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1.
Oncotarget ; 9(46): 28016-28029, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29963259

RESUMO

The stability, binding, and tissue penetration of variable new-antigen receptor (VNAR) single-domain antibodies have been tested as part of an investigation into their ability to serve as novel therapeutics. V13 is a VNAR that recognizes vascular endothelial growth factor 165 (VEGF165). In the present study V13 was used as a parental molecule into which we introduced mutations designed in silico. Two of the designed VNAR mutants were expressed, and their ability to recognize VEGF165 was assessed in vitro and in vivo. One mutation (Pro98Tyr) was designed to increase VEGF165 recognition, while the other (Arg97Ala) was designed to inhibit VEGF165 binding. Compared to parental V13, the Pro98Tyr mutant showed enhanced VEGF165 recognition and neutralization, as indicated by inhibition of angiogenesis and tumor growth. This molecule thus appears to have therapeutic potential for neutralizing VEGF165 in cancer treatment.

2.
Mar Drugs ; 16(4)2018 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-29614715

RESUMO

Variable new antigen receptor domain (vNAR) antibodies are novel, naturally occurring antibodies that can be isolated from naïve, immune or synthetic shark libraries. These molecules are very interesting to the biotechnology and pharmaceutical industries because of their unique characteristics related to size and tissue penetrability. There have been some approved anti-angiogenic therapies for ophthalmic conditions, not related to vNAR. This includes biologics and chimeric proteins that neutralize vascular endothelial growth factor (VEGF)165, which are injected intravitreal, causing discomfort and increasing the possibility of infection. In this paper, we present a vNAR antibody against human recombinant VEGF165 (rhVEGF165) that was isolated from an immunized Heterodontus francisci shark. A vNAR called V13, neutralizes VEGF165 cytokine starting at 75 µg/mL in an in vitro assay based on co-culture of normal human dermal fibroblasts (NHDFs) and green fluorescence protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) cells. In the oxygen-induced retinopathy model in C57BL/6:Hsd mice, we demonstrate an endothelial cell count decrease. Further, we demonstrate the intraocular penetration after topical administration of 0.1 µg/mL of vNAR V13 by its detection in aqueous humor in New Zealand rabbits with healthy eyes after 3 h of application. These findings demonstrate the potential of topical application of vNAR V13 as a possible new drug candidate for vascular eye diseases.


Assuntos
Produtos Biológicos/farmacocinética , Doenças Retinianas/tratamento farmacológico , Tubarões , Anticorpos de Domínio Único/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Tópica , Animais , Produtos Biológicos/imunologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/uso terapêutico , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Olho/irrigação sanguínea , Olho/metabolismo , Fibroblastos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Soluções Oftálmicas/farmacocinética , Soluções Oftálmicas/uso terapêutico , Oxigênio/toxicidade , Coelhos , Proteínas Recombinantes/metabolismo , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/patologia , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos de Domínio Único/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
J Cell Mol Med ; 16(12): 3009-21, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22947336

RESUMO

To further contribute to the understanding of multiple myeloma, we have focused our research interests on the mechanisms by which tumour plasma cells have a higher survival rate than normal plasma cells. In this article, we study the expression profile of genes involved in the regulation and protection of telomere length, telomerase activity and apoptosis in samples from patients with monoclonal gammopathy of undetermined significance, smouldering multiple myeloma, multiple myeloma (MM) and plasma cell leukaemia (PCL), as well as several human myeloma cell lines (HMCLs). Using conventional cytogenetic and fluorescence in situ hybridization studies, we identified a high number of telomeric associations (TAs). Moreover, telomere length measurements by terminal restriction fragment (TRF) assay showed a shorter mean TRF peak value, with a consistent correlation with the number of TAs. Using gene expression arrays and quantitative PCR we identified the hTERT gene together with 16 other genes directly involved in telomere length maintenance: HSPA9, KRAS, RB1, members of the Small nucleolar ribonucleoproteins family, A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins, and 14-3-3 family. The expression levels of these genes were even higher than those in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which have unlimited proliferation capacity. In conclusion, the gene signature suggests that MM tumour cells are able to maintain stable short telomere lengths without exceeding the short critical length, allowing cell divisions to continue. We propose that this could be a mechanism contributing to MM tumour cells expansion in the bone marrow (BM).


Assuntos
Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Homeostase do Telômero/genética , Telômero/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Instabilidade Cromossômica , Células-Tronco Embrionárias/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucemia Plasmocitária/genética , Leucemia Plasmocitária/metabolismo , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/metabolismo , Plasmócitos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Transcriptoma , Proteínas ras/genética , Proteínas ras/metabolismo
4.
Ann Surg Oncol ; 19(7): 2367-79, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22395973

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary tumor of the central nervous system in adults. Patients with GBM have few treatment options, and their disease is invariably fatal. Molecularly targeted agents offer the potential to improve patient treatment; however, the use of these will require a fuller understanding of the genetic changes in this complex tumor. METHODS: We analyzed a series of 32 patients with GBM with array comparative genomic hybridization in combination with gene expression analysis. We focused on the recurrent breakpoints found by spectral karyotyping (SKY). RESULTS: By SKY we identified 23 recurrent breakpoints of the 202 translocations found in GBM cases. Gains and losses were identified in chromosomal regions close to the breakpoints by array comparative genomic hybridization. We evaluated the genes located in the regions involved in the breakpoints in depth. A list of 406 genes that showed a level of expression significantly different between patients and control subjects was selected to determine their effect on survival. Genes CACNA2D3, PPP2R2B, SIK, MAST3, PROM1, and PPP6C were significantly associated with shorter survival (median 200 days vs. 450 days, P≤0.03). CONCLUSIONS: We present a list of genes located in regions of breakpoints that could be grounds for future studies to determine whether they are crucial in the pathogenesis of this type of tumor, and we provide a list of six genes associated with the clinical outcome of patients with GBM.


Assuntos
Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Cariotipagem Espectral , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Glioblastoma/mortalidade , Humanos , Cariotipagem , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Taxa de Sobrevida
5.
Stem Cells ; 29(2): 179-92, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21732477

RESUMO

Human sarcomas have been modeled in mice by expression of specific fusion genes in mesenchymal stem cells (MSCs). However, sarcoma models based on human MSCs are still missing. We attempted to develop a model of liposarcoma by expressing FUS (FUsed in Sarcoma; also termed TLS, Translocated in LipoSarcoma)-CHOP (C/EBP HOmologous Protein; also termed DDIT3, DNA Damage-Inducible Transcript 3), a hallmark mixoid liposarcoma-associated fusion oncogene, in wild-type and p53-deficient mouse and human adipose-derived mesenchymal stem/stromal cells (ASCs). FUS-CHOP induced liposarcoma-like tumors when expressed in p53(-/-) but not in wild-type (wt) mouse ASCs (mASCs). In the absence of FUS-CHOP, p53(-/-) mASCs forms leiomyosarcoma, indicating that the expression of FUS-CHOP redirects the tumor genesis/phenotype. FUS-CHOP expression in wt mASCs does not initiate sarcomagenesis, indicating that p53 deficiency is required to induce FUS-CHOP-mediated liposarcoma in fat-derived mASCs. In a human setting, p53-deficient human ASCs (hASCs) displayed a higher in vitro growth rate and a more extended lifespan than wt hASCs. However, FUS-CHOP expression did not induce further changes in culture homeostasis nor initiated liposarcoma in either wt or p53-depleted hASCs. These results indicate that FUS-CHOP expression in a p53-deficient background is sufficient to initiate liposarcoma in mouse but not in hASCs, suggesting the need of additional cooperating mutations in hASCs. A microarray gene expression profiling has shed light into the potential deregulated pathways in liposarcoma formation from p53-deficient mASCs expressing FUS-CHOP, which might also function as potential cooperating mutations in the transformation process from hASCs.


Assuntos
Adipócitos/metabolismo , Lipossarcoma/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição CHOP/metabolismo , Proteína Supressora de Tumor p53/deficiência , Adipócitos/citologia , Animais , Diferenciação Celular , Proliferação de Células , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Lipossarcoma/genética , Lipossarcoma/patologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteína FUS de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição CHOP/genética , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética
6.
Genomics ; 86(2): 117-26, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15961272

RESUMO

Here we report a large, extensively characterized set of single-nucleotide polymorphisms (SNPs) covering the human genome. We determined the allele frequencies of 55,018 SNPs in African Americans, Asians (Japanese-Chinese), and European Americans as part of The SNP Consortium's Allele Frequency Project. A subset of 8333 SNPs was also characterized in Koreans. Because these SNPs were ascertained in the same way, the data set is particularly useful for modeling. Our results document that much genetic variation is shared among populations. For autosomes, some 44% of these SNPs have a minor allele frequency > or =10% in each population, and the average allele frequency differences between populations with different continental origins are less than 19%. However, the several percentage point allele frequency differences among the closely related Korean, Japanese, and Chinese populations suggest caution in using mixtures of well-established populations for case-control genetic studies of complex traits. We estimate that approximately 7% of these SNPs are private SNPs with minor allele frequencies <1%. A useful set of characterized SNPs with large allele frequency differences between populations (>60%) can be used for admixture studies. High-density maps of high-quality, characterized SNPs produced by this project are freely available.


Assuntos
Mapeamento Cromossômico , Genoma Humano , Polimorfismo de Nucleotídeo Único , Alelos , Bases de Dados Genéticas , Frequência do Gene , Genótipo , Humanos , Análise de Sequência de DNA
7.
Mol Pharmacol ; 67(6): 1909-19, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15755908

RESUMO

The lipid and metabolic disturbances associated with human immunodeficiency virus (HIV) protease inhibitor therapy in AIDS have stimulated interest in developing new agents that minimize these side effects in the clinic. The underlying explanation of mechanism remains enigmatic, but a recently described link between endoplasmic reticulum (ER) stress and dysregulation of lipid metabolism suggests a provocative integration of existing and emerging data. We provide new evidence from in vitro models indicating that proteasome inhibition and differential glucose transport blockade by protease inhibitors are proximal events eliciting an ER stress transcriptional response that can regulate lipogenic pathways in hepatocytes or adipocytes. Proteasome activity was inhibited in vitro by several protease inhibitors at clinically relevant (micromolar) levels. In the intact cells, protease inhibitors rapidly elicited a pattern of gene expression diagnostic of intracellular proteasome inhibition and activation of an ER stress response. This included induction of transcription factors GADD153, ATF4, and ATF3; amino acid metabolic enzymes; proteasome components; and certain ER chaperones. In hepatocyte lines, the ER stress response was closely linked to moderate increases in lipogenic and cholesterogenic gene expression. However, in adipocytes where GLUT4 was directly inhibited by some protease inhibitors, time-dependent suppression of lipogenic genes and triglyceride synthesis was observed in coordination with the ER stress response. These results further link ER stress to dyslipidemia and contribute to a unifying mechanism for the pathophysiology of protease inhibitor-associated lipodystrophy, helping explain differences in clinical metabolic profiles among protease inhibitors.


Assuntos
Retículo Endoplasmático/metabolismo , Inibidores da Protease de HIV/farmacologia , Hiperlipidemias/metabolismo , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Inibidores de Proteassoma , Estresse Fisiológico/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Humanos , Hiperlipidemias/enzimologia , Hiperlipidemias/genética , Camundongos , Proteínas de Transporte de Monossacarídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ratos Wistar , Estresse Fisiológico/genética
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