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1.
Molecules ; 25(7)2020 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-32225092

RESUMO

The novel prenyl transferase-mediated, site-specific, antibody-drug conjugate LCB14-0110 is comprised of a proprietary beta-glucuronide linker and a payload (Monomethyl auristatin F, MMAF, an inhibitor for tubulin polymerization) attached to human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab. A LC-MS/MS method was developed to quantify the antibody-conjugated drug (acDrug) for in vitro linker stability and preclinical pharmacokinetic studies. The method consisted of affinity capture, enzymatic cleavage of acDrug, and LC-MS/MS analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2), with the equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 19.17~958.67 ng/mL for acDrug. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. The overall recovery was 42.61%. The dilution integrity was for a series of 5-fold dilutions with accuracy and precision values ranging within ±25%. The stability results indicated that acDrug was stable at all stability test conditions (short-term: 1 day, long-term: 10 months, Freeze/Thaw (F/T): 3 cycles). This qualified method was successfully applied to in vitro linker stability and pharmacokinetic case studies of acDrug in rats.


Assuntos
Cromatografia Líquida , Imunoconjugados/química , Imunoconjugados/farmacocinética , Neopreno , Espectrometria de Massas em Tandem , Transferases , Animais , Monitoramento de Medicamentos , Estabilidade de Medicamentos , Humanos , Estrutura Molecular , Neopreno/química , Ratos , Transferases/química , Trastuzumab/química , Trastuzumab/farmacocinética
2.
G3 (Bethesda) ; 7(12): 3887-3899, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29025917

RESUMO

In the nematode Caenorhabditis elegans, RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) spreads systemically to cause gene silencing throughout the organism and its progeny. We confirm that Caenorhabditis nematode SID-1 orthologs have dsRNA transport activity and demonstrate that the SID-1 paralog CHUP-1 does not transport dsRNA. Sequence comparison of these similar proteins, in conjunction with analysis of loss-of-function missense alleles, identifies several conserved 2-7 amino acid microdomains within the extracellular domain (ECD) that are important for dsRNA transport. Among these missense alleles, we identify and characterize a sid-1 allele, qt95, which causes tissue-specific silencing defects most easily explained as a systemic RNAi export defect. However, we conclude from genetic and biochemical analyses that sid-1(qt95) disrupts only import, and speculate that the apparent export defect is caused by the cumulative effect of sequentially impaired dsRNA import steps. Thus, consistent with previous studies, we fail to detect a requirement for sid-1 in dsRNA export, but demonstrate for the first time that SID-1 functions in the intestine to support environmental RNAi (eRNAi).


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Transporte de RNA/genética , RNA de Cadeia Dupla/genética , Animais , Animais Geneticamente Modificados/genética , Caenorhabditis elegans/genética , Inativação Gênica , Mucosa Intestinal/metabolismo , Interferência de RNA
3.
Korean J Intern Med ; 29(3): 281-90, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24851060

RESUMO

Pulmonary fibrosis is a fatal progressive disease with no effective therapy. Transforming growth factor (TGF)-ß1 has long been regarded as a central mediator of tissue fibrosis that involves multiple organs including skin, liver, kidney, and lung. Thus, TGF-ß1 and its signaling pathways have been attractive therapeutic targets for the development of antifibrotic drugs. However, the essential biological functions of TGF-ß1 in maintaining normal immune and cellular homeostasis significantly limit the effectiveness of TGF-ß1-directed therapeutic approaches. Thus, targeting downstream mediators or signaling molecules of TGF-ß1 could be an alternative approach that selectively inhibits TGF-ß1-stimulated fibrotic tissue response while preserving major physiological function of TGF-ß1. Recent studies from our laboratory revealed that TGF-ß1 crosstalk with epidermal growth factor receptor (EGFR) signaling by induction of amphiregulin, a ligand of EGFR, plays a critical role in the development or progression of pulmonary fibrosis. In addition, chitotriosidase, a true chitinase in humans, has been identified to have modulating capacity of TGF-ß1 signaling as a new biomarker and therapeutic target of scleroderma-associated pulmonary fibrosis. These newly identified modifiers of TGF-ß1 effector function significantly enhance the effectiveness and flexibility in targeting pulmonary fibrosis in which TGF-ß1 plays a significant role.


Assuntos
Pulmão/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Animais , Desenho de Fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Hexosaminidases/antagonistas & inibidores , Hexosaminidases/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/patologia , Terapia de Alvo Molecular , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Receptor Cross-Talk , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo
4.
J Biol Chem ; 287(50): 41991-2000, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23086930

RESUMO

Dysregulated amphiregulin (AR) expression and EGR receptor (EGFR) activation have been described in animal models of pulmonary fibrosis and in patients with idiopathic pulmonary fibrosis. However, the exact role of AR in the pathogenesis of pulmonary fibrosis has not been clearly defined. Here, we show that a potent profibrogenic cytokine TGF-ß1 significantly induced the expression of AR in lung fibroblasts in vitro and in murine lungs in vivo. AR stimulated NIH3T3 fibroblast cell proliferation in a dose-dependent manner. Silencing of AR expression by siRNA or chemical inhibition of EGFR signaling, utilizing AG1478 and gefitinib, significantly reduced the ability of TGF-ß1 to stimulate fibroblast proliferation and expression of α-smooth muscle actin, collagen, and other extracellular matrix-associated genes. TGF-ß1-stimulated activation of Akt, ERK, and Smad signaling was also significantly inhibited by these interventions. Consistent with these in vitro findings, AR expression was impressively increased in the lungs of TGF-ß1 transgenic mice, and either siRNA silencing of AR or chemical inhibition of EGFR signaling significantly reduced TGF-ß1-stimulated collagen accumulation in the lung. These studies showed a novel regulatory role for AR in the pathogenesis of TGF-ß1-induced pulmonary fibrosis. In addition, these studies suggest that AR, or AR-activated EGFR signaling, is a potential therapeutic target for idiopathic pulmonary fibrosis associated with TGF-ß1 activation.


Assuntos
Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Pulmão/metabolismo , Sistema de Sinalização das MAP Quinases , Fibrose Pulmonar/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Anfirregulina , Animais , Proliferação de Células , Família de Proteínas EGF , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica/genética , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ligantes , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/genética
5.
PLoS One ; 7(9): e45023, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23028742

RESUMO

Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast Schizosaccharomyces pombe, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI(50); 10 µM) and induced cell elongation and septum formation in wild-type S. pombe. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in S. pombe. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in S. pombe deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Naftoquinonas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Técnicas de Silenciamento de Genes , Haploinsuficiência/genética , Heterozigoto , Humanos , Células MCF-7 , Modelos Biológicos , Mutação/genética , Naftoquinonas/química , Naftoquinonas/uso terapêutico , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Interferente Pequeno/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos
6.
Proc Natl Acad Sci U S A ; 106(36): 15279-84, 2009 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-19706407

RESUMO

Mechanical signals regulate blood vessel development in vivo, and have been demonstrated to regulate signal transduction of endothelial cell (EC) and smooth muscle cell (SMC) phenotype in vitro. However, it is unclear how the complex process of angiogenesis, which involves multiple cell types and growth factors that act in a spatiotemporally regulated manner, is triggered by a mechanical input. Here, we describe a mechanism for modulating vascular cells during sequential stages of an in vitro model of early angiogenesis by applying cyclic tensile strain. Cyclic strain of human umbilical vein (HUV)ECs up-regulated the secretion of angiopoietin (Ang)-2 and PDGF-betabeta, and enhanced endothelial migration and sprout formation, whereas effects were eliminated with shRNA knockdown of endogenous Ang-2. Applying strain to colonies of HUVEC, cocultured on the same micropatterned substrate with nonstrained human aortic (HA)SMCs, led to a directed migration of the HASMC toward migrating HUVECs, with diminished recruitment when PDGF receptors were neutralized. These results demonstrate that a singular mechanical cue (cyclic tensile strain) can trigger a cascade of autocrine and paracrine signaling events between ECs and SMCs critical to the angiogenic process.


Assuntos
Comunicação Autócrina/fisiologia , Células Endoteliais/fisiologia , Neovascularização Fisiológica/fisiologia , Comunicação Parácrina/fisiologia , Estresse Mecânico , Fenômenos Biomecânicos , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Veias Umbilicais/citologia
7.
Dev Dyn ; 225(4): 522-35, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12454928

RESUMO

For over a century, amphibian embryos have been a source of significant insight into developmental mechanisms, including fundamental discoveries about the process of induction. The recently developed transgenesis for Xenopus offers new approaches to these poorly understood processes, particularly when undertaken in the quickly maturing species Xenopus tropicalis, which greatly facilitates establishment of permanent transgenic lines. Several X. tropicalis transgenic lines have now been generated, and experiments demonstrating the value of these lines to study induction in embryonic tissue recombinants and explants are presented here. A revised protocol for transgenesis in X. tropicalis resulting in a significant increase in the percentage of transgenic animals that reach adulthood is presented, as well as improvements in tadpole and froglet husbandry, which have facilitated the raising of large numbers of adults. Working transgenic populations have been rapidly expanded, and some transgenes have been bred to homozygosity. Established lines include those bearing the promoter regions of Pax-6, Otx-2, Rx, and EF1alpha coupled to fluorescent reporter genes. Multireporter lines combining, in a single animal, up to three gene promoters coupled to different fluorescent reporters have also been established. The value of X. tropicalis transgenic lines for the study of induction is demonstrated by showing activation of Pax-6 by noggin treatment of Pax-6/GFP transgenic animal caps, illustrating how reporter lines allow a rapid, in vivo assay for an inductive response. An experiment showing lens induction in gamma-crystallin/GFP transgenic lens ectoderm when it is recombined with mouse optic vesicle demonstrates conservation of inducing signals from amphibians and mammals. It also shows how the warmer culture temperatures tolerated by X. tropicalis embryos can be used in assays of factors produced by mammalian cells and tissues. The many applications of transgenic reporter lines and other lines designed to target gene expression in particular tissues promise to bring significant new insights to the classic issues first defined in amphibian systems.


Assuntos
Animais Geneticamente Modificados , Indução Embrionária , Xenopus/embriologia , Xenopus/genética , Animais , Proteínas de Transporte , Linhagem Celular , Olho/embriologia , Proteínas do Olho , Feminino , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas de Homeodomínio/metabolismo , Cariotipagem , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Neurônios/metabolismo , Oócitos/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras , Fatores de Tempo , Proteína Vermelha Fluorescente
8.
Mech Dev ; 117(1-2): 235-41, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12204263

RESUMO

Analysis of gene function in vertebrates is facilitated by gain-of-function studies, such as injection of synthetic mRNA in amphibian embryos. This approach is hampered by lack of spatial and temporal control of expression of the introduced gene product. An additional level of control is obtained by nuclear-transfer-mediated transgenesis, but functional analyses are complicated by variability and background abnormalities in primary transgenic embryos. The GAL4/UAS system permits establishment of stable lines and elimination of nuclear-transfer-associated abnormalities, through generation of separate UAS-'effector' and GAL4 'transactivator' transgenic lines. When the GAL4 DNA-binding domain is combined with a steroid hormone ligand-binding domain, this system allows full temporal regulation of transgene expression by introduction of an exogenous steroid analogue, the progesterone antagonist RU486. We show here that by crossing stable Xenopus tropicalis transgenic lines, one bearing a UAS-enhanced cyan fluorescent protein (ECFP) reporter construct, and the other with a GAL4-progesterone receptor fusion driven by a retina-specific promoter, reporter expression in the resulting embryos can be induced with RU486 in a tissue-specific manner. These results suggest that the inducible binary system, in which the target gene expression can be controlled in a stage- and tissue-specific pattern, should be readily applicable for gene function studies at all stages of development.


Assuntos
Xenopus/embriologia , Xenopus/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Masculino , Microinjeções , Mifepristona/farmacologia , Fenótipo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas de Saccharomyces cerevisiae/genética , Distribuição Tecidual , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Proteínas de Xenopus/genética
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