same. The results of the research to date indicate that in melanoma both the MAPK and the AKT signaling pathways may represent promising therapeutic targets.


Assuntos
Melanoma/patologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais/fisiologia , Apoptose , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases , Humanos , Melanoma/fisiopatologia , Invasividade Neoplásica/fisiopatologia , Células Tumorais Cultivadas
12.
Mol Microbiol ; 49(3): 717-30, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12864854

RESUMO

The COP9 signalosome (CSN) is a conserved multiprotein complex involved in regulation of eukaryotic development. The deduced amino acid sequences of two Aspergillus nidulans genes, csnD and csnE, show high identities to the fourth and fifth CSN subunits of higher eukaryotes. The csnD transcript is abundant during vegetative growth as well as development and the corresponding protein accumulates in the nucleus. Strains deleted for either csn gene are viable and show identical mutant phenotypes at conditions that allow development: hyphae appear partly red and contain cells of reduced size. Additionally, light dependence of propagation onset is affected. The Delta csn mutants are capable of initiating the sexual cycle and develop primordia, but maturation to sexual fruit bodies is blocked. This developmental arrest could not be overcome by overexpression of the sexual activator velvet (VEA). We conclude that the COP9 signalosome in A. nidulans is a key regulator of sexual development, and its proposed structural and functional conservation to the CSN of higher eukaryotes enables studies on this regulatory complex in a genetically amenable organism.


Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Proteínas Fúngicas/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Complexos Multienzimáticos/fisiologia , Sequência de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/efeitos da radiação , Aspergillus nidulans/ultraestrutura , DNA Complementar/genética , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos , Luz , Dados de Sequência Molecular , Morfogênese , Complexos Multienzimáticos/genética , Mutagênese , Peptídeo Hidrolases , Fenótipo , Pigmentos Biológicos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Reprodução , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de Transcrição/química , Transcrição Gênica
13.
Curr Genet ; 42(4): 209-19, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12589472

RESUMO

The non-proteinogenic amino acid, alpha-aminoadipate, defines the biosynthetic branch-point of lysine and penicillin biosynthesis in the filamentous fungus, Aspergillus nidulans. Regulation of both pathways was analysed in response to amino acid limitation. The lysF-encoded homoaconitase acts upstream of the alpha-aminoadipate branch point, whereas the lysA gene product, saccharopine dehydrogenase, catalyses the ultimate step of the lysine-specific branch. The lysA gene from A. nidulans was identified and isolated. Amino acid starvation resulted in significantly increased transcription of lysA but not lysF. Starvation-dependent changes in transcription levels of lysA were dependent on the presence of the central transcriptional activator of the cross-pathway control (CPCA). The effect of amino acid starvation under penicillin-producing conditions was analysed in A. nidulans strains with reporter genes for the penicillin-biosynthesis genes, acvA and ipnA, and genetically altered activity of the cross-pathway control. Overproduction of CPCA decreased expression of ipnAand acvA reporter genes and even more drastically reduced penicillin production. This work suggests that, upon amino acid starvation, the cross-pathway control overrules secondary metabolite biosynthesis and favours the metabolic flux towards amino acids instead of penicillin in A. nidulans.


Assuntos
Aspergillus nidulans/metabolismo , Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Lisina/biossíntese , Penicilinas/biossíntese , Sequência de Aminoácidos , Aspergillus nidulans/genética , Proteínas de Bactérias/genética , Carboxiliases/genética , Dados de Sequência Molecular , Sacaropina Desidrogenases/genética , Análise de Sequência de Proteína , Homologia de Sequência
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